MicroRNA-218 is upregulated in gastric cancer after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy and increases chemosensitivity to cisplatin.

AIM
To investigate the molecular mechanisms of miRNA in advanced gastric cancers (AGCs) before and after cytoreductive surgery (CRS) + hyperthermic intraperitoneal chemotherapy (HIPEC).


METHODS
A miRNA microarray containing human mature and precursor miRNA sequences was used to compare expression profiles in serum samples of 5 patients with AGC before and after CRS + HIPEC. The upregulation of miR-218 was confirmed by real-time reverse transcription polymerase chain reaction and its expression was analyzed in SGC7901 gastric cancer cells.


RESULTS
miRNA microarray chip analysis found that the level of miR-218 expression was upregulated more than 8 fold after CRS + HIPEC. Furthermore, miR-218 increased gastric cancer cell chemosensitivity to cisplatin in vitro and inhibited gastric cell tumor growth in nude mice in vivo (0.5 vs 0.78, P < 0.05).


CONCLUSION
Our results indicated that targeting miR-218 may provide a strategy for blocking the development of gastric cancer and reverse the multi-drug resistance of gastric cell lines.


INTRODUCTION
Gastric cancer is the second leading cause of cancer death in the world, with often a poor prognosis due to the advanced stage of the disease at diagnosis. The peritoneum is the most common site of metastasis in patients with gastric cancer [1] . Although surgery and chemotherapy can be curative for early-stage disease, the prognosis for patients with advanced disease is poor. In China, advanced gastric cancer (AGC) is the third leading cause of cancer deaths, with 300000 deaths per year [2] . In addition to hematogenous and lymphatic metastasis, patients with AGC may have free cancer cells dissemination in the peritoneal cavity, leading to peritoneal carcinomatosis (PC). PC may be present in 5%-20% of patients undergoing gastrectomy of AGC. The prognosis and survival of patients with gastric originated PC are extremely poor [3,4] .
Patients completed the first session of HIPEC in the operating room under general anesthesia. At the first session, 5-fluorine and cisplatin were added into the 0.9% sodium chloride (3500-5000 mL) as perfusion liquid. HIPEC was then performed for 60 min with a velocity of 450-600 mL/min and an inflow temperature of 43 ± 0.2 ℃.
Several non-randomized comparative studies have suggested that CRS + HIPEC is superior to CRS alone in patients with AGC [14,15] . Although CRS + HIPEC is an accepted method of treatment, its exact molecular mechanisms have not been fully elucidated.
Since the molecular mechanisms of miRNA expression in AGCs treated with CRS + HIPEC remain elusive, further research is needed to examine the contribution of miRNAs to the mechanisms of carcinogenesis and their association with patient prognosis. We therefore utilized miRNA microarray chips to analyze the expression profiles of miRNAs in five patients with AGC being treated with CRS + HIPEC. We found that miR-218 was upregulated by more than 8 fold, which was confirmed by polymerase chain reaction (RT-PCR). These findings indicate the molecular basis of CRS + HIPEC effects in patients with AGC.
To further define the function of miR-218, it was upregulated in human gastric cancer cells (SGC7901), and cell vitality and chemosensitivity were then studied.

Patients
Five patients with gastric PC, 4 men, and 1 woman, aged 46-75 years (median, 50.3 ± 1.6 years) were recruited from January to June 2012 (Table 1) and treated with CRS + HIPEC. Eligibility criteria included the following: (1) no prior chemotherapy or chemotherapy administered more than 6 mo ago; (2) an Eastern Cooperative Oncology Group performance score of 0-2; (3) adequate bone marrow, hepatic, and renal functions; and (4) 18-75 years of age. All diagnoses of AGC were confirmed histologically. Pre-and post-operative serum samples were collected from the 5 patients. All patients provided written informed consent, and the study protocol was approved by the ethics committee of Guangzhou Medical College and was in accordance with the Helsinki Declaration on ethical principles for medical research involving human subjects.

CRS and HIPEC
All CRS and HIPEC procedures were performed by the same team of surgical oncologists and anesthesiologists. Abdominal exploration was performed according to previously reported methods [29] . The volume and character of ascites were also recorded. After evaluation, patients were scheduled for CRS and HIPEC [30] . HIPEC used to manage peritoneal surface malignancies was in accordance with the standard protocol of our center. Hy-perthermic perfusion was performed with 4 L of heated saline supplied with 60 mg of cisplatin and 1000 mg of 5-FU. Human serum samples were collected from the 5 AGC patients 1 h before and 1 h after CRS + HIPEC, which were quickly frozen in liquid nitrogen and stored at -80 ℃.

