epithelial ovarian cancer ( eoc ) is a primary cause of mortality among patients with gynecological malignancies .
although several eoc histotypes have been identified ( e.g. , serous , endometrioid , clear - cell , and mucinous ) , all eoc patients still receive similar cytotoxic chemotherapeutic regimens regardless of histotype , and their survival outcomes have not significantly improved over the past three decades . moreover , accumulated evidence suggests that eoc histotypes should be considered distinct disease states originating from different ovarian cell types .
given this heterogeneity in eoc histotypes , the development of screening and therapeutic strategies based on histotype - specific biomarkers may be critical to improving future clinical outcomes .
based on this paradigm shift in our understanding of eoc , re - analysis of eoc cases based on histotypes has led to the molecular characterization of specific subtypes .
moreover , mutations in brca1/2 and other dna repair genes ( e.g. , palb2 , rad51 , rad50 , bard1 , chk2 , and brip1 ) have been shown to occur more frequently in serous eoc than in other eoc histotypes .
in addition , an analysis of almost 500 serous eoc cases confirmed that these tumors are characterized by widespread dna copy number aberrations .
these findings led bowtell et al . to postulate that serous eoc cells evolve from initial mutations to dna repair genes followed by dna copy number aberrations and subsequent malignant transformation .
based on this previous evidence , we hypothesized that the cyclin - dependent kinase 16 ( cdk-16 , pctaire-1 , pctk1 )  which has been shown to be upregulated in several transformed cancer lines    may be upregulated in serous eoc cells .
therefore , here we comparatively examined the mrna and protein expression of cdk-16 in samples resected from serous eoc patients and normal controls .
this study was approved by the ethics committee ( irb ) of the gynecology and obstetrics hospital of guizhou medical university ( approval no : 201528 ) .
all subjects recruited for this study provided written informed consent prior to participation . from january 2013 to march 2015 , tissue samples were consecutively collected from primary ovarian cancer surgery patients undergoing radical surgical resection of their ovarian tumors at the gynecology and obstetrics hospital of guizhou medical university .
independent staff gynecologic pathologists determined the subtypes and grades of all resected tumors , while independent staff gynecologic oncologists scored tumor stage .
inclusion criteria were as follows : ( i ) primary surgical patient , ( ii ) serous eoc histopathology subtype , ( iii ) complete records regarding pre - operative chemotherapy and past medical history , and ( iv ) signed informed consent for inclusion in this study .
exclusion criteria were as follows : ( i ) non - primary surgical patients , ( ii ) non - serous eoc histopathology subtypes , ( iii ) missing or incomplete records regarding pre - operative chemotherapy or past medical history , ( iv ) chemotherapy of any kind prior to tumor resection , ( v ) previous history of any malignancy , or ( vi ) lack of informed consent .
prior to patient recruitment , pilot testing was performed to obtain preliminary data on cdk16 expression in serous eoc patients in order to determine a minimum sample size . on this basis , minimum sample sizes of 34 and 37 were calculated based on the cdk16 mrna and protein expression testing , respectively . after application of the inclusion and
exclusion criteria , a total of 70 han chinese serous eoc patients were finally recruited into this study . during the same time period , healthy ovaries from 40 patients that had underwent oophorectomy for prolapses or uterine fibromas
each sample was split in two portions : one for histologic assays and one for rna isolation .
samples were placed in a medium at a suitable temperature for micro - dissection and hematoxylin and eosin ( h&e ) staining to ascertain the epithelial portion . from a frozen state , 23 eoc and 12 control samples were carefully dissected out , and then a mechanical rotary homogenizer ( qiagen , valencia , ca , usa ) was used to homogenize the samples .
a trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) was applied to extract the total rna content followed by rna purification with an rneasy cleanup kit ( qiagen ) .
an agilent 2100 bioanalyser ( santa clara , ca , usa ) was used to assess rna integrity . using a superscript ii rt rnaseh - reverse transcriptase kit ( invitrogen life technologies ) , 1 mg rna was reverse - transcribed in a 20-ml volume .
quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) was conducted in triplicate for the cdk-16 gene on an abi prism 7000 sequence detection system with taqman universal pcr master mix and the anti - human cdk-16 assay on demand probe ( hs00178837_m1 ) ( all from applied biosystems , applera , uk ) .
cdna levels were normalized from with an endogenous anti - human gapdh control probe ( hs02758991_g1 , applied biosystems ) .
reverse transcription volume ( 5 l ) was conducted in a total pcr reaction volume of 25 l .
