The Effect Of SiRNA Silencing YKL-40 Gene Expression On The In Vitro Biological Characteristics Of Endometrial Carcinoma HEC-1A Cells

Objective: The purpose of this experiment was to screen from endometrial cancer cell lines which has high expression of YKL-40 gene.We detected the expression of YKL-40 m RNA in HEC-1A、HEC-1B、ishikawa and RL-952 endometrial cancer cell lines through q PCR assay.Then we inhibited the expression of YKL-40 gene through RNAi technology on HEC-1A cells.Methods: 1.The quantitative real time-PCR(q PCR)assay was performed to investigate the levels of YKL-40 m RNA in four endometrial cancer cell lines.2.To inhibit the expression of YKL-40 gene through RNAi technology on HEC-1A cells.3.The YKL-40 gene RNA interference vector was transform into endometrial cancer HEC-1A cells by lentivirus.4.There were three groups in our experiment: siRNA experimental group(E.G)was transfected with YKL-40 siRNA,mock-treatment group(mock)was transfected with transfection reagent only,and the blank control group(blank)was left untransfected.The cells in the logarithmic growth phase were used for the experiments.5.Constructing and establishing of YKL-40 siRNA lentiviral vector in HEC-1A stable transfected cell line: The cells were cultured in medium supplemented with puromycin and the puromycin-resistant cells were used for our subsequent experiments.6.The m RNA expression of YKL-40 in each group was measured using quantitative real time-PCR(q PCR)assay after transfected with YKL-40 siRNA.7.The western blotting assay was performed to investigate the YKL-40 protein after transfected with YKL-40 siRNA.Results: 1.The expression of YKL-40 m RNA in four strains of endometrial cancer : The expression of YKL-40 gene was highly in the HEC-1A cell line other than HEC-1B,ishikawa and RL-952 cell lines(HEC-1A: 1.0052±0.13;HEC-1B: 0.0024±0.00;ishikawa: 0.0017±0.00;RL-952: 0.0032±0.00)(P<0.05).2.The sequencing confirmed that the YKL-40 sh RNA gene was successfully constructed.And the corresponding lentiviral vector titer is 1×108PFU /ml.3.Detection of fluorescence in transfected cells: The transfected cells were observed under a fluorescence microscope.The HEC-1A cells that were successfully transfected with siRNA exhibited a green fluorescence.4.Constructing and establishing of YKL-40 siRNA lentiviral vector in HEC-1A stable transfected cell line: The cells were cultured in medium supplemented with puromycin and the puromycin-resistant cells were transformed successfuly.5.Interference effects of YKL-40 siRNA: After infection with lentivirus,the expression of YKL-40 in the experiment group was lower compared to thatin the blank control group and mock-treatment group(F=6.875,P=0.015).No significant difference was seen between the blank control group and mock-treatment group(P>0.05).6.The expression of YKL-40 protein after tansfected with YKL-40 siRNA in the western blotting assay: No significance was seen between the experimental group,blank group and the mock-treatment group(P=0.061).Objective: The purpose of this study is to explore the effects of YKL-40 gene RNA interference(siRNA)on the biological behaviors of endometrial cancer HEC-1A cells and the sensitivity of endometrial cancer HEC-1A cells to cisplatin-based chemotherapy.Methods: 1.The puromycin-resistant endometrial cancer HEC-1A cells were used for our experiments.2.The m RNA expression of YKL-40 in each group was measured using quantitative real time-PCR(q PCR)assay after treated with cisplatin.3.The proliferation of HEC-1A cells were also determined using MTT assay after treated with cisplatin.4.The biological behavior including cell proliferation,migration,invasion and apoptosis were detected by MTT,transwell,and flow cytometer(FCM),respectively.Results: 1.YKL-40 gene controls the proliferation potential of HEC-1Acells: MTT assays indicated that si-YKL-40 inhibited the HEC-1A cell proliferation compared to the blank control group and mock-treatment group(P<0.05).2.YKL-40 gene promotes the migratory potential of HEC-1A cells : Cell migration assays showed that si-YKL-40(133±14)inhibited the HEC-1A cell migration abilities compared to the blank control group(179±19)and mock-treatment group(178±11)(F=14.494,P=0.001).3.YKL-40 gene regulates the invasive ability of HEC-1A cells: The Matrigel invasion assays revealed that the invasion ability of HEC-1A cells in the experimental group was significantly inhibited,the invasion number(143±13)was lower than that of the blank control group(227±18)and mock-treatment group(238±26)(F=33.476,P<0.001).4.YKL-40 gene controls the proliferation ability of HEC-1A cells after treated with cisplatin:The proliferation ability of HEC-1A cells significantly inhibited when the cells were addding different concentration of cisplatin48 hours.The experiment group(transfection after siRNA)was sinificanty inhibited than the mock-treatment group and the blank control group(P<0.005),while no difference between the mock-treatment group and the blank control group(P>0.05).5.The m RNA expression of YKL-40 was up-regulated when treated HEC-1A cells with cisplatin(P=0.000).6.YKL-40 gene inhibits apoposis of HEC-1A cells after treated with cisplatin:HEC-1A cells apoptosis rate increased after the inhibition of YKL-40expression(E.G: 38.07±4.88,blank: 13.3±1.01,mock: 12.5±0.17),when treated with cisplatin for 48 hours.FCM revealed that the average cell apoptosis rate increased after the inhibition of YKL-40 gene expression(P=0.000,P=0.000).Conclusion:YKL-40 gene involves the proliferation,migration,invasion and anti-apoptosis of HEC-1A cells.The sensitivity of HCE-1A cells to cisplatin was elevated by silencing YKL-40 gene.And YKL-40 gene have the ability of anti-apoptosis in HCE-1A cells.YKL-40 gene is hopeful to be a potential small molecular target for the treatment of endometrial cancer.