RNA isolation
Total RNA was isolated from samples using TRIzol kits (Invitrogen, Carlsbad, CA, United States) according to the manufacturer's protocol. Yield and quality of RNA were assessed by ultraviolet absorbance at 260 nm and 280 nm. miRNA microarrays miRNA was isolated from the total RNA sample using mirVana miRNA Isolation Kits (Ambion Inc., Austin, TX, United States). The miRNA quantity was assessed by a fluorometer (Bio-Rad, Hercules, CA, United States) and by 1% formaldehyde agarose gel electrophoresis. Expression of miRNAs was assayed using microRNA chips (Exiqon, Vedbaek, Denmark). Serum samples were sent to KangChen-Biotech (Shanghai, China) for array hybridization, background subtraction, and normalization. Total RNA (5 μg) was labeled with Cy3 modified RNA linker at the 3'-end using T4 RNA ligase. Briefly, the reaction was carried out in 3 μL volumes at 37 ℃ for 30 min containing 2.0 μL RNA, 1.0 μL of CIP buffer, and CIP (Exiqon) mixture. The reaction was terminated by heating at 80 ℃ for 3 min. Images were analyzed with Genepix Pro 6.0 (Axon). Data were then collected from three independent experiments and miRNA with at least 2-fold changes were selected for further analysis.
DNA was amplified with an initial denaturation at 94 ℃ for 3 min, followed by 35 cycles of 94 ℃ for 15 s and 59.8 ℃ for 15 s (gain set at 10 for SYBR Green).
Each experiment was carried out in triplicate. The amplification efficiency was assessed based on the slope of a linear regression model. Ten-fold dilutions of each cDNA were used as a PCR template.

Generation of SGC7901 cells with stable expression of miR-218
SGC7901 cells were first transfection with pCR-miR-218 or pCR-miR-NC, and then selected in 1 mg/mL G418 (Invitrogen) supplied complete DMEM. miR-218 or miR-NC stable overexpressed cells were obtained after 3-4 wk of selection. The chemosensitivity of these cells were evaluated using a WST-1 kit (Roche) according to the manufacturer's instruction at the indicated times. Each experiment was carried out in triplicate.

Anchorage-independent colony formation assay
The soft agar colony formation assay was used to monitor anchorage-independent growth. Briefly, agar plates were prepared using 1% agarose (Sigma, Saint Louis, MI, United States). Plates were stored at 4 ℃ and pre-warmed in 37 ℃ before use. Top agar (0.5%) containing 10 3 cells were then added onto the well. After 3-4 wk of incubation, colonies were assessed after methanol fixation and 0.1% crystal violet staining. Colonies with a diameter greater than 2 mm were counted. Each experiment was carried out in triplicate.
In vitro assay of chemosensitivity to cisplatin 6 × 10 3 cells were first seeded in each well of 96-well plates. After overnight incubation, cisplatin was added into each well, resulting in the final concentration of 0 to 256 μmol/L. Cell viability was determined using WST-1 kit after 72 h. IC50 was obtained from three independent 11349 August 28, 2014|Volume 20|Issue 32| WJG|www.wjgnet.com

Real-time PCR validation of microarray results
Results of the microarray were further validated by quantitative RT-PCR, including the results of miR-218, miR-135a, miR-409, and miR-96. The RT-PCR results were consistent with the microarray data ( Figure 2).

Upregulation of miR-218 suppressed in the proliferation of gastric cancer
MiR-218 was reported to be downregulated in gastric cancers [31] and was upregulated after CRS + HIPEC.
To explore the roles of miR-218 in drug resistance, SGC7901 gastric cell line with a stable overexpression of miR-218 were established by transfecting miR-218 plasmids and then undergoing cisplatin-based selection. Meanwhile, the SGC7901/miR-NC cell line was established as a control. The overexpression of miR-218 in SGC7901/miR-218 cells was confirmed by RT-PCR ( Figure 3C). Cell growth was measured using WST-1 kit at indicated time points.
The results showed that upregulation of miR-218 markedly inhibited the proliferation of SGC7901 cells ( Figure 3A). The anchorage independent colony formation assay also demonstrated the same results ( Figure 3B).

