The Synergistic Effect of Triterpenoids and Flavonoids—New Approaches for Treating Bacterial Infections?

Currently, the pharmaceutical industry is well-developed, and a large number of chemotherapeutics are being produced. These include antibacterial substances, which can be used in treating humans and animals suffering from bacterial infections, and as animal growth promoters in the agricultural industry. As a result of the excessive use of antibiotics and emerging resistance amongst bacteria, new antimicrobial drugs are needed. Due to the increasing trend of using natural, ecological, and safe products, there is a special need for novel phytocompounds. The compounds analysed in the present study include two triterpenoids ursolic acid (UA) and oleanolic acid (OA) and the flavonoid dihydromyricetin (DHM). All the compounds displayed antimicrobial activity against Gram-positive (Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, and Listeria monocytogenes ATCC 19115) and Gram-negative bacteria (Escherichia coli ATCC 25922, Proteus hauseri ATCC 15442, and Campylobacter jejuni ATCC 33560) without adverse effects on eukaryotic cells. Both the triterpenoids showed the best antibacterial potential against the Gram-positive strains. They showed synergistic activity against all the tested microorganisms, and a bactericidal effect with the combination OA with UA against both Staphylococcus strains. In addition, the synergistic action of DHM, UA, and OA was reported for the first time in this study. Our results also showed that combination with triterpenoids enhanced the antimicrobial potential of DHM.

Introduction
Collectively, humans are gradually reverting to harnessing the benefits of nature, especially plants. To maintain and improve our health, we prefer natural products, including therapeutics. Unfortunately, the excessive and inappropriate use of chemotherapeutic agents has harmful effects on human health. Moreover, the increase in the number of bacterial strains resistant to many commercially available antibiotics has drawn the attention of researchers to search for alternative healing compounds. Many have been tested as new candidate antibacterial drugs, for example, antibacterial peptides produced by microorganisms [1], bacteriophages [2], and phytocompounds [3].
Plants are an abundant source of biologically active compounds, which can be potential therapeutics or precursors for the development of new drugs. Some plants may even produce phytocompounds with antimicrobial and antioxidant activities [4]. Selected substances can inhibit the growth of popular pathogens, such as Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa. Moreover, isolated components from various parts of plants showed antiviral, antitumor, anti-inflammatory, antiplatelet, and prostaglandin-inhibitory activities [5,6]. Furthermore, an increase in environmental pollution, especially from the use of antibiotics, has led to the search for safer medicinal products.
Among the main components of plant extracts that have been used in natural medicine for millennia are triterpenoids, which are the most abundant group of terpenoids found in dicotyledonous plants. Although they do not play a significant role in primary metabolism, they are involved in the adaptation of plants for survival. Some triterpenoids function as specific chemical weapons against competitive plants, pathogens, or herbivores. Previous studies have described the antioxidant, antimicrobial, antiallergic, antidiabetic [7], fungicidal [8], antiparasitic [9], and anticancer [10,11] potential of triterpenes. Other studies have also reported their anti-inflammatory [12], analgesic, hepatoprotective [13,14], cardiotonic, and sedative activity [15]. Triterpenoids are synthesised in plants via squalene cyclisation, which is a C30 hydrocarbon [16]. Based on their structural skeleton, triterpenes are classified into several groups: cucurbitanes, cycloartanes, dammaranes, euphanes, fiedelanes, holostanes, hopanes, isomalabaricanes, lanostanes, lupanes, oleananes, protostanes, tirucallanes, and ursanes [17]. The representative pentacyclic triterpenoids are oleanolic acid (OA) and ursolic acid (UA). They are commonly found in nature in free acid form or as an aglycone precursor for a triterpenoid called saponin [14]. The chemical structures of these phytocompounds are shown in Figure 1A-C. These pentacyclic triterpenoids affect the expression of bacterial genes involved in biofilm formation, peptidoglycan turnover, and cell autolysis [18]. In addition, OA and UA along with their derivatives have strong antimutagenic effects [19]. Owing to their similar structures, UA and OA often occur simultaneously. In addition, the activities of these triterpenes also include their potential to enhance bacterial susceptibility to other compounds [20]. Dihydromyricetin (DHM) belongs to the flavonoid family, whose chemical structure is shown in Figure 1C. It is a major secondary metabolite of the plant Ampelopsis grossedentata, which is widely distributed in the mountainous areas of China and has been used in herbal medicine for centuries [21]. Previous studies have shown many valuable properties of this compound, including antioxidant [21], anti-inflammatory [22], antibacterial [23][24][25][26], neuroprotective [27], anticancer [28], and metabolic regulation of glucose and lipids [29]. It is apparent that DHM has a wide range of prospects in the food industry as an antioxidant and antibacterial agent. However, the antimicrobial mechanism of this compound has not been adequately investigated [30]. Previous reports have confirmed that hydroxylation at positions 5 and 7 of their structure can play a key role in the antibacterial activity of flavanols, and that hydroxylation of the B and C rings can enhance it [31].
However, there are many contrasting data in the literature. The reason for this may be the test methods used, the purity of the phytocompound, and/or the bacterial strain used.
In addition to the possibility of using phytochemicals in medicine, their application in the food industry is also of interest, where food-borne diseases still pose a threat. In recent years, synthetic preservatives have been commonly used in food because of their low cost and high antimicrobial activity. However, increasing consumer demand for safe, unprocessed foods and prolonged storage time is mobilizing the food industry to introduce natural antimicrobial components as synthetic preservative replacements.
The aim of this study was to investigate the antibacterial activity of phytocompounds (UA, OA, and DHM) individually and in various combinations. Finally, the cytotoxic activity of the phytocompounds was determined in vitro using mammalian cells.