the pcr reaction was cycled as follows : 95c for 10 min , 95c for 15 sec ( 40 cycles ) , and 60c for 1 min .
fold - changes were calculated using the comparative threshold cycle ( ct ) method . to evaluate cdk-16 protein expression , semi - quantitative immunohistochemical staining
was conducted on the eoc and normal tissue samples as previously described with minor modifications .
the sections were slide - mounted , dried for 16 h at 37c , xylene - deparaffinized , rehydrated through an alcohol gradient , and finally lightly washed with faucet water .
low - grade  or  high - grade  by h&e staining ( sigma - aldrich , st . louis , mo , usa ) .
then , 0.3% hydrogen peroxide in methyl alcohol was applied to block endogenous peroxidase activity .
thereafter , the sections were lightly washed in distilled water . microwaving the sections ( three cycles at 750 w for 5 min per cycle ) in 1.0-mm ethylenediaminetetracetic acid ( edta ) buffer ( ph 8.0 ) was performed for epitope retrieval . following light washing with phosphate buffer ,
sections were treated with rabbit anti - human cdk-16 antibody ( diluted 1:200 in phosphate buffer with 1% bovine serum albumin ( bsa ) , sigma - aldrich ) in a humidified chamber for 2 h at 22c .
visualization was conducted with 3 , 3-diaminobenzidine ( dab ) plus hydrogen peroxide as horseradish peroxidase substrates ( staining brown ) . as a control
sections were lightly washed in faucet water , hematoxylin - counterstained , and lightly washed again in running faucet water for 15 min . an alcohol gradient and xylene
were applied to dehydrate the sections , which were then permount - mounted ( fisher scientific , pittsburgh , pa , usa ) .
microscopy was used to assess cdk-16 protein localization and expression as previously described with minor modifications .
then , section thickness was assessed in three unique regions at a 600 magnification in order to calculate the mean section thickness therefrom . then
, a 600 magnification under oil immersion was used for cell counts , which were calculated with a grid size of 800800 m and a frame size of 6060 m within a 7-m dissector height .
therefrom , we calculated the mean  standard deviation ( sd ) of cdk-16 + cells .
differences in mrna and protein expression between serous eoc cells and normal tissue cells were analyzed by kruskal - wallis and analysis of variance ( anova ) testing .
this study was approved by the ethics committee ( irb ) of the gynecology and obstetrics hospital of guizhou medical university ( approval no : 201528 ) .
from january 2013 to march 2015 , tissue samples were consecutively collected from primary ovarian cancer surgery patients undergoing radical surgical resection of their ovarian tumors at the gynecology and obstetrics hospital of guizhou medical university .
independent staff gynecologic pathologists determined the subtypes and grades of all resected tumors , while independent staff gynecologic oncologists scored tumor stage .
inclusion criteria were as follows : ( i ) primary surgical patient , ( ii ) serous eoc histopathology subtype , ( iii ) complete records regarding pre - operative chemotherapy and past medical history , and ( iv ) signed informed consent for inclusion in this study .
exclusion criteria were as follows : ( i ) non - primary surgical patients , ( ii ) non - serous eoc histopathology subtypes , ( iii ) missing or incomplete records regarding pre - operative chemotherapy or past medical history , ( iv ) chemotherapy of any kind prior to tumor resection , ( v ) previous history of any malignancy , or ( vi ) lack of informed consent .
prior to patient recruitment , pilot testing was performed to obtain preliminary data on cdk16 expression in serous eoc patients in order to determine a minimum sample size . on this basis , minimum sample sizes of 34 and 37 were calculated based on the cdk16 mrna and protein expression testing , respectively .
after application of the inclusion and exclusion criteria , a total of 70 han chinese serous eoc patients were finally recruited into this study . during the same time period , healthy ovaries from 40 patients that had underwent oophorectomy for prolapses or uterine fibromas
each sample was split in two portions : one for histologic assays and one for rna isolation .
samples were placed in a medium at a suitable temperature for micro - dissection and hematoxylin and eosin ( h&e ) staining to ascertain the epithelial portion . from a frozen state , 23 eoc and 12 control samples were carefully dissected out , and then a mechanical rotary homogenizer ( qiagen , valencia , ca , usa ) was used to homogenize the samples .
a trizol reagent ( invitrogen life technologies , carlsbad , ca , usa ) was applied to extract the total rna content followed by rna purification with an rneasy cleanup kit ( qiagen ) .
