Effects Of FEN1Gene Silencing On Proliferation And Apoptosis Of Gastric Cancer Cell Line SGC-7901

Backgroud:Flap endonuclease1(FEN1),a structure-specific5’nuclease,is a member of the Rad2structure-specific nuclease family.As aDNA repair protein,it plays pivotal roles in the maturation of Okazakifragments, long-patch base excision repair, restarting of stalled replicationforks and telomere maintenance.FEN1possesses5’ endonuclease,5’exonuclease and gap-endonuclease activities, which render it an essentialnode in maintaining genome fidelity.Objective:To investigate the association between the expression levelof FEN1and gastric cancer and to explore the role of FEN1incarcinogenesis and the progression of gastric cancer.Methods:1. The mRNA and protein expression of FEN1in42matched pairs ofhuman gastric tumor tissues and corresponding normal tissues weremeasured by semiquantitative reverse transcription-PCR andimmunohistochemical staining. The differential expression of FEN1between gastric cancer tissues and their matched normal tissues and the association between FEN1expression and clinicopathologicalcharacteristics was statistically analyzed.2. The specific si RNA targeting the FEN1gene was constructed andwas transfected into gastric cancer cell line SGC-7901usingLipofectamine2000after screening.Two groups of cells was transfectedin the study,including FEN1siRNA group (the SGC-7901cells transfectedwith FEN1siRNA) and the negative control group (transfected with NCsiRNA).Western blot analysis was used to evaluate the protein expressionof FEN1in SGC-7901human gastric cancer cells in order to verify thetransfection efficiency of FEN1siRNA.3. The cells transfected with FEN1siRNA were used as theexperimental group,while cells transfected with NC siRNA were used ascontrol group.Cell proliferation was analyzed by MTS assay. The apoptosisof the cells was determined by flow cytometry.Results:1. Semiquantitative RT-PCR showed that Of the42samples,32(76%)displayed a higher FEN1expression in the tumor tissues compared to thecorresponding normal tissues.The relative mRNA expression of FEN1inthe tumor tissues and matched normal tissues was1.32±0.26and1.02±0.34,respectively and the difference between the2groups (corresponding tonormal and tumor tissues) was statistically significant (P<0.01). As isshown from immunohistochemistry that higher expression of FEN1was observed in the tumor tissues in comparison to the corresponding normaltissues (P<0.01).In addition,The outcomes of immunohistochemistryintimated that the expression of FEN1positively correlates with the degreeof differentiation (P=0.027), lymphatic metastasis (P=0.001), tumor size(P=0.026) and TNM stage (P=0.020) in gastric cancer, but has nosignificant correlation with age and gender, as shown in our42cases ofgastric cancer.2. FEN1siRNA specifically targeting the FEN1gene was constructedand screened successfully then transfected into the SGC-7901celllines.The obvious down-regulation of FEN1expression was observed inthe SGC-7901cells transfected with FEN1siRNA compared to the cells inthe negative control group (transfected with NC siRNA; P<0.01).3. The proliferation of the SGC-7901cells transfected with FEN1siRNA was determined to be markedly suppressed in comparison to thecells transfected with the NC siRNA (P<0.05). The average apoptotis rateof the SGC-7901cells transfected with FEN1siRNA [(25.54±1.47)%]wasmarkedly increased in comparison to the cells in the negative control group[(8.86±2.04)%; P<0.01].Conclusion:1. The expression level of FEN1was evidently greater in gastrictumor tissues compared with the corresponding normal tissues and theoverexpression of FEN1is associated with tumor size, lymphatic metastasis, degree of differentiation and TNM stage in gastric cancer,whereas it is not associated with age and gender.It suggest that FEN1maybe a more precise maker for identifying gastric cancer with clinical dataand selecting patients in different situations.2. FEN1expression in the SGC-7901cells was markedly decreasedfollowing transfection with FEN1siRNA,which makes preparation for thesubsequent experiments.3. The down-regulation of FEN1in SGC-7901gastric cancer cellssuppressed proliferation and induced apoptosis,which suggest that FEN1may be an new and an effective therapeutic target in human gastric cancer.