Antimicrobial Activity of Phytocompunds
Selected phytocompounds were tested for antimicrobial activity against six bacterial strains (S. aureus, S. epidermidis, L. monocytogenes, E. coli, P. hauseri, and C. jejuni) using the microdilution method. The antibacterial potential of UA, OA, and DHM is shown in Figures 2-4 and Table S1.   Triterpenoids showed great activity toward Gram-positive strains. The addition of UA (20 µg/mL) reduced the growth of the Gram-positive strains S. aureus and S. epidermidis by 80%. In the case of the Gram-negative strain, L. monocytogenes, a higher concentration (40 µg/mL) of the compound resulted in 60% growth inhibition ( Figure 2). Moreover, we also noted that OA was more effective against Gram-positive bacteria ( Figure 3). These similar effects of the tested triterpenoids may be due to their related structures. However, higher concentrations of the compound had to be added, compared to UA. Previous studies have demonstrated high antibacterial activity of UA [15,32,33] and its derivatives [34] against S. aureus. Methicillin-resistant S. aureus (MRSA) infections are a serious problem in hospitalised patients. Olean-27-carboxylic acid-type triterpenes possess antibacterial activity against various MRSA strains as well as quinolone-resistant S. aureus [35]. In addition, the antimicrobial potential of UA and OA against L. monocytogenes have also been confirmed [36,37]. However, Panizzi et al. [38] described the lack of OA (from Geum rivale) activity against S. aureus, E. coli, and P. aeruginosa. Similarly, Calis et al. [39] did not observe antibacterial activity of OA (from Cyclamen mirabile) against Gram-positive (S. aureus and E. faecalis) and Gram-negative (P. aeruginosa and E. coli) bacteria. The diversity of the antimicrobial properties of UA and OA has also been observed against Mycobacterium tuberculosis, which is the most common cause of deaths worldwide [40,41]. In addition, bacteria, such as E. faecalis [42], Streptococcus pneumoniae [33], E. faecium, Bacillus subtilis, and B. cereus [32,42,43] are sensitive to OA treatment. Cunha et al. [43] investigated the antimicrobial effect of OA against Streptococcus strains (S. mutans, S. sanguis, S. mitis, and S. salivarius) and determined that both the hydroxy and carboxy groups in triterpenes were responsible for OA antimicrobial activity. It has been proven that the tested pentacyclic triterpenoids affect peptidoglycan structure, gene expression, and biofilm formation [44,45] in bacteria. Another mechanism of action can be associated with the induction of stress response. Grudniak et al. [46] showed that E. coli treated with OA altered the synthesis of DnaK, thus inducing a heat-shock response in this species. When Zhou et al. [47] used TEM to analyse the morphological changes in MRSA cells after treating them with UA and oxacillin, they observed cell membrane disintegration, cell lysis, and cytoplasmic content release.
In the present study, the triterpenoids showed less activity toward Gram-negative bacteria. The addition of UA (50 µg/mL) limited the growth of E. coli, P. hauseri, and C. jejuni by 20-30%, whereas that of OA (50 µg/mL) inhibited the growth of the Gram-negative strains by 10-30%. Some studies have described poor UA activity against E. coli [48]. Mallavadhani et al. [49] showed moderate activity of UA and its lipophilic 3-O fatty acid ester chains (C12-C18) against Gram-negative strains (E. coli, S. typhi, and P. syringae). Interestingly, it has been reported that UA (50 mg/mL) could be a therapeutic agent for treating Helicobacter pylori infections [50].
Based on the results shown in Figures 2 and 3, the triterpenoids were more active against Gram-positive bacteria than Gram-negative bacteria. This may be due to the structural differences in the cell walls of these bacterial classes. Cells of Gram-negative bacteria are surrounded by an additional outer membrane, which provides them with a hydrophilic surface that functions as a permeability barrier against many substances, including natural compounds [51,52]. Moreover, the intrinsic resistance in Gram-negative bacteria is supported by efflux pumps which pump many compounds, such as toxins and antibiotics from the periplasm to the outside of the cell [53,54].
The next part of our experiment investigated the antibacterial potential of DHM against the tested bacterial strains. The results showed that with an increase in DHM concentration, the inhibition level increased. The strain most sensitive to the action of DHM was S. epidermidis. At a concentration of 45-50 µg/mL, growth inhibition was 65%. Other Gram-positive bacteria showed moderate sensitivity to the phytocompounds, but only at higher concentrations (35-50 µg/mL). Wu et al. [23] has previously described the antibacterial potential of DHM against S. aureus. Moreover, we observed that the Gramnegative strains were weakly susceptible to DHM at each concentration range. Addition of DHM at a concentration of 50 µg/mL caused a 26%, 13%, and 24% growth reduction of E. coli, P. hauseri, and C. jejuni, respectively. Xiao et al. [55] demonstrated that DHM has great antibacterial activity against tested food-borne bacteria (S. aureus, B. subtilis, E. coli, S. paratyphi, and P. aeruginosa). Moreover, SEM analysis (E. coli and S. aureus) suggested that DHM induces aggregation, shrivelling, and adhesion of bacteria. Cui et al. [56] performed SEM analysis of E. coli treated with DHM and reported that the cell had a wrinkled surface and was lysed at both ends. Apart from disturbing the integrity of the cellular membrane, DHM can also inhibit the respiratory metabolism of bacteria [55].

Combined Antimicrobial Activity of Selected Phytocompounds
Given the issue of multidrug-resistant strains, new ways to eliminate these pathogens must be developed, such as combinatorial therapy using phytocompounds. Therefore, studying the synergistic effects of the compounds has become a key step in phytochemical studies [57,58]. Therefore, we tested the antimicrobial synergy of the selected phytocompounds against the test strains. The results are presented in Figure 5. The most representative synergistic effect was observed by the treatment of 20 µg/mL OA in combination with 20 µg/mL UA against both the Staphylococcus strains, with bactericidal effect ( Figure 5, Table 1, Table S2). Based on the previous experiment, we found that DHM had the weakest effect on the tested bacteria ( Figure 4, Table 1). Our results showed that combination with triterpenoids (UA or OA) enhanced the antimicrobial potential of DHM. Coadministration of UA and DHM (20 µg/mL each) considerably reduced the growth of Gram-negative strains. The addition of both UA and DHM reduced bacterial growth by 60% for E. coli and approximately 45% for P. hauseri and C. jejuni (Figures 2 and 4, Table 1). However, when UA and DHM were tested individually, bacterial growth was inhibited by only 7% and 6%, respectively ( Figure 5, Table 1). In the case of Gram-negative strains, treatment with phytocompounds (10 and 20 µg/mL) alone did not inhibit bacterial growth. Thus, only a combination of these compounds yielded satisfactory results.
We concluded that the most promising results of synergistic effects were obtained in a combination of UA and OA. The data also indicated the possibility of reducing the dosage of the compounds.

Assessment of the Cytotoxic Activity of Selected Phytocompounds
A safe antibacterial agent should be nontoxic to eukaryotic cells and show robust activity against microorganisms. The cytotoxic activity of the phytocompounds was studied in several variants at 20 µg/mL concentration ( Figure 6). The effect of UA, OA, and DHM (individual or simultaneous addition to the bacterial culture) on the viability of human fibroblasts was assessed via the MTT assay. The percentage of viable cells was computed relative to that of the control (cells incubated without phytocompounds), the viability of which was considered 100%. After 24 h of incubation of the cells with the phytocompounds alone, cell viability slightly decreased to 90% after OA and DHM treatment and to 74% after UA treatment. Fibroblast viability reduced to 72%, 74%, and 85% in the cell cultures supplemented with two of the compounds (synergistic effect): UA+OA, UA+DHM, and OA+DHM, respectively. Wójciak-Kosior et al. [59] analysed the cytotoxic activity of UA and OA against human skin fibroblasts and reported higher cytotoxic activity of UA compared to that of OA. A similar relationship was observed in the present study. Zhang et al. [60], however, reported no cytotoxic effect of UA against HCT-8 and Bell-7402 cell lines. DHM has been shown to exhibit selective cytotoxicity against non-small-cell lung cancer cells (A549 and H1975) but not against normal cells (WI-38) [61] (Kao et al., 2017). DHM has also been shown to be noncytotoxic to normal hepatocytes [62,63], but it inhibited hepatocellular carcinoma (HCC) cell proliferation and triggered apoptosis in a p53-dependent manner [63].

Determination of Antimicrobial Activity
The antimicrobial activity of UA, OA, and DHM was evaluated using the microdilution method for aerobic and anaerobic bacterial strains according to the CLSI documents M07 (11th Edition) [64] and M11 (9th Edition) [65], respectively. The antimicrobial activity of the phytocompounds was determined against aerobic bacteria (S. aureus ATCC 6538, S. epidermidis ATCC 12228, E. coli ATCC 25922, and P. hauseri ATCC 15442) and anaerobic bacteria (L. monocytogenes ATCC 19115 and C. jejuni ATCC 33560). The growth of the aerobic and anaerobic bacterial strains that were either treated with the phytocompounds or left untreated was evaluated in 96-well microtiter plates in Mueller-Hinton broth and Brucella broth supplemented with hemin, vitamin K 1 , and laked horse blood, respectively. The phytocompounds were supplemented in a range of 5-50 µg/mL, and their antimicrobial potential was determined. They were diluted in the appropriate growth medium before administration. An inoculum of bacteria grown in the Mueller-Hinton or Brucella broths was added to each well to achieve a final density of 5 × 10 5 CFU/mL and 1 × 10 6 CFU/mL for the aerobic and anaerobic strains, respectively. The microtiter plates were then incubated for 24 h at 37 • C (aerobic strains) and for 48 h at 37 • C (anaerobic strains). The plates inoculated with anaerobic strains were incubated in jars where anaerobic conditions were achieved using GasPak envelopes and monitored with a disposable BBL dry anaerobic indicator strip (Becton Dickinson). After incubation, the optical density was measured spectrophotometrically at 620 nm. Experiments for each type of phytocompound were performed in triplicate, leading to the analysis of six independent experiments. The antimicrobial activity of the tested compounds was calculated as the percentage of bacterial growth inhibition (SD) compared to that of the biotic control (bacteria incubated in the medium).