an agilent 2100 bioanalyser ( santa clara , ca , usa ) was used to assess rna integrity . using a superscript ii rt rnaseh - reverse transcriptase kit ( invitrogen life technologies ) , 1 mg rna was reverse - transcribed in a 20-ml volume .
quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) was conducted in triplicate for the cdk-16 gene on an abi prism 7000 sequence detection system with taqman universal pcr master mix and the anti - human cdk-16 assay on demand probe ( hs00178837_m1 ) ( all from applied biosystems , applera , uk ) .
cdna levels were normalized from with an endogenous anti - human gapdh control probe ( hs02758991_g1 , applied biosystems ) .
reverse transcription volume ( 5 l ) was conducted in a total pcr reaction volume of 25 l .
the pcr reaction was cycled as follows : 95c for 10 min , 95c for 15 sec ( 40 cycles ) , and 60c for 1 min .
fold - changes were calculated using the comparative threshold cycle ( ct ) method . to evaluate cdk-16 protein expression , semi - quantitative immunohistochemical staining
was conducted on the eoc and normal tissue samples as previously described with minor modifications .
the sections were slide - mounted , dried for 16 h at 37c , xylene - deparaffinized , rehydrated through an alcohol gradient , and finally lightly washed with faucet water .
low - grade  or  high - grade  by h&e staining ( sigma - aldrich , st .
then , 0.3% hydrogen peroxide in methyl alcohol was applied to block endogenous peroxidase activity .
thereafter , the sections were lightly washed in distilled water . microwaving the sections ( three cycles at 750 w for 5 min per cycle ) in 1.0-mm ethylenediaminetetracetic acid ( edta ) buffer ( ph 8.0 ) was performed for epitope retrieval . following light washing with phosphate buffer ,
sections were treated with rabbit anti - human cdk-16 antibody ( diluted 1:200 in phosphate buffer with 1% bovine serum albumin ( bsa ) , sigma - aldrich ) in a humidified chamber for 2 h at 22c .
visualization was conducted with 3 , 3-diaminobenzidine ( dab ) plus hydrogen peroxide as horseradish peroxidase substrates ( staining brown ) . as a control
sections were lightly washed in faucet water , hematoxylin - counterstained , and lightly washed again in running faucet water for 15 min .
an alcohol gradient and xylene were applied to dehydrate the sections , which were then permount - mounted ( fisher scientific , pittsburgh , pa , usa ) .
microscopy was used to assess cdk-16 protein localization and expression as previously described with minor modifications .
then , section thickness was assessed in three unique regions at a 600 magnification in order to calculate the mean section thickness therefrom .
then , a 600 magnification under oil immersion was used for cell counts , which were calculated with a grid size of 800800 m and a frame size of 6060 m within a 7-m dissector height .
therefrom , we calculated the mean  standard deviation ( sd ) of cdk-16 + cells .
differences in mrna and protein expression between serous eoc cells and normal tissue cells were analyzed by kruskal - wallis and analysis of variance ( anova ) testing .
from january 2013 to march 2015 , tissue samples were consecutively collected from primary ovarian cancer surgery patients undergoing radical surgical resection of their ovarian tumors at the gynecology and obstetrics hospital of guizhou medical university .
independent staff gynecologic pathologists determined the subtypes and grades of all resected tumors , while independent staff gynecologic oncologists scored tumor stage .
inclusion criteria were as follows : ( i ) primary surgical patient , ( ii ) serous eoc histopathology subtype , ( iii ) complete records regarding pre - operative chemotherapy and past medical history , and ( iv ) signed informed consent for inclusion in this study .
exclusion criteria were as follows : ( i ) non - primary surgical patients , ( ii ) non - serous eoc histopathology subtypes , ( iii ) missing or incomplete records regarding pre - operative chemotherapy or past medical history , ( iv ) chemotherapy of any kind prior to tumor resection , ( v ) previous history of any malignancy , or ( vi ) lack of informed consent .
prior to patient recruitment , pilot testing was performed to obtain preliminary data on cdk16 expression in serous eoc patients in order to determine a minimum sample size . on this basis , minimum sample sizes of 34 and 37 were calculated based on the cdk16 mrna and protein expression testing , respectively .
after application of the inclusion and exclusion criteria , a total of 70 han chinese serous eoc patients were finally recruited into this study . during the same time period , healthy ovaries from 40 patients that had underwent oophorectomy for prolapses or uterine fibromas
each sample was split in two portions : one for histologic assays and one for rna isolation .
samples were placed in a medium at a suitable temperature for micro - dissection and hematoxylin and eosin ( h&e ) staining to ascertain the epithelial portion .
