| Objective:Cervical cancer is the most common malignant tumor in the female reproductive system.The population of cervical cancer has been younger year by year recent years,which seriously endangers women’s health.With the research achievements in the field of modern molecular biology such as tumor molecular pathology and gene detection,various genes or proteins involved in the occurrence and development of cervical cancer have been found one after another,but there are no targeted drugs for the treatment of cervical cancer.The relationship with FLCN and cervical cancer is not clear,although FLCN is a gene closely related to the occurrence and development of a variety of tumors.The purpose of this study was to explore the expression of folliculin(FLCN)gene in cervical squamous cell carcinoma and its effect on the proliferation and apoptosis of cervical squamous cell carcinoma SiHa cells,so as to lay an experimental foundation for further study of the mechanism of FLCN in the occurrence and development of cervical squamous cell carcinoma and molecular targeted therapy of cervical squamous cell carcinoma.Methods:(1)The cervical tissues of 29 patients with cervical squamous cell carcinoma(cervical squamous cell carcinoma group)and 27 patients who underwent total hysterectomy due to gynecological benign tumors(control group)were collected from December 2017 to November 2018 in the affiliated Hospital of Jiangsu University.Real-time fluorescence quantitative polymerase chain reaction(Quantitative Real-time PCR,qRT-PCR)and immunohistochemistry were used to detect the expression of FLCN mRNA and protein in cervical squamous cell carcinoma and normal cervical tissues.(2)Using liposome transfection method,NC siRNA,FLCN siRNA-1703 and FLCN siRNA-630 were transfected into cervical squamous cell carcinoma SiHa cells at preset concentrations,and the growth of cells in each group was observed under microscope.QRT-PCR was used to detect the level of FLCN mRNA in differentknockdown groups,and the optimal concentration of siRNA fragment was selected for follow-up test.(3)The blank plasmid and FLCN-eGFP plasmid were transfected into cervical squamous cell carcinoma SiHa cells by liposome transfection,and the growth of each group was observed under microscope.QRT-PCR and cellular immunofluorescence techniques were used to detect the levels of FLCN mRNA and protein in the cell lines after different transfection treatments.(4)The proliferation activity of cells in each group was detected by CCK-8method,and the apoptosis and proliferation of cells in each group were observed by TUNEL and phosphorylated histone H3(PH3)staining.Results:(1)Compared with the control group,the expression levels of FLCN mRNA and protein in cervical squamous cell carcinoma were significantly lower(all P < 0 05).(2)The results of qRT-PCR showed that compared with the control group,the relative expression level of FLCN mRNA in each siRNA transfection group was down-regulated to some extent.The interference effect of FLCN siRNA-630 is the best at the concentration of 50 nmol/L,which can be down-regulated by about 70%.Therefore,we chose to transfect 50 nmol/L FLCN siRNA-630 into SiHa cells for follow-up experiments.(3)48 hours after transfection of FLCN-eGFP plasmid,the expression of FLCNmRNA was significantly higher than that of the blank control group.After immunofluorescence staining,there were more green fluorescence signals after transfection of FLCN-eGFP.It was proved that FLCN gene was indeed transfected into SIHA cells,and the transfection efficiency was about 40% by calculation(P <0.01).(4)Compared with the negative control group,the positive proportion of PH3 protein increased((6.24 ±1.20)vs(0.85 ± 0.05),P <0.05] and the proportion of TUNEL positive cells decreased((6.24 ± 1.20)vs(0.85 ± 0.05),P < 0.05)in FLCNsiRNA-630 group,suggesting that cell proliferation was enhanced and apoptosis decreased.(5)Compared with the blank control group,the proliferation activity of cells in FLCN overexpression group was decreased,the proportion of PH3 protein positive cells in SiHa cells decreased((0.67±0.11)vs(7.64±0.55),P<0.01)and the proportion of TUNEL positive cells increased((0.67 ±0.11)vs(7.64 ±0.55),P < 0.001)in SiHa cells,suggesting that cell proliferation was weakened and apoptosis was enhanced.Conclusion:(1)The expression of FLCN gene is low in cervical squamous cell carcinoma.(2)FLCN gene can inhibit the proliferation and promote the apoptosis of SiHa cells. |