Determination of Cytotoxic Activity of Phytocompounds
The cytotoxic potential of phytocompounds against human cells was examined using the fibroblast BJ ATCC CRL-2522 cell line. Cells were suspended in Dulbecco's modified Eagle medium (DMEM) containing foetal bovine serum (10%) and antibiotics (penicillin 100 IU/mL and streptomycin 100 µg/mL) at a final density of 1 × 10 5 cells/well. The final volume of the culture was 100 µL. The fibroblasts were incubated in 96-well microplates in a humidified atmosphere at 37 • C and 5% CO 2 for 24 h. After incubation, exhausted DMEM was replaced with fresh medium and phytocompounds were supplemented at 10-20 µg/mL. Adequate control cultures that were untreated with the selected phytocompounds were also prepared and the plates were incubated under the same conditions. After 24 h of incubation, the media were removed from the cells and the wells were supplemented with 500 µg/mL MTT. Following a 2 h incubation under the same conditions, the solution was removed from the wells, which were then refilled with 100 µL of sterile DMSO to dissolve the formazan crystals. The viability of fibroblasts was calculated based on the spectrophotometric quantification of the cultures at λ = 550 nm using a SpectraMax i3x multimode microplate reader (Molecular Devices Ltd., Wokingham, Berkshire, UK). The results are presented as the mean values of the percentages of fibroblast viability in control cultures untreated with phytocompounds with the SD. The experiment, with n = 4, was performed in triplicate.
In addition, the combined effects of the tested compounds on the bacteria were investigated. This allowed for the selection of the best combination of two compounds, which could allow for reduction in dosage.

Conclusions
Nature is a rich source of plant resources that possess bioactive phytocompounds. As such, medicinal phytocompounds are one of the best sources for obtaining new therapeutics that would be clinically effective, biodegradable, and safe for human use. The present work enabled us to document the antibacterial activity of UA, OA, and DHM individually and in combination with each other to test for synergy. Triterpenoids (UA and OA) proved to be successful antibacterial agents, particularly in Gram-positive strains, and considering the cytotoxicity results, all the tested phytocompounds would be safe for use in the pharmaceutical industry.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.


Association of MTHFR 677C>T polymorphism with pregnancy outcomes in IVF/ICSI-ET recipients with adequate synthetic folic acid supplementation

(HCY) can impair female fertility. Folic acid (FA) supplementation is necessary in pregnant women who are undergoing in-vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) - embryo transfer (ET), and especially in women with MTHFR rs1801133 C-to-T mutations. At present, affordable and accessible synthetic FA is mainly used. However, some studies have suggested that 5-methylenetetrahydrofolate (5-MTHF), a type of active FA, may be more suitable for women with the MTHFR 677C>T polymorphism, since it is safer and more effective. This retrospective study aimed to evaluate whether the MTHFR rs1801133 gene polymorphism is related to the pregnancy outcomes of IVF/ICSI-ET recipients after sufficient supplementation with FA instead of 5-MTHF. Data on 692 women undergoing IVF/ICSI-ET and taking adequate FA were collected. Participant characteristics were compared using the Kruskal-Wallis test and Pearson chi-square test. Logistic regressions were used to calculate the odds ratio (OR) and 95% confidence interval (95% CI), after adjusting for age, BMI, method of fertilization, method of embryo transfer and number of embryos transferred. An additive model (T/T vs. C/C), dominant model (C/T + T/T vs. C/C), and recessive model (T/T vs. C/T + C/C) were evaluated. Analysis revealed that MTHFR rs1801133 in IVF/ICSI-ET women with adequate FA supplementation was not associated with the pregnancy rate but with age (OR = 0.91, 95% CI = 0.88, 0.94, P < 0.001) and BMI (OR = 0.95, 95% CI = 0.90, 0.997, P = 0.037). In 349 clinically pregnant women, no association of the MTHFR 677C>T with pregnancy outcomes was found in the additive model, dominant model, or recessive model. Of the 273 women with positive pregnancy outcomes, 34 had a preterm delivery. MTHFR 677C>T was not associated with a preterm delivery after adjusting for age and BMI. The current results indicated that MTHFR polymorphism rs1801133 was not related to the pregnancy rate or pregnancy outcomes of women undergoing IVF/ICSI-ET with adequate synthetic FA supplementation, suggesting that simple supplementation with less expensive and readily available FA, rather than expensive 5-MTHF, appeared to be appropriate.

T h e m e t h y l e n e t e t r a h y d r o f o l a t e r e d u c t a s e (MTHFR) gene is located at the end of the short arm of chromosome 1 (chr1:11796321), and is 2.2 kb long. One of the most common single nucleotide polymorphisms (SNPs) in the MTHFR gene is rs1801133 (677C>T). A systematic review and meta-analysis indicated that the overall T allele frequency of MTHFR rs1801133 was 36.9% in Chinese (6), and 78.4% of Chinese people have homozygous or heterozygous mutations (7); this figure exceeds that in many other countries. The C-to-T transition results in a missense mutation that changes alanine (Ala) to valine (Val), which reduces the thermal stability and activity of MTHFR (8). The enzyme activity of individuals with homozygous TT mutation was about 30% of that in individuals with the wild-type (CC) genotype, whereas individuals with the heterozygous genotype (CT) had about 65% of the wild-type enzyme activity (9). Subsequently, the mutated genotype reduced the capacity to convert 5,10-methylenetetrahydrofolate (5,10-MTHF) to 5-methylenetetrahydrofolate (5-MTHF), the predominant circulating form of folate, thus decreasing the utilization of folate (10) (Figure 1). Several studies have suggested that the MTHFR gene is a major genetic factor for adverse pregnancy outcomes (APOs) (11)(12)(13)(14). Moreover, intake of FA from supplements has been found to reduce the risk of spontaneous abortion and pregnancy complications (15)(16)(17).
In-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and embryo transfer (ET) are assisted reproductive techniques (ARTs). Several studies have indicated that women after IVF/ICSI-ET have a higher spontaneous miscarriage rate than those after natural conception (18,19). In addition, IVF/ICSI-ET was found to be associated with preterm delivery (20). With the implementation of the three-child policy and the increase in women of advanced maternal age in China, the need for and use of ART is also estimated to be increasing.
FA supplementation is necessary in pregnant women who are undergoing ART, and especially in women with MTHFR rs1801133 C-to-T mutations. Therefore, the choice of the type of FA supplementation is not only a family-related but also a social issue that needs to be addressed. At present, synthetic FA is mainly used. Synthetic FA is a form of FA extracted and synthesized from the chemical raw material L-N-paminophenylmethylcool glutamic acid or 2,4,5-triamino -6-hydroxypyrimidine sulfate, and it has the advantages of good stability and affordability. China has been implementing a folate fortification policy since 2009, providing free supplements of synthetic FA to women of childbearing age.
Nevertheless, studies have pointed out that the conversion of synthetic FA to 5-MTHF is limited in women with the MTHFR rs1801133 CT or TT genotype (21,22), and FA is inactive until reduced into the bioactive folate derivative, 5-MTHF (23). Active FA, 5-MTHF, is the main circulating form of folate in the body, and it can bypass the MTHFR block. However, whether active FA is better than synthetic FA in preventing APOs remains a subject of controversy (24,25). Folate levels in women are still low even in countries with mandatory folate fortification (21). Since China is a developing country with a large population, affordable and accessible synthetic FA is more readily available than 5-MTHF, which is more expensive. Therefore, the aim of the current study was to evaluate whether MTHFR rs1801133 gene polymorphism was related to the pregnancy outcomes of IVF/ICSI-ET subjects after taking sufficient synthetic FA supplements.

