Clinical Significance And Function Of Extracellular Matrix Protein SERPINA3 In Endometrial Carcinoma

Background and objective:Endometrial cancer(EC)is the most common cancer of the female reproductive system,the pathogenesis of endometrial cancers is complex.The etiology is not clear.Due to different level of economic development of regions and their respective not the same incidence.Now,it is considered that endometrial cancer is closely related to lifestyle.Endometrial cancer is a woman of the world's sixth most common malignancy in 2008 about 288,000 new cases.It is the most common gynecologic malignancy of the female reproductive tract,the fourth most common cancer of women,the incidence rate second only to breast cancer,lung cancer,colorectal cancer.In China,with the development of improved social and economic conditions,the incidence of endometrial cancer is also increased year by year,now second only to the cervical cancer,ranking second in the female reproductive system cancer.The most important prognostic factors at diagnosis are:stage,grade,depth of invasive disease,lymphovascular space invasion(LVSI)and histological subtype.Traditionally,endometrial cancer has been classified into two types.Type ? endometrial carcinoma comprises the endometrioid adenocarcinomas that express the estrogen receptor(ER)and progesterone receptor(PR)and usually low grade and rarely metastasize.The prognosis of type ? cancers is favorable if diagnosed at an early stage,with a 5-year survival rate higher than 80%.Type ?endometrial carcinomas are those of non-endometrioid histology,in particular serous or clear-cell morphology.These tumors are considered to be of high histological grade,arise in the background of atrophic endometrium and do not seem to be related to the ER pathway.Despite the higher prevalence of type cancers,type ?tumors account for a high proportion of endometrial cancer-related deaths.Comparing with a higher incidence of type ? cancers,which is accounting for 70%-80%of the total number of endometrial cancer,the type ? endometrial cancer only accounting 15%-20%of endometrial cancer,accounting proportion of the total mortality which is as high as 40%.In spite of advances in radiotherapy,surgery and chemotherapeutic strategies,the prognosis of women with recurrent or advanced endometrial cancer is still poor with a median overall survival(OS)of approximately 7-10 months.However,the mechanisms which involved in the evolution of this tumor were not clear.Now the change in the matrix surrounding the tumor microenvironment may play an important role in tumor evolution.Early in the 19th century,stephen Paget firstly proposed the "seed and soil"hypothesis,the hypothesis is the foundation of concept of the tumor microenvironnent.Paget forecasted that as the "seed" of tumor cells if they can settle in the "soil" suitable for their growth,namely distant tissues and organs,tumor cells must synergies with its surrounding factors affecting.1975,Beatrice Mintz Karl and Illmensee formally proposed the concept of the tumor microenvironment,the malignant tumor cells were injected into the developing Blastocytes,and they were surprised to find that Blastocytes mice develop into completely normal mice.Thus they speculated that tumor cells are totipotent cells in a developmental,changed the microenvironment of tumor cells can induce its reversal of normal cells.Currently changes in tumor microenvironment,which is important for the progression,had been widely appreciated in endometrial cancer.As an important component of the microenvironment,the extracellular matrix proteins play a very important role in the cancer process,but their role and mechanism of them had been reported rarely in endometrial cancer.In this study,we detected and analyzed the relationship between the extracellular matrix protein SERPINA3 expression and clinicopathological characteristics and biological behavior of endometrial cancer at histology and cellular level.This study wanted to provide new starting point for revealing the pathogenesis of endometrial cancer and improving the outcome of endometrial cancer,and to provide clues for exploring endometrial cancer therapeutic targets.Methods1,Cell cultureHuman EC cell lines AN3CA,KLE,HEC-1A ECC-1,and Ishikawa were purchased from Cell Bank of the Chinese Academy of Sciences.AN3CA,KLE,Ishikawa Cells were cultured in Dulbecco's modified Eagle medium(DMEM)/F12;HEC-1A Cells were cultured in McCoy'5A;ECC-1 Cells were cultured in RPMI-1640;and all of these cells were supplemented with 10%(v/v)fetal bovine serum(FBS),100U/ml penicillin and 100ug/ml streptomycin,and incubated at 37? in a humidified incubator under 5%CO2 condition.2,Clinical tissue samplesWe recruited consecutive patients with endometrial carcinomas to a discovery cohort,from October 2004 to May 2013.The fresh endometrial specimens were immediately frozen at-80? until RNA extraction.217 human endometrial tissue samples in tissue microarrays as well as the fresh specimens were obtained from Department of Gynecology,Changzhou Maternal and Child Care Hospital and Department of Gynecology and Fengxian Hospital,Southern Medical University.The cases of endometrial carcinomas were selected in this study only if follow up was obtained and clinical data were available.All patients with endometrial carcinomas underwent a modified radical hysterectomy or complete hysterectomy,bilateral salpingo-oophorectomy and pelvic lymphadenectomy with or without para-aortic lymph node sampling.None of them had received radiotherapy,chemotherapy,hormone therapy or other related anti-tumor therapies before surgery.The diagnosis and histologic classification of the endometrial carcinomas was made using the criteria proposed by World Health Organization.Immunohistochemical staining3,Construction of tissue microarraySelects and marks the representative tumor or normal endometrial tissue regions on the corresponding HE slice.Then selects two points from each tissues.Target tissue 1s removed from the desired target donor paraffin block with a suction needle.Sort by chip design positioning in the recipient paraffin block loading.All tissue samples were fixed in phosphate-buffered neutral formalin