from a frozen state , 23 eoc and 12 control samples were carefully dissected out , and then a mechanical rotary homogenizer ( qiagen , valencia , ca , usa ) was used to homogenize the samples . a trizol reagent ( invitrogen life technologies , carlsbad , ca
, usa ) was applied to extract the total rna content followed by rna purification with an rneasy cleanup kit ( qiagen ) .
an agilent 2100 bioanalyser ( santa clara , ca , usa ) was used to assess rna integrity . using a superscript ii rt rnaseh - reverse transcriptase kit ( invitrogen life technologies ) , 1 mg rna was reverse - transcribed in a 20-ml volume .
quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) was conducted in triplicate for the cdk-16 gene on an abi prism 7000 sequence detection system with taqman universal pcr master mix and the anti - human cdk-16 assay on demand probe ( hs00178837_m1 ) ( all from applied biosystems , applera , uk ) .
cdna levels were normalized from with an endogenous anti - human gapdh control probe ( hs02758991_g1 , applied biosystems ) .
reverse transcription volume ( 5 l ) was conducted in a total pcr reaction volume of 25 l .
the pcr reaction was cycled as follows : 95c for 10 min , 95c for 15 sec ( 40 cycles ) , and 60c for 1 min .
to evaluate cdk-16 protein expression , semi - quantitative immunohistochemical staining was conducted on the eoc and normal tissue samples as previously described with minor modifications .
the sections were slide - mounted , dried for 16 h at 37c , xylene - deparaffinized , rehydrated through an alcohol gradient , and finally lightly washed with faucet water .
low - grade  or  high - grade  by h&e staining ( sigma - aldrich , st .
thereafter , the sections were lightly washed in distilled water . microwaving the sections ( three cycles at 750 w for 5 min per cycle ) in 1.0-mm ethylenediaminetetracetic acid ( edta ) buffer (
ph 8.0 ) was performed for epitope retrieval . following light washing with phosphate buffer ,
sections were treated with rabbit anti - human cdk-16 antibody ( diluted 1:200 in phosphate buffer with 1% bovine serum albumin ( bsa ) , sigma - aldrich ) in a humidified chamber for 2 h at 22c .
visualization was conducted with 3 , 3-diaminobenzidine ( dab ) plus hydrogen peroxide as horseradish peroxidase substrates ( staining brown ) . as a control
sections were lightly washed in faucet water , hematoxylin - counterstained , and lightly washed again in running faucet water for 15 min .
an alcohol gradient and xylene were applied to dehydrate the sections , which were then permount - mounted ( fisher scientific , pittsburgh , pa , usa ) .
microscopy was used to assess cdk-16 protein localization and expression as previously described with minor modifications .
then , section thickness was assessed in three unique regions at a 600 magnification in order to calculate the mean section thickness therefrom .
then , a 600 magnification under oil immersion was used for cell counts , which were calculated with a grid size of 800800 m and a frame size of 6060 m within a 7-m dissector height .
therefrom , we calculated the mean  standard deviation ( sd ) of cdk-16 + cells .
differences in mrna and protein expression between serous eoc cells and normal tissue cells were analyzed by kruskal - wallis and analysis of variance ( anova ) testing .
cdk-16 mrna expression was significantly higher in serous eoc tumor cells as compared to normal control ovarian cells ( p<0.01 ; table 1 , figure 1 ) .
the target amplified gene was confirmed as human cdk-16 ( hs00178837_m1 , applied biosystems ) ( data not shown ) .
although there was no significant correlation between cdk-16 mrna expression and serous eoc stage ( p=0.0794 ) , there was a significant correlation between cdk-16 mrna expression and serous eoc grade ( p<0.0001 ) ( table 1 , figure 1 ) .
similarly , cdk-16 protein expression was significantly higher in serous eoc tumor cells as compared to normal control ovarian cells ( p<0.01 ; table 1 ; figures 2 , 3 ) .
moreover , there were significant correlations between cdk-16 protein expression and serous eoc stage ( p<0.0001 ) and grade ( p<0.0001 ) ( table 1 , figure 3 ) .
the human cdk-16 gene maps to the x chromosome ( xp11.3p11.23 ) and belongs to the cmgc protein kinase family that includes all cyclin - dependent kinases ( cdks ) , mitogen - activated protein kinases ( mapks ) , glycogen synthase kinase , and cdk - like kinases [ 1113 ] .