Study population
The study population was 692 women who underwent IVF/ICSI-ET at Zhoushan Women's and Children's Health Hospital from 2016 to 2020. All of the women had adequate synthetic FA supplementation depending on their genotype, and they sought the treatment due to Figure 1. Possible mechanism of MTHFR rs1801133 affecting folic acid and pregnancy outcomes. MTHFR rs1801133 will lead to a missense mutation that changes Ala to Val, which can decrease the thermal stability and reduce the activity of MTHFR. The enzyme activity of individuals with homozygous TT mutation is about 30% of that in individuals with the wild-type (CC) genotype, whereas individuals with the heterozygous genotype (CT) have about 65% of the wild-type enzyme activity. Subsequently, the mutated genotype reduces the capacity to convert 5,10-MTHF to 5-MTHF, the predominant circulating form of folic acid, thus decreasing the utilization of folic acid.

Determination of pregnancy
Twelve days after transplantation, a urine pregnancy test or a blood HCG quantitative test was conducted. For HCG-positive patients, the first vaginal ultrasound was performed 21 days after transplantation to exclude ectopic pregnancy. Ultrasound was performed again after 28 days to determine the number of embryos and their development.

FA supplementation
Patients were encouraged to take FA (Silian, China), instead of 5-MTHF, depending on their genotype. For the CC genotype: 400 μg/day FA was taken three months before pregnancy, 400 μg/day FA was taken in early pregnancy (0-12 weeks), and food supplementation was considered in middle/late pregnancy (13-40 weeks), but no extra supplementation was needed. For the CT genotype: FA was supplemented 400 μg/day three months before pregnancy, 800 μg/day in early pregnancy (0-12 weeks), and 400 μg/day in middle/late pregnancy (13-40 weeks). For the TT genotype: FA was supplemented 800 μg/day three months before pregnancy, 800 μg/day in the early pregnancy (0-12 weeks), and 400 μg/day in middle/ late pregnancy (13-40 weeks).

Statistical analysis
Characteristics of participants with different genotypes were compared using the Kruskal-Wallis test for continuous variables and the chi-square test or Fisher's exact test for discrete variables. Genotype and allele frequencies were calculated. Observed genotype frequencies in different genotypes were separately tested for deviation from the Hardy-Weinberg equilibrium (HWE) using the exact test. Logistic regressions were used to calculate the odds ratio (OR) and 95% confidence interval (95% CI), after adjusting for age, body mass index (BMI), method of fertilization, method of embryo transfer and number of embryos transferred. An additive model (T/T vs. C/C), dominant model (C/T + T/T vs. C/C), and recessive model (T/T vs. C/T + C/C) were evaluated to assess the strength of association between MTHFR polymorphism rs1801133 and pregnancy outcomes. All significance tests were two-sided; a P value of < 0.05 was considered to be statistically significant. Data analyses were performed using the software platform SPSS v.26.0 (IBM, Armonk, NY, USA).

Participant characteristics
The age range of the 692 women was between 22 and 49 years of age, and the range of their BMI was fallopian tube or ovulation disorders or rare, weak, or abnormal sperm of their husbands.

Genotype determination
The extraction of whole-genome DNA from blood samples was performed using QIAquick PCR purification kits (QIAGEN, Germany). The MTHFR rs1801133 genotype was determined using a fluorescence PCR detection kit (PCR-fluorescence probe) designed by Osama Medicine (Shenzhen, Guangdong, China). Genotyping was performed under the following conditions: a 25-μL whole blood sample, a 16-μL PCR reaction system, reaction conditions of 45 cycles, 95℃ denaturation for 15 s, and 60℃ annealing/extension for 1 min, as recommended by the manufacturer. After the reaction was completed, the end point fluorescence in sample wells was read on the ABI 7900 fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA), and the genotyping results for each sample were determined using the ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Ovarian stimulation and oocyte retrieval
A controlled ovarian hyperstimulation protocol was tailored to the individual. According to comprehensive factors such as age of the woman, ovarian preparation, and outcomes of previous ovulation induction protocols, a long luteal phase protocol, a long early follicular phase protocol, an overlength protocol, a short protocol, an antagonist protocol, a natural cycle protocol, or a microstimulation protocol was adopted. When the diameter of two dominant follicles was ≥ 18 mm and that of three follicles ≥ 16mm, human chorionic gonadotropin (HCG) (a recombinant HCG alfa solution for injection, Merck Serono Sweden; chorionic gonadotrophin for injection, Livzon China, 6500/8500 IU) was injected at 9 PM on the same night. If the follicle diameter was ≥ 14 mm and urine luteinizing hormone (LH) was positive or blood LH > 10 mIU/ml, the oocyte was retrieved 24 hours after the immediate injection of HCG. Oocyte retrieval was performed under ultrasound guidance using a K-OPSD oocyte retrieval needle (Cook, Australia).

IVF/ ICSI-ET
The oocytes and sperm are then fertilized using IVF or ICSI and cultured in vitro on cleavage stage/blastocyst culture medium (Vitrolife Sweden). Embryo transfer was performed using a K-JETS catheter (Cook, Australia) 3-5 days after oocyte retrieval. Women younger than 35 years of age or who had received IVF/ ICSI-ET for the first time underwent single embryo transfer, while women older than 35 or who had failed IVF/ ICSI-ET several times received 2-3 embryos. between 32.66 kg/m 2 and 16.02 kg/m 2 . Based on the genotype of MTHFR rs1801133, 316 (45.66%) women were classified as wildtype homozygotes (CC), 226 (32.66%) women were classified as heterozygotes (CT), and 150 (21.68%) women were classified as mutated homozygotes (TT). There were no differences found regarding age (P = 0.34), BMI (P = 0.53), method of fertilization (P = 0.83), method of embryo transfer (P = 0.44), or number of embryos transferred (P = 0.82) among the three genotypes (Table 1).

Pregnancy outcomes
The pregnancy outcomes were compared among the different genotypes. Results revealed no significant differences in pregnancy rates of wildtype homozygotes (CC, 47.15%), heterozygotes (CT, 39 Table 2). The proportion of preterm deliveries by women with a positive pregnancy outcome is also shown in Table 2. 3.3. Association of the MTHFR rs1801133 genotype with a successful pregnancy All DNA samples were successfully genotyped for rs1801133, and this SNP deviated from HWE (P < 0.01) in women receiving IVF/ICSI-ET. The proportions of each genotype among 385 pregnant and 307 non-    Table 3. Logistic regression was used to analyze the association between theMTHFR rs1801133 genotype and a successful pregnancy, after adjusting for age, BMI, method of fertilization, method of embryo transfer, and number of embryos transferred. An additive model, dominant model, and recessive model were all assessed (Table 3). Results indicated that MTHFR genetic polymorphism rs1801133 was not associated with a successful pregnancy but with age (OR = 0.91, 95% CI = 0.88-0.94, P < 0.001) and BMI (OR = 0.95, 95% CI = 0.90, 0.997, P = 0.04) ( Table 3) (Figure 2).

Association of the MTHFR rs1801133 genotype with pregnancy outcomes
Of 385 pregnant women, 349 were clinically pregnant, and 36 were biochemically pregnant. The proportions of genotypes in positive and adverse pregnancy outcomes are shown in Table 4. Results indicated that after adjusting for covariates, MTHFR rs1801133 was not associated with pregnancy outcomes in the additive model, dominant model, or recessive model. However, a younger age (OR = 0.92, 95% CI = 0.86-0.98, P = 0.01) was positively associated with pregnancy outcomes (Table 4) (Figure 2).

Association of the MTHFR rs1801133 genotype with preterm delivery
The association of MTHFR rs1801133 with a preterm delivery was further analyzed in 273 women with positive pregnancy outcomes. The genotype proportions in preterm deliveries and normal deliveries are shown in Table 5. MTHFR rs1801133 was not associated with a preterm delivery after adjusting for age and BMI (Table 5) (Figure 2).

Figure 2. Flow diagram of the study population and results.
A total of 692 women were included. They all received sufficient folic acid supplementation, and their genotype of MTHFR rs1801133 was determined. These participants were divided into two groups depending on whether they were pregnant or not. Then, the participants who were pregnant were divided into two groups, those with a positive pregnancy and those with an adverse pregnancy. The participants with a positive pregnancy were divided into two groups, those with a preterm delivery and those with a normal delivery.

Discussion
This study found no association between MTHFR polymorphism rs1801133 and pregnancy outcomes of women undergoing IVF/ICSI-ET with adequate synthetic FA supplementation. MTHFR rs1801133 fits the additive model, but results were also analyzed in a dominant model and a recessive model. Age was found to be related to the pregnancy rate (OR = 0.91, 95% CI = 0.88-0.94, P < 0.001) and pregnancy outcome (OR = 0.92, 95% CI = 0.86-0.98, P = 0.01), which was consistent with the results of previous studies (18,26). Older women may have an increased risk of impaired oocyte quality and chromosomal abnormalities and decreased endometrial receptivity (27)(28)(29). In addition, BMI also had an impact on the pregnancy rate (OR = 0.95, 95% CI = 0.90, 0.997, P = 0.04). Obese women often have impaired folliculogenesis, ovulation, and conception, resulting in decreased reproductive potential (30).  The conversion of 5,10-MTHF to 5-MTHF, a cosubstrate for the re-methylation of HCY to methionine, requires the protein encoded by the MTHFR gene. The T allele of rs1801133 will lead to decreased activity of MTHFR, thus affecting the conversion of 5,10-MTHFR to 5-MTHF, and reduce the amount of folate circulating in the blood. In addition, the T allele of rs1801133 can also reduce the rate of FA utilization (8). Studies have been conducted to evaluate the association between MTHFR genetic polymorphism rs1801133 and pregnancy outcomes. Some have found that the mutated T allele of MTHFR rs1801133 was associated with a higher risk of adverse outcomes, including spontaneous abortion, premature birth, and stillbirth (2,11,(31)(32)(33).
Folate deficiency and its resulting HCY accumulation can impair female fertility; possible mechanisms for this include reduced cell division, increased apoptosis, overproduction of inflammatory cytokines, impaired nitric oxide (NO) metabolism, oxidative stress, and defective methylation reaction (34). Moreover, maternal demand for folate increases during pregnancy, and a maternal folate deficiency often leads to APOs (35). Previous in vivo experiments have found that in the absence of maternal folate, placental mTOR signaling and amino acid transporter activity are inhibited (36) as well as the uterine decidualization (37) and decidual angiogenesis in pregnant mice (38), subsequently causing placental dysplasia and dysfunction and ultimately resulting in fetal growth restriction. A case-control study in Venezuela confirmed the association between maternal folate deficiency and an increased risk of a preterm delivery at the end of the third trimester and in labor (39). Another case-control study in Sweden suggested that the increased risk of early spontaneous abortion was also associated with low plasma folate levels (40).
Taking high doses of FA can prevent developmental delay and placental abnormalities (41) that may reduce the risk of low birth weight and premature birth (36). FA supplementation is especially necessary in patients undergoing IVF/ICSI-ET because of the inherent risk of an adverse pregnancy. Patients taking FA supplements were found to have significantly reduced HCY levels in follicle fluid, indicating that the recovered oocytes would be more mature and of better embryo quality (42).
However, studies in recent years have indicated that 5-MTHF is safer than FA. 5-MTHF is comparable to FA in reducing HCY, and it is comparable to or more effective than FA in maintaining serum and plasma folate levels (43). Compared to FA, 5-MTHF is less likely to cause unmetabolized folic acid (UMFA) syndrome or mask a B12 deficiency (43).
A Chinese study analyzed MTHFR polymorphism in normal pregnant patients and found that active FA (5-MTHF) had a significant therapeutic effect for patients with MTHFR rs1801133 C-to-T mutations.
In patients without such a mutation, the therapeutic effect of 5-MTHF did not differ significantly from that of FA (25). A possible explanation for this is that 5-MTHF does not require the already reduced activity of MTHFR in individuals with the MTHFR 677C>T polymorphism to convert 5,10-MTHFR to 5-MTHF to become effective, but it can function directly.
5-MTHF appears to be more advantageous in women with the MTHFR 677C>T polymorphism than FA. However, a randomized, double-blind, placebocontrolled trial found that there was no difference in the abortion rate between women with MTHFR 677C>T polymorphism taking 5-MTHF and those taking FA; that is, there was no beneficial effect of 5-MTHF compared to FA supplementation (44). The current study also found that for women undergoing IVF/ICSI-ET, taking a sufficient amount of FA, rather than 5-MTHF, starting 3 months before conception can lead to a result where the MTHFR 677C>T genotype is irrelevant to the pregnancy outcome.
Although 5-MTHF has a slight advantage in increasing serum folate levels for the population with the MTHFR 677C>T polymorphism, previous studies found no difference in the effect on HCY in groups taking FA or 5-MTHF (44,45). Pharmacokinetic studies have also attempted to explain the possible reason why 5-MTHF did not appear superior to FA in improving pregnancy outcomes. On each of the four mornings following the start of dosing (7.5 mg/day), the serum total folate level of 5-MTHF was 23 to 55% higher than that of FA. Interestingly, 12 days later, when both groups continued to take a dose of 0.4 mg/day, serum total folate levels in the 5-MTHF and FA groups were indistinguishable (21). One of the advantages of 5-MTHF is that it can replenish the body's reserves more quickly in women with a folate deficiency. However, the quick replenishment for women undergoing IVF/ICSI-ET seems unnecessary since they usually start taking a full, genotypic dose of FA three months before conception.
For developing countries like China, affordable and more accessible synthetic FA seems to be cost-effective for most women than expensive 5-MTHF at the current stage. Because, according to the current results, the use of synthetic FA alone, in a sufficient amount and with enough time, can nullify the association between the MTHFR 677C>T genotype and pregnancy outcomes in IVF/ICSI-ET recipients. However, the related mechanism and whether FA alone is enough to eliminate the effects of the MTHFR 677C>T polymorphism still needs to be investigated further.
In summary, the current study did not find that MTHFR polymorphism rs1801133 was related to the pregnancy rate or pregnancy outcomes of women undergoing IVF/ICSI-ET with adequate synthetic FA supplementation, suggesting that simple supplementation with less expensive and readily available synthetic FA, rather than expensive 5-MTHF, appeared to be appropriate.


A novel epithelial-mesenchymal transition (EMT)-related gene signature of predictive value for the survival outcomes in lung adenocarcinoma

Lung adenocarcinoma (LUAD) is a remarkably heterogeneous and aggressive disease with dismal prognosis of patients. The identification of promising prognostic biomarkers might enable effective diagnosis and treatment of LUAD. Aberrant activation of epithelial-mesenchymal transition (EMT) is required for LUAD initiation, progression and metastasis. With the purpose of identifying a robust EMT-related gene signature (E-signature) to monitor the survival outcomes of LUAD patients. In The Cancer Genome Atlas (TCGA) database, least absolute shrinkage and selection operator (LASSO) analysis and cox regression analysis were conducted to acquire prognostic and EMT-related genes. A 4 EMT-related and prognostic gene signature, comprising dickkopf-like protein 1 (DKK1), lysyl oxidase-like 2 (LOXL2), matrix Gla protein (MGP) and slit guidance ligand 3 (SLIT3), was identified. By the usage of datum derived from TCGA database and Western blotting analysis, compared with adjacent tissue samples, DKK1 and LOXL2 protein expression in LUAD tissue samples were significantly higher, whereas the trend of MGP and SLIT3 expression were opposite. Concurrent with upregulation of epithelial markers and downregulation of mesenchymal markers, knockdown of DKK1 and LOXL2 impeded the migration and invasion of LUAD cells. Simultaneously, MGP and SLIT3 silencing promoted metastasis and induce EMT of LUAD cells. In the TCGA-LUAD set, receiver operating characteristic (ROC) analysis indicated that our risk model based on the identified E-signature was superior to those reported in literatures. Additionally, the E-signature carried robust prognostic significance. The validity of prediction in the E-signature was validated by the three independent datasets obtained from Gene Expression Omnibus (GEO) database. The probabilistic nomogram including the E-signature, pathological T stage and N stage was constructed and the nomogram demonstrated satisfactory discrimination and calibration. In LUAD patients, the E-signature risk score was associated with T stage, N stage, M stage and TNM stage. GSEA (gene set enrichment analysis) analysis indicated that the E-signature might be linked to the pathways including GLYCOLYSIS, MYC TARGETS, DNA REPAIR and so on. In conclusion, our study explored an innovative EMT based prognostic signature that might serve as a potential target for personalized and precision medicine.

Introduction
Lung adenocarcinoma (LUAD), as the predominant histological type of lung cancer, has biological characteristics of strong aggressiveness and heterogeneity (1)(2)(3). In spite of optimized treatment methods including surgery, chemotherapy, radiotherapy, immunotherapy and targeted therapy, prognosis of LUAD patients remains dismal, because of cancer progression (4, 5). Collectively, it is imperative to distinguish populations at a high-risk of LUAD for early intervention and improving clinical outcome. At present, the combination of clinicopathological features and TNM staging system is the consensus criterion for determining treatment options and predicting relapse of LUAD, however this criterion restrains the provision of optimal clinical care to patients (6). Hence, identifying reliable biomarkers for optimizing the prognosis of LUAD is urgently needed.
Metastases are the primary cause of LUAD-associated mortality (7,8). Migratory tumor cells escape from the primary site, remodel the basement membrane to engage with peritumoural stroma, undergo intravasation, endure shear stress in circulation and adapt to the tumor microenvironment of distant metastasis (9,10). In total, the metastatic process generally involves three distinct phases: dissemination, dormancy and colonization (11). Epithelial mesenchymal transition (EMT) of cancer cells is a fundamental event during the multistep process participated in cancer metastasis (12,13). Moreover, EMT activation confers on tumor cells to acquire plasticity with more aggressive phenotype, which exerts a decisive function on the malignant cancer progression (14,15). Accumulating evidence has revealed that EMT process was closely implicated in tumorigenicity, angiogenesis and drug resistance (16)(17)(18). EMT results in a series of changes during epithelial tumor cells transforming into mesenchymal cells, including loss of tight junctions, cell polarity, cytoskeletal reorganization, and increase of cell viability (19). EMT is controlled or induced by a number of factors such as exosomal There is a significant link between the aberrant expression of genes related with EMT and poor clinical outcomes in LUAD patients (13,23).
Since the advent of next generation sequencing, research on bioinformatic analysis has flourished (24; 25). For instance, The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database, such public databases are desirable to access transcriptomic information, that advances the efficient methods to select gene signatures (26-28). Numerous studies have attempted to construct the risk model to get biological characteristics or prognostic appraisal in malignant tumors, which had potential clinical impact (29)(30)(31). Interestingly, EMT-related gene signature (E-signature) could reveal the prognostic consequences in various types of cancer (32)(33)(34). Nevertheless, the existence of heterogeneity in samples among diversified studies on various tumours resulting in different risk models (35). Recently, some researchers reported the prognostic significance of E-signature, however they did not validate the biological functions of EMT-related genes with in vitro experiments in LUAD and their studies remains rudimentary to some extent (36)(37)(38).
In the current study, candidate genes involved in EMT process were identified based on TCGA-LUAD training dataset and validated using cell-based assays in vitro and clinical tissue samples. Then, the E-signature which can accurately predict the prognosis of LUAD patients was developed by us. Meanwhile, the nomogram affirmed the feasibility in clinical application of the E-signature. Subgroup analysis suggested that E-Signature could be conducive to identify patients with adverse events at high risk. The Esignature is closely related to multiple pathways associated with cancer progression, as well. In conclusion, the E-signature could be used as an inspiring molecular indicator for evaluation of clinical prognosis in LUAD patients.

LASSO Cox regression analysis
To yield the independent prognostic EMT factor, univariate analysis and multivariate analysis were showed with P< 0.05 as a threshold. least absolute shrinkage and selection operator (LASSO) analysis was capable for reducing the dimension (39). 80% TCGA samples after preprocessing were randomized to the training dataset. our prognostic model preserved the advantage of subset shrinkage and maintained a high accuracy rate based on the penalty parameter l (40). Risk score was calculated from expression of related gene and associated coefficient. Analytical formula for risk score assessment was derived on the basis of the EMT related gene signature = o n i=1 ðcoef i Â Expri), in this formula, Expr i represents gene expression of patient, and coef i represents the multivariate regression coefficient.

Survival analysis
According to median risk scores, all LUAD samples were divided into two groups, involving in high-risk and low-risk groups in different cohorts. Kaplan Meier (KM) curves were plotted to compare the prognostic difference (41). The area under the curve (AUC) which performed from receiver operating characteristic (ROC) curve analysis for overall

Establishment and validation of the nomogram
The significant variables from the multivariate models were introduced to draw the graphical nomogram by utilizing "rms" and "nomogramEx" packages (43). The calibration curves for probability of OS showed that match condition between prediction by nomogram and actual observation (44). Decision curve analysis (DCA) was utilized to evaluate the ability of the predictive model in view of clinical applicability (44).

Gene set variation analysis (GSVA)
Single-sample gene set enrichment analysis (ssGSEA) scores were gained by "ClusterProfiler" and "GSVA" R package to recognize the correlation between risk scores and enriched biological processes (45). The results with a cut-off criterion of P value< 0.05 were statistical significance.

