The Effects And Mechanisms Of TPRC3 On Migration And Invasion In Ovarian Cancer Cells

Ovarian cancer is one of the three malignant tumors common in female genital organs,and its mortality rate is the first in gynecological malignant tumor,which seriously threatens the life and health of women.Because the ovaries in deep pelvic,and the lack of a reliable and effective prevention and early detection method of epithelial ovarian sex(epithelial ovarian cancer,EOC)patients with ovarian discovered 70-80%for middle-late already,found widespread metastasis,the five-year survival rate is only 20%to 40%.However,the extensive metastasis of ovarian malignant tumors in the late stage is caused by the unpredictable and uncontrollable migration of ovarian cancer cells and the unpredictable and uncontrollable invasion ability.Therefore,in the research field of malignant tumors,the metastasis and invasion of ovarian cancer and other malignant biological behaviors have been a hot topic.Biological behaviour and molecular mechanism of the development of malignant tumor is the basis of the transformation of epithelial cells of the stroma(epithelial-m esenchym al transition,EMT).EMT is refers to the epithelial cells in morphology ectomesenchymal cells to phenotypic change and migration ability of a basic process,is the embryo physiology development,wound healing and the basis for the development of malignant epithelial tumors occur.Force phenotypic changes in the epithelial cells,and the migration and invasion to the regulation of initiating factor lies in the induction of external factors,genetic mutation,membrane receptors and signal channel change in form or function change,intracellular signal transduction.And transient receptor potential channels TRP is located on the cell membrane by multiple super family,composed of nonselective cation channels of Ca2+have appear a gender,increase Ca2+can lead to different physiological and pathological changes including cell proliferation,differentiation,apoptosis,etc.TRPC is a widely studied membrane channel in recent years,its main mediated Ca2+internal flow to participate in a variety of pathophysiological process,studies have shown that the kind of malignant tumor in more plays an important role in occurrence and development.TRPC1 and TRPC3 are mainly distributed in ovarian tissue.Previous studies have shown that TRPC3 can promote the proliferation and tumorigenesis of ovarian cancer cells,while the mechanisms of invasion and metastasis of ovarian cancer have not been reported.In this study,TRPC3 expression in different epithelial ovarian cancer cells and its role in migration and invasion were studied from three aspects.Observation of TRPC3inhibitory effect,and through the siRNA TRPC3 gene after cut influence on ovarian cancer cell migration,invasion ability,and to further explore TRPC3 in epithelial ovarian cancer(epithelial ovarian cancer,EOC)molecular mechanism of malignant biological behavior may involve,for clinical treatment of invasion and metastasis of ovarian recurrence and possible therapeutic targets.Objective:1.To clarify the differences in the expression levels of TRPC3 in SKOV3,ES-2 and HEY-T30 ovarian cancer cells and their relationship with the migration and invasion of ovarian cancer cells.2.To clarify the role of TRPC3 in regulating the migration and invasion of ovarian cancer cell line ES-2.3.To explore the mechanism of TRPC3 in the invasion and metastasis of epithelial ovarian cancer cells.Methods:1.Among the three human EOC cell lines of SKOV3,ES-2 and HEY-T30,1 x 10~5 cells were selected for Transwell migration experiment(without stromal colloid compartment)and Transwell invasion experiment(including stromal colloid compartment).Crystal violet staining was performed on the cells passing through the small chamber and count was performed under the microscope to analyze the differences in migration and invasion ability of three human EOC cells.2.Cultivate people EOC cell line SKOV3,ES-2,HEY-T30,select the logarithmic growth phase cells,cells extracted total RNA and proteins of cells,using method of qRT-PCR and Western Blot to test TRPC3 mRNA and protein expression level of three different human EOC cell lines.3.Choose high expression of TRPC3 EOC cell line ES-2,After transfection of TRPC3 siRNA and Control siRNA,extracting total cell mRNA and total cell protein and using method of qRT-PCR and Western Blot to detect TRPC3 mRNA and protein expression level,verify its interference efficiency.4.After transfection of human EOC cell line es-2 with TRPC3 siRNA and Control siRNA interference,1 x 105 cells were selected for Transwell migration experiment (without stromal colloid compartment)and Transwell invasion experiment(including stromal colloid compartment).The cells passing through the Transwell chamber were stained with crystal violet and counted under microscope,and the effect of downregulation of TRPC3 on migration and invasion ability of human EOC cells was analyzed.5.After transfection of human EOC cell line ES-2 with TRPC3 siRNA and Control siRNA,the total protein of cells was extracted.Western Blot method was used to detect the protein expression levels of EMT-related moleculeβ-catenin and Snail.6.After transfection of human EOC cell line ES-2 with TRPC3 siRNA and Control siRNA,the total protein of cells was extracted.Western Blot method was used to detect the protein expression level of BMPR2 in BMP/BMPR2 related signaling pathways.Results:1.Results of both Transwell migration experiment and Transwell invasion experiment showed that the migration and invasion ability of ES-2 cell lines were significantly higher than those of the other two strains in SKOV3,ES-2 and HEY-T30 human EOC cell lines.2.Both qRT-PCR and Western Blot test results showed that the mRNA and protein expression levels of TRPC3 in ES-2 cell lines were significantly higher than those in the other two strains in SKOV3,ES-2 and HEY-T30 human EOC cell lines.3.Both qRT-PCR and Western Blot test results showed that after transfection of TRPC3 siRNA,the expression levels of TRPC3 mRNA and protein in EOC cell line ES-2 were significantly lower than those in the control group,and the results confirmed that interference was effective.4.Transwell migration experiment(excluding chamber rubber matrix)rubber matrix and Transwell invasion experiment method(containing chamber rubber matrix),according to the results of transfection TRPC3 siRNA,EOC cell line ES-2 migration and invasion to the Transwell little room the lower cells was significantly less than the control group.5.Western Blot test results showed that after transfection of TRPC3 siRNA,the protein expression levels of EMT-related molecules calledβ-catenin and Snail in EOC cell lines were significantly lower than those in the control group.6.Western Blot test results showed that after transfection of TRPC3 siRNA,the protein expression level of BMPR2 in the BMP/BMPR2 signaling pathway of EOC cell line ES-2 was significantly higher than that of the control group.Conclusions:Three different people epithelial ovarian cancer cell lines in,including ES-2 cell lines migration,invasion ability was significantly higher in SKOV3,and HEY-T30 cell lines,and ES-2 cell lines TRPC3 expression is significantly higher than in the other two groups,prompted the TRPC3 may have relationship with ovarian malignant biological behavior.The migration and invasion ability of cell line ES-2 were significantly inhibited after the downregulation of TRPC3 gene,and detect the EMT related molecularβ-catenