Effect Of RNA Interference Mediated SNCG Gene Downregulation On Hunman Endometrial Carcinoma Cell Line HEC-1A

Endometrial cancer is one of the three major malignant tumors of the female genital tract,of which prevalence is nearly behind Cervical Cancer.In recent years,the prevalence of endometrial cancer in China has increased significantly and tends to younger.It is becoming a big threat to the women’s health among malignant diseases in female genital tract.Our prevoius research showed that the expression γ-Synuclein protein in endometrial carcinoma was significantly higher than that in normal endometrial tissues,and closely related to the clinical stage,lymph node metastasis and depth of myometrial invasion.The overall survival rate of γ-Synuclein positive group was significantly lower than that of negative group [1-2],suggesting thatγ-Synuclein expression may be associated with the development,invasion,metastasis and the prognosis of endometrial carcinoma.In order to further elucidate the mechanism of SNCG gene in promoting the malignant progression of endometrial carcinoma cells,we constructed the stable transfected endometrial cancer lines low-expressing SNCG protein using the RNA interfering techinque targeting SNCG gene.The effects of SNCG gene on biological behaviors of endometrial carcinoma cell lines were investigated usingCCK8,cloning,scratches,invasion,cell apoptosis and cell cycle experiments.PART Ⅰ Screening of human endometrial carcinoma cell lines with high expression of SNCG【Objective】Firstly,we screen from endometrial carcinoma cell lines which has high expression of SNCG in protein.And then,those who has comparatively high expression of SNCG protein will be cell basis for the construction of sh RNA Lentiviral vector for the next step of our study.【Method】Through cell culture techniques,develop Ishikawa,HEC-1B,HEC-1A and other human endometrial cancer cell,which would be collected in the proliferative phase of various strains of endometrial cancer cell samples for experiments.Using quantitative Western Blot detection of protein expression of SNCG in all cell lines.【Results】By cell culture and Western Blot techniques,we filter out the higher expression of SNCG cell HEC-1A,and its protein expression of SNCG and the remaining two have significant differences(P<0.05).【Conclusion 】 Human endometrial cancer cell line HEC-1A in the expression of SNCG protein has a relatively high level of silence as I experiment next SNCG cell basis.PART Ⅱ Constructing and establishing of SNCG sh RNA lentiviral vector in HEC-1A stable transfected cell line【 Objective 】 To inhibit the gene expression of SNCG through RNAi technology on HEC-1A which would silence SNCG genes of Endometrial Carcinoma HEC-1A cell line stably.【Method】1.By RNAi-related software design and synthesis of three specific SNCG sh RNA sequences of the gene and construction of sh RNA Lentiviral transfection of HEC-1A cells.2.Using RT-PCR and Western Blot technology to detect the effect in inhibition of SNCG gene,so as to filter out the best efficiency in silence target。3.Take the best target sequences into Lentiviral sh RNA Lentiviral transfection of HEC-1A cells.4.HEC-1A cells were transfected with SNCG gene to obtain stable cell lines,and the inhibitory effect on SNCG gene was detected by RT-PCR and Western Blot.【Results】1.The best interference target sequence which was screened by RT-PCR and Western Blot:CCAAGGAGAATGTTGTACA。2.The sequencing confirmed that the sh RNA gene was successfully constructed.And the corresponding lentiviral vector titer is 8x108TU/ml。3.The HEC-1A cells were transfected with lentiviral vector and then screened by puromycin.The growth state of the cells before and aftertransfection was observed by inverted microscope,and the interference efficiency was verified by RT-PCR and Western Blot.Stable HEC-1A cell lines were obtained by the above techniques.【 Conclusion 】 Lentiviral vector of human SNCG gene can effectively inhibit the expression of SNCG gene in endometrial carcinoma cell line HEC-1A,and lay the foundation of cell model for the following experiments.PART Ⅲ Effect of RNA interference mediated SNCG gene downregulatation in human endometrial cell line HEC-1A【Objective】Chapter on the construction of stable expression of SNCG silenced in human endometrial cancer cell line HEC-1A as cell model of this chapter.Through the observation of their cell proliferation,migration,invasion,and changes of cell cycle and apoptosis to explore SNCG gene silencing effect on endometrial cancer cell line HEC-1A.