Collection of LUAD tissues and cell culture
Under the premise that ethical clearance and approval have been obtained, 5 pairs of fresh tumor samples and matched normal tumor-adjacent samples were dissected from 5 LUAD patients who underwent lobectomy but did not receive radiotherapy and chemotherapy at Harbin Medical University Cancer Hospital between May 2021 and June 2021.
Human LUAD cell lines (A549 and NCI-H1299) were cultured with RPMI-1640 including 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and the culture environment needed to be maintained at 37°C and containing 5% carbon dioxide.
When the density of A549 and H1299 cells reached 60-70% in 6-well dishes, the transfection mixture was prepared by fully mixing 120mL riboFECT ™ CP Buffer, 12mL riboFECT ™ CP Reagent and 5mL siRNA, and incubated at room temperature for 15min. Finally, 3mL RPMI-1640 containing 10% FBS was added into the 6-well dishes. Follow-up experiments were carried out after ensuring the efficiency of knockout.

Western blotting
Lysate of tissues or cells was centrifuged for 15 min (4°C, 14000rpm) and we measured protein concentrations using the BCA protein analysis kit. Electrophoretic separation and electrotransfer of protein samples with the comparable quality, membrane blocking and incubate overnight (primary antibody, 4°C). PVDF membranes containing proteins are incubated with secondary antibodies for 1 hour at room temperature. The bands on membrane were exposed by CL Xposure film (Thermo Fisher Scientific).

Detection of cancer cell migration and invasion
Use the tip of a 10 mL pipette to form a scratch on a six-well plate covered with LUAD cells. By comparing the rate of wound healing and taking pictures under the microscope, the cell migration rate was finally counted. Transwell assay was conducted with the Corning Inc. transwell chamber. The invasion experiment required the participation of the matrigel matrix involved. Cell suspension arranged in the upper chamber contained 2x10 4 cells, besides, ingredient of the lower chamber was 600mL RPMI-1640 complemented with 10% FBS. The fixation of methanol and staining of 0.1% crystal violet for observing the migration and invasion efficiency after 24h or 48h, respectively. Fields were randomly selected in each membrane for capturing.

Statistical methods
Data processing was focused on GraphPad Prism 8.0.2 software. Typicality of data in the study could be reflected with at least three independent experiments completed. Continuous data conforming to a normal distribution were analyzed by Student's t-test, and subgroup differences were counted using the c2 test. All results are expressed as mean ± SEM (* P< 0.05, ** P< 0.01, *** P< 0.001).

Results
The identification and validation with in vitro experiments of 4 prognostic EMTrelated genes comprising the E-signature The 200 genes linked to EMT were acquired from the "HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSI TION" gene list in the MsigDB. Univariate Cox analyses systematically deduced 68 genes significantly relevant to prognosis based on TCGA-LUAD training cohort. To exploit an optimal model for testing risk, the LASSO analysis was used in its broadest sense to summary the results of each dimensionality reduction and count the occurrence frequency and standard deviation distribution of each probe. Standard deviation (SD) calculations may comprehensively describe the distribution characteristics of data, which is generally understood to measure the deviation degree (46). Four EMTrelated genes, including dickkopf-like protein 1 (DKK1), lysyl oxidase-like 2 (LOXL2), matrix Gla protein (MGP) and slit guidance ligand 3 (SLIT3), were comprehensively screened depending on the following criteria ( Figure 2A). SD of candidate mRNAs was greater than the median and frequency was well over 500 ( Figure 2A). Patients with high DKK1 and LOXL2 expression had the shorter overall survival (OS) according to a Kaplan-Meier analysis, whereas ones with high expression of MGP or SLIT3 had longer OS ( Figure 2B). Based on TCGA-LUAD transcriptome data, differential expression analysis showed that with the comparison of normal samples, DKK1 and LOXL2 expression were significantly increased in tumor tissues, whereas MGP and SLIT3 were markedly overexpressed in non-tumoral tissues ( Figure 2C). The same results were achieved from the collected clinical tissue samples by western blotting (Figure 2D).
The functions and roles of these four genes in LUAD metastasis and EMT remain to be clarified (47-50). We used cell-based assays in vitro to examine the effect of these four genes on EMT and metastasis, respectively. In A549 and H1299 cells, Gene-specific siRNAs were used with three independent siRNAs for knockdown of each gene. Successful knockdown of these four genes was validated by Western blotting in A549 ( Figure 3A) and NCI-H1299 cell lines ( Figure S1A). Moreover, as illustrated in Figure 3A and Figure S1A, knockdown of DKK1 or si-LOXL2 enhanced the expression of E-cadherin and inhibited the mesenchymal markers' expression, including N-cadherin and Vimentin. MGP or SLIT3 knockdown led to downregulation of epithelium-derived markers and upregulation of mesenchymal markers ( Figure 3A; Figure S1A). Furthermore, wound healing and Transwell assays revealed silencing of DKK1 and LOXL2 suppressed the ability of migration and invasion in LUAD cells, but silencing of MGP and SLIT3 had the opposite effect ( Figures 3B, C; Figures S1B, C). Our experimental data indicated that the four EMT-related genes could regulate metastasis and EMT program.

Construction of the prognostic Esignature in LUAD
Based on multivariate Cox regression analysis, the independent prognostic signature still composed of four EMT genes was generated. Scoring formula as follows: Risk Score = 0.308×exp DKK1 +0.299×exp LOXL2 -0.084×exp MGP -0.165×exp SLIT3 ( Figure 4A). The statistical correlation between risk model and 4 genes expression was assessed by the Pearson correlation metric ( Figure 4B). Risk score was converted into Z-score, taking 0 served as the optimal boundary value to divide the samples into two groups, in which z score of the high-risk subgroup was greater than 0, and the rest belonged to the low-risk subgroup. The permutation of risk scores, survival status and four genes expression levels were displayed ( Figure 4C). These results demonstrated that the risk E-signature was a deleterious indicator of prognosis.
Furthermore, based on TCGA training dataset, the prognostic accuracy of E-signature was appraised by timedependent ROC analysis, the AUC was 0.73 (1-year), 0.7 (2year) and 0.69 (3-year), respectively ( Figure 4D). Referring to the training set, in comparison with the high-risk group, the low-risk group revealed a significantly longer OS ( Figure 4D). The ROC and KM curve also revealed that our model exhibited good sensitivity and specificity in predicting TCGA-LUAD recurrence free survival (RFS, Figure 4E). Compared with recently published lung cancer prognostic models in the literatures ( Figure 4F) including Zhu (51), Ma (52), Zhang (53) and Zhang M (54), our E-signature had better specificity by ROC curve analysis especially at 1-year ( Figure 4F). Similarly, the Esignature predicted OS better than either the known signatures alone, with a better calibration and classification accuracy ( Figure 4G).  The flow chart in our study.