MiR-218 increased chemosensitivity of gastric cancer cells to cisplatin via its target mTOR inhibitor
To investigate whether miR-218 plays an important role in the drug resistance of gastric cancer, gastric tumor cell lines, SGC7901/miR-NC, SGC7901/miR-218, or SGC7901/miR-218 using mTOR inhibitor rapamycin and different concentrations of cisplatin (0 to 256 μmol/L) for 72 h ( Figure 4A). Cell viability was measured using the WST-1 kit. The results showed that overexpression of miR-218 increased sensitivity of SGC7901 cells to cisplatin, while rapamycin can further enhance chemosensitivity. Cisplatin at 10 μmol/L showed a remarkable inhibition of cell proliferation ( Figure 4B).

MiR-218 impaired in vivo tumor growth
The chemosensitization role of miR-218 was further explored using ectopic transplantation in a nude mice model. Stable cell lines, SGC7901/miR-218, and SGC7901/ miR-NC were injected into nude mice subcutaneously. Tumor growth in the miR-218 group was reduced significantly when compared to the control group ( Figure  5A and B). The tumor weights of the xenograft in the miR-218 group were greater than those in the miR-NC group ( Figure 5C). These data indicated the suppressor role of miR-218 in gastric cancer.

DISCUSSION
PC of gastrointestinal or primary origin is considered invariably fatal, with only palliative treatment possible. To date, no standard treatment has been developed for PC from AGC, although the original trial of CRS + HIPEC resulted in a mean overall survival of 7.2 ± 4.6 mo with acceptable morbidity [32] . This new treatment modality has experiments. Cisplatin at twice the concentration of IC50 were used in the following studies.

In vivo tumorigenesis study
Twenty four BALB/c nude mice (female, 6-9 wk, Guangzhou Laboratory Animal Center, Chinese Academy of Sciences, Guangzhou, China) were used in the study. Mice were maintained in a special pathogen-free (SPF) house with 12 h alternating light and dark cycles, and were given adequate nutrition and water ad libitum. 6 × 10 6 of cells suspended in FBS-free DMEM were injected into each side of the posterior flank of nude mice subcutaneously. Mice were sacrificed and tumors were collected 30 days after implantation. Tumor volume was calculated as follows: volume = 0.5 × length × width 2 . Experimental protocols were reviewed and approved by the Animal Ethics Committee of Guangzhou Medical College.

Statistical analysis
Quantitative data were presented as mean ± SD. Comparisons between the quantitative data were made using the t test. SPSS 16.0 was used in the statistical analysis and P < 0.05 was considered statistically significant.
In the present study, we focused on the effect of miR-218 on AGC metastasis and found that miR-218 was a tumor suppressor in AGC metastasis. Furthermore, miR-218 increased chemosensitivity to cisplatin in vitro and in vivo by inducing apoptosis. Cisplatin is currently used in the treatment of many types of advanced cancer, including gastric cancer, due to the broadest-spectrum anticancer capability [37] . Currently, the most effective systemic treatment for metastatic gastric cancer consists of cisplatin-based combination chemotherapy [38] . However, chemoresistance is still the most major and frequent obstacle to effective treatment [39] . Our data show that miR-218 overexpression can reverse the MDR of cisplatin and can increase the chemosensitivity of gastric cancer cells. When compared to the mTOR inhibitor rapamycin, the effect is better than miR-218 alone. Therefore, miR-218 may be used as a biomarker to confirm the extensiveness of tumor resection and to evaluate the efficacy of HIPEC.
The key finding in our study is that miR-218 suppressed the proliferation of tumor cells and inhibited the mTOR signaling pathway in gastric cancer. In addition, upregulation of miR-218 increased chemosensitivity of gastric cancer cells to cisplatin via its target mTOR.
In conclusion, we have identified several miRNAs whose expression was affected before and after CRS + HIPEC in AGC. The upregulation of miR-218 can inhibit AGC cell invasion and metastasis, as well as further reverse the MDR of cisplatin. The results indicate that restoration of miR-218 may be a rational therapeutic strategy for the treatment of AGC in the future. Impor-

Background
Gastric cancer is the second leading cause of cancer death in the world, with often a poor prognosis due to the advanced stage of the disease at diagnosis.

Research frontiers
2010 French guidelines designated cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) as the standard method for treating colorectal PCs, and evaluated the effectiveness of CRS + HIPEC treatment for other primary tumors, including gastric cancer, ovarian, and sarcoma.

Applications
To further define the function of miR-218, it was upregulated in human gastric cancer cells (SGC7901), and the cell vitality and chemosensitivity were then studied.

Peer review
This is an interesting study with both in vivo and in vitro data. The discovery of miR-218 was based on microarray results; hence the clinical significance is good.