【 Method 】 1.Using SNCG-KD1 recombinant lentivirus and transfected human endometrial cancer cells.The subjects were divided into three groups:group SNCG-KD1: sh RNA lentiviral vector SNCG after low expression of endometrial carcinoma cell line HEC-1A;control group: high expression of SNCG in endometrial carcinoma HEC-1A cell line;Empty vector transfection group.2.CCK8 assay was used to detect the proliferation of those three groups of cells before and after the analysis of SNCG gene silencing effect on the survival12 rate of HEC-1A cells.3.Through the cell colony formation,before and after the analysis of SNCG gene silencing effect on HEC-1A cell proliferation.4.The migration ability of the three groups of cells was detected by scratch assay.The effects of SNCG gene silencing on the migration ability of HEC-1A cells were analyzed5.The invasion ability of each group of cells was detected by invasion chamber method,and the effect of SNCG gene silencing on invasion ability of HEC-1A cells in vitro was analyzed.6.Three groups were detected by flow cytometry cell cycle changes before and after the analysis of SNCG gene silencing effect on HEC-1A cell cycle.7.The effect of SNCG gene silencing on apoptosis of HEC-1A cells was detected by detecting the apoptosis of the cells through Annexin-V&PI.【Results】1.CCK8 test results show that: There was no significant difference in cell doubling time between empty vector transfection group and the control group(P > 0.05).SNCG-KD1 lentivirus transfected HEC-1A cells,the doubling time of cells in SNCG-KD1 group compared with the control group decreased significantly(P < 0.05).2.The colony formation assay showed that The number of empty vector transfection cell clone group staining(%)and the control group had no significant difference(P>0.05).After lentivirus transfected HEC-1A cells,endometrial cancer cells of SNCG-KD1 group was lower than that of control group the number of cells,the difference was statistically significant(P<0.05).The number of empty vector transfection group is comparatively higher thanthose of SNCG-KD1 group and the difference was statistically significant(P <0.05).3.Cell scratch assay showed that There is no significant difference in 8hours,24 hours migration rate(%)between empty vector transfection Group and the control group(P>0.05).The result of sh RNA lentivirus transfected HEC-1A cells in endometrial cancer cells in SNCG-KD1 group than in the control group 24 hours reduced migration rate(%)decreased,and the difference was statistically significant(P<0.05).The 24 hours migration rate in SNCG-KD1 group in scratch was decreased,compared with the empty vector group,whose difference was statistically significant(P < 0.05).4.There is no statistical difference between empty vector group and the control group in migration rate(%)(P>0.05).Transwell assay showed that migration rate(%)of SNCG-KD1 group is lower than that of the control group decreased the rate(%)decreased,and the difference between them was statistically significant(P<0.05).The difference was statistically significant when experimental group compared with the empty transfected group in migration rate the invasion assay(P < 0.05).5.The ratio of the difference in the control group and empty vector group was not statistically significant(P> 0.05).The the cell cycle experimental results showed that the ratio of G1,G2/M phase of SNCG-KD1 group was increasing compared with the control group,and their difference was statistically significant(P < 0.05).6.The empty group of total cell apoptosis rate(%)and that of the control group had no significant difference(P > 0.05).The results showed that apoptosis in endometrial cancer cells SNCG-KD1 group than in the control group the totalapoptosis rate(%)decreased,the difference was statistically significant(P <0.05).The total apoptosis rate(%)of group SNCG-KD1 increased than the empty vector group,and their difference was statistically significant(P < 0.05).【Conclusion】In vitro cultured SNCG gene silenced human endometrial cancer cell line malignant biological behavior such as tumor cell proliferation,migration,invasion ability was significantly reduced,and endometrial cancer HEC-1A cell line SNCG stable silence,Tumor cell cycle arrests in G1 and G2 /M phase,we suggesting that abnormal overexpression of SNCG gene may be closely related to the cell cycle of human endometrial carcinoma cell line HEC-1A in vitro,which can be presumably predicted in endometrial cancer cells SNCG gene may be a potential target for molecular therapy of endometrial cancer.