cdk-16 is one of three members of the pctaire family that contain a highly - conserved pstaire motif which plays a key role in cyclin binding . despite this well - conserved cyclin - binding motif and cdk s widely - acknowledged role in cell cycle regulation ,
rather , cdk-16 is expressed in post - mitotic cells and has been associated with vesicle trafficking , neurite outgrowth , and spermatogenesis . of specific relevance to the current study is cdk-16 s vital role in spermatogenesis , as ovarian cells share some homology with testicular cells and are capable of reorganizing into testicular structures under the proper conditions . in the testis ,
cdk-16 expression is upregulated during spermatid differentiation but is remarkably absent in mature spermatozoa . specifically , cdk-16 is sequestered within the cytoplasmic residual body , which is shed after spermatogenesis is completed . in vivo ,
cdk-16-knockout mice develop normally , but male mice are infertile with spermatozoa displaying a bent morphology , malformed heads , thinning and elongation of the annulus , and impaired motility .
in contrast , targeting of other cdks or their associated cyclins led to arrest in spermatogonium proliferation or disruption of meiosis .
these findings suggest that cdk-16 deficiency does not affect meiosis but adversely affects terminal differentiation of sperm cells . here
, we found that cdk-16 mrna and protein expression was significantly higher in serous eoc tumor cells compared to normal control ovarian cells .
moreover , cdk-16 mrna and protein expression were significantly higher in high - grade serous eoc tumor cells as compared to low - grade serous eoc tumor cells .
based on the previous findings in the testis in which cdk-16 is required for proper spermatid differentiation , we speculate that cdk-16 upregulation in serous eoc cells may represent a negative feedback loop to promote ovarian cell differentiation in malignantly - transformed serous eoc cells . this hypothesis is supported by abundant evidence of negative feedback loops involving cdks and cyclins that play regulatory roles in human cancers [ 1821 ] .
since no other study has yet examined cdk-16 expression in ovarian cancer cells , further research is necessary to support this hypothesis .
third , serous eoc was only compared to normal control tissue , not benign ovarian tumors .
therefore , larger , multi - center studies that include women of different ethnicities with benign tumors would provide more robust conclusions .
third , due to the short duration of this study , we did not correlate cdk-16 expression with specific histopathological grades nor with clinical outcomes .
fourth , we did not examine eoc morphology ( beyond the low - grade versus high grade classification ) , the levels of other cdk s or cyclins , or the potential mechanism(s ) underlying cdk-16 s effect on eoc cells .
cdk-16 mrna and protein expression was significantly higher in serous eoc tumor cells compared to normal control ovarian cells .
moreover , cdk-16 mrna and protein expression were significantly higher in high - grade serous eoc tumor cells as compared to low - grade serous eoc tumor cells .
cdk-16 upregulation in serous eoc cells may represent a negative feedback loop to promote ovarian cell differentiation in malignantly - transformed serous eoc cells .
 backgroundas cdk-16 has been shown to be upregulated in several transformed cancer lines , we hypothesized that the cyclin - dependent kinase 16 ( cdk-16 ) may be upregulated in serous epithelial ovarian cancer ( eoc ) cells . 
 therefore , we comparatively examined the mrna and protein expression of cdk-16 in samples resected from serous eoc patients and normal controls.material/methodstissue samples were collected from 70 serous eoc patients and 40 normal controls . 
 quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) was conducted to assess mrna expression . 
 cdk-16 protein expression was assessed by semi - quantitative immunohistochemical staining . 
 differences in mrna and protein expression between serous eoc cells and normal tissue cells were tested with the kruskal - wallis test and analysis of variance ( anova).resultsboth cdk-16 mrna and protein expression were significantly higher in serous eoc tumor cells as compared to normal control ovarian cells ( p<0.01 ) . 
 although there was no significant correlation between cdk-16 mrna expression and serous eoc stage ( p=0.0794 ) , there was a significant correlation between cdk-16 mrna expression and serous eoc grade ( p<0.0001 ) . 
 moreover , there were significant correlations between cdk-16 protein expression and serous eoc stage ( p<0.0001 ) and grade ( p<0.0001).conclusionscdk-16 upregulation in serous eoc cells may represent a negative feedback loop to promote ovarian cell differentiation in malignantly - transformed serous eoc cells . further in - depth investigation on cdk-16 s role in serous eoc is needed . 
Background
Material and Methods
Ethics statement
Sample size determination
Patient recruitment
Ovarian tissue sampling
Reverse transcription of extracted RNA
Gene expression assay
Semi-quantitative immunohistochemistry
Statistical analysis
Results
Discussion
Conclusions