Establishment of a nomogram based on the E-signature
clinicopathological characteristics, such as T stage, N stage, M stage, TNM stage, age, gender, and smoking history, and risk score were incorporated as covariates into univariate and multivariate Cox regression analyses. Risk score, T stage and N stage were demonstrated the independent prognostic factors by forest plot for OS ( Figures 5A).
The nomogram could intuitively and effectively display the influence of the risk model on the prognostic outcome (55). A multi-scale nomogram was constructed on account of independent prognostic factors, which effectively predicted 1-year, 2-year and 3year OS probabilities of LUAD patients. The scores corresponding to the parameters were calculated to obtain the total points. Thus, high total score was significantly correlated to worse outcome ( Figure 5B). Furthermore, calibration curve implied well performance of the nomogram at predicting survival capacity in LUAD patients ( Figure 5C). ROC curve analysis and Decision Curve Analysis (DCA) showed that, compared with other independent variables, the nomogram model classifier had the distinctly superior accuracy and net benefit rate (Figures 5D, E). The identification 4 prognostic EMT-related genes comprising the E-signature. (A) The standard deviation distribution of all mRNAs. In the left panel, location corresponding to the red columns were standard deviation (SD) of genes with frequency greater than 500, where SD was used as the abscissa and the vertical axis represented the number of genes. The right panel showed that the gene frequency distribution chart obtained by LASSO analysis. The green baseline meant that frequency was equal to 500, the part exceeded the green line was genes with frequency greater than 500.

Correlation between the prognostic model and clinicopathologic features
To further clarify the clinical implication of the EMT signature, we tested the relationship between risk score and clinicopathologic variables by the Chi-square test. LUAD patients were classified into different subgroups under diverse clinical properties, including T stage, N stage, M stage, tumor stage, gender, age, smoking history, status of EGFR. Notably, there was a gradual upward trend in the proportion of high-risk score patients with increase of malignant grade of pathological T stage, pathological N stage, pathological M stage and tumor stage. Regrettably, other clinicopathological characteristics showed no obvious difference in the distribution ( Figure 6A). Subsequently, the risk model was also applied to each subgroup for KM survival analysis. We observed that subgroup with highrisk presented dramatically worse OS, suggesting that our specific signature had a precise predictive value ( Figure 6B).

Functional annotation and enrichment analysis of the E-signature
The association between risk score and EMT biomarkers was assessed based on the TCGA database ( Figure 7A). In order to further observe biological functions of the risk model, ssGSEA method was applied to derive scores of multifarious molecular pathways. The heatmap of hierarchical cluster analysis showed E-signature was enriched in "EPITHELIAL MESENCHYMAL TRANSITION" and other carcinogenic pathways, while metabolic-related pathways such as" HEME METABOLISM" and "BILE ACID METABOLISM" were inversely regulated by our signature ( Figure 7B). Moreover, the correlation map visualized the KEGG pathways extracted by correlation coefficient > 0.3 statistically significant associated with the Esignature ( Figure 7C). Taken together, the constructed specific signature played a compelling role in promoting tumor development.

Discussion
LUAD is the accepted common classification of lung cancer, accompanied by high prevalence and fatality (2,56). In cancer patients, metastasis is primary cause of shorten survival and high mortality, and often has already occurred at the time of diagnosis (57,58). During the procedure of the classical invasion-metastasis cascade, transformation from tumor epithelial cells to mesenchymal cells with the invasion and migration capacity (59). Subsequently, mesenchymal cells locally invade the surrounding matrix and extracellular matrix (ECM), transport and stay in distant organ tissues, eventually extravasate and proliferate to form metastasis (60). The induction and ultimate success of this process depends on EMT and its key regulators (59). Regulation of EMT markers expression, for instance, Ncadherin, Vimentin and E-cadherin, ultimately affects tumor progression, metastasis and drug resistance (61,62). To date, considerable studies have shown that the marked association of sophisticated regulation of EMT with poor prognosis of lung cancer patients (63, 64). To break down this barrier in clinical settings, molecular biological characteristics should be adequately considered, and circulating tumor markers, TNM staging and other indicators are not accurate enough in predicting the survival of LUAD patients (65,66). Similarly, single-gene biomarkers are unable to achieve a satisfying prediction result (67). Therefore, a multigene panel might be a promising and reliable method for precision and individualized treatment of LUAD patients.
With rapid advancements in high-throughput sequencing, abundant studies have used data from large communal databases, for example, TCGA and GEO databases, to construct the prognostic signatures for identification of patient risk stratification, guidance of treatment regimens, precise prognostic assessment, and improvement of clinical efficacy (68, 69). Previous studies have reported risk-prediction models based on EMT in LUAD (37,38). Although the conventional analytical methods used were similar, the genes required for construction of the A B D E C FIGURE 5 The prognostic nomogram and decision curve analysis (DCA) of the risk score based on E-signature. (A) The forest plot showed that the signature was independent from other risk factors for prognostic prediction. (B) Nomogram predicted risk of secondary progression. Each variable axis, containing T stage, N stage and risk score, corresponded to the characteristic attribute score of single sample. The final score was summarized on the total score axis and the likelihood of 1-, 2-, and 3 years OS is determined on the survival axis. (C) The nomogram yielded an accurate predictive capability that was extremely close to actual survival was presented by the calibration plots. X-axis represented the predicted value of survival probability and y-axis represented actual survival possibility. (D) ROC curves represented that the E-signature ranked first among all the parameters. (E) DCA curves graphically verified the E-signature brought more net benefit of survival than other clinical indexes. Solid lines indicated net benefit of the predictive model within the threshold probabilities range. The black and grey line respectively represented the hypothesis that none or all patients would experience. A B FIGURE 6 Correlation between the current prognostic E-signature and clinicopathologic attributes. signatures were fundamentally different, mainly due to the different databases and screening processes. In terms of bioinformatics analysis, our study not only plotted KM curves, ROC curves and nomogram to improve the accuracy and specificity of prognosis prediction, but also generated calibration curves for the nomogram to further prove effective clinical utility of E-signature. Secondly, the DCA (decision curve) was drawn for clarifying clinical feasibility of the risk model and prognostic significance of our signature was emphasized by comparing with known prognostic models (51)(52)(53). In addition, compared with the previously reported EMT models, we conducted in vitro experiments in-depth to verify a dramatically correlation between our signature and EMT procedure. Collectively, the specific Esignature we developed could predict the cancer process more comprehensively and accurately, with more stable and robust predictive performance and superior clinical practice (37,38).
In this study, the E-signature was found to be composed of DKK1, LOXL2, MGP and SLIT3. It is reported that DKK1 (a secreted protein) contains the cysteine-rich domain and is a member of the family of Dickkopf, (70). DKK1 has emerged as an indispensable regulatory factor in multiple cancers and commonly existed as an inhibitor of the Wnt pathway (71)(72)(73). Nevertheless, DKK1 acting as a tumor promoter, which played a critical role in the cancer progression (74)(75)(76). LOXL2 belonging to the LOX family, has the typical function of catalyzing the cross-link of elastin and collagen in the ECM and has attracted much attention in cancer biology (48). Plenty of studies have revealed that LOXL2 participated in tumor progression, metastasis, poor prognosis and chemoradiotherapy resistance in varied cancers, e.g., lung cancer, breast cancer, pancreatic cancer and colorectal cancer (77-80). MGP, an extracellular matrix protein, whose well-defined function is still unknown, and currently acts as a double-edged sword in cancers (49, [81][82][83][84]. Upregulation of MGP promoted cancer proliferation, migration and invasion, that was linked with unfavorable prognosis (49, [81][82][83]. Whereas, MGP reversed chemotherapy resistance and received favorable survival outcomes in estrogen receptor positive breast cancer (84). SLIT3, a secreted protein, is widely distributed in normal tissues and mainly participates in the Slit/Robo pathway (85). SLIT3 has rarely been reported in human cancers, which could inhibit the progression of thyroid cancer (86).

Conclusion
In summary, we developed and validated a trustworthy and powerful signature, which could serve as an independent and promising biomarker to optimize prognosis and surveillance protocols for individual LUAD patients.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.