and routinely embedded in paraffin,and then cut:into 5-?m-thick sectioris.Tissue sections were incubated with 0.3%hydrogen peroxide/phosphate-buffered saline for 30 minutes and blocked with 10%BSA,then were detected with primary polyclonal antibodies overnight at 4?.After incubated with the second antibody labeled by HRP for 1 hour at room temperature,the sections were treated with diaminobenzidine and counterstained with haematoxylin.All the sections were observed and photographed with a microscope and scored was conducted according to the ratio and intensity of positive-staining cells All the expression levels were quantified two-blindly by two independent pathologists.Quantitative real-time PCRTotal RNA was extracted from cells and tissues using Trizol reagent and reverse transcribed by PrimeScript RT reagent kit according to the manufacturer's instruction.The quantitative real-time polymerase chain reaction(qRT-PCR)was subsequently performed with SYBR Premix Ex Taq using an ABI7300 instrument.The relative expression was analyzed by the comparative cycle threshold method(??Ct method)which was normalized to 18s RNA.Western-blottingWhole cell lysates were prepared by lysis buffer.Proteins were separated by SDS-PAGE and were transferred to nitrocellulose membranes.Then the electroblotted membranes were blocked in phosphate-buffered saline/Tween-20 containing 1%BSA.The primary antibodies were used.After incubating with the IRDye 680 anti-mouse or IRDye 800 anti-rabbit secondary antibodies for 1 hour at room temperature,the bands were detected by an Odyssey infrared imaging system.siRNA transfectionOne day before transfection,the cells in logarithmic growth phase(1×105)were seeded in 6-well culture plates,cell confluency of 40%to 50%.Transfection was performed by using the Lipofectamine RNAiMAX Reagent according to the manufacturer's instruction.The siRNA diluted with dilution Mate after mixing to form a transfection complex was added to cultured cells,culture medium was changed 6-8 hrs.Lentivirus production and cell transfectionShort hairpin RNA(shRNA)sequences were cloned into pLKO.1-puro vectors.shRNA containing plasmids were packaged into lenti-virus and transducted into target cell lines.The efficiency was tested by qRT-PCR or western blotting.Cell viability assayCells were seeded into a 96-well plate.10 ?L Cell Counting Kit-8(CCK-8,WST-8)was added to each well after 24h,48h and 72h,respectively.In viable cells,WST-8 was metabolized to produce a colorimetric dye that can be detected at 450 nm using a microplate reader.The experiment was performed in triplicate and repeated twice.migration assay in vitroWithing serum-free medium mixed the cell suspension.The chamber was placed in 24 well plates,cell suspension was implanted into the upper chamber,the lower chamber containing of complete medium plus 10%FBS.Cell Cycle assaycells grown for 24h.Cells were then incubated with 1 mM thymidine to synchronize cells at the G1/S boundary.The cells were then treated with serum-deprived culture medium.Next,the cells were trypsinized,washed and fixed with cold 70%ethanol.The cells were then washed twice and incubated with Rnase A and propidium iodide(PI).Cells were subsequently analyzed by flow cytometry.Fluorescence Activated Cell Sorter(FACS)Apoptosis Assay.With serum-free cell culture medium,at a specific time with EDTA-free trypsin digestion cells were collected,Pl-Annxin V double staining,flow cytometry apoptosis.Statistical analysisData were presented as the means ± standard error of the mean(SEM).Statistical analyses were done using SPSS 16.0 for windows(IBM).The chi-square test,or student's t-test were used for comparison between groups.Values of P<0.05 were considered statistically significant.ResultsPart ? Clinical significance of expression of SERPINA3 in endometrial cancer1.1 SERPINA3 expression is elevated in endometrial cancer tissues1.2 SERPINA3 is associated with adverse clinical characteristics by immunohisto--chemical analysis in tissue microarrayPart ? Function of SERPINA3 expression in endometrial cancer2.1 SERPINA3 highly expressed cell lines were transfected with small interfering RNA(siRNA)to knockdown SERPINA3 expression.SERPINA3 were successfully decreased after siRNA transfection2.2 Knockdown of SERPINA3 inhibited EC cells viability in vitro2.3 Knockdown of SERPINA3 expression arrested cell cycle at G2/M phase2.4 Knockdown of SERPINA3 induced endometrial cancer cells apoptosis2.5 Interference expression of SERPINA3,endometrial cancer cells migration was significantly reduced.2.6 AKT and ERK1/2 were inactivated by SERPINA3 knockdown in EC cellsConclusionThis study collected the clinical tissue samples,small sample screening of candidate gene and constructing of tissue microarray to further validation.Then using artificial changes in gene expression and recombinant proteins in eukaryotic expression technologies to study the clinical significance of upregulation of two extracellular matrix proteins and their ftunctional effects in endometrial cancer,and to investigate them play signal transduction pathways,through these studies we obtained the following conclusions:1.We found that SERPINA3 was highly expressed in EC.In an immunohistochemical tissue mcroarray analysis(TMA),we observed that SERPIINA3 protein expression was up-regulated in EC tissues and was closely associated with adverse clinicopathological behaviors.By in vitro cells experiments,we found that silencing of SERPINA3 significantly inhibited EC cells proliferation with cells cycle arrested in G2/M phase and led to apoptosis.Further investigations indicated that the growth-promoting and apoptosis-inhibition effects of SERPINA3 might be ascribed to the activation of MAPK/ERKI/2 and PI3K/AKT signaling pathways.In this study,we detected and analyzed the relationship between the extracellular matrix protein SERPINA3 expression and clinicopathological characteristics and biological behavior of endometrial cancer at histology and cellular level.This study wanted to provide new starting point for revealing the pathogenesis of endometrial cancer and improving the outcome of endometrial cancer,and to provide clues for exploring endometrial cancer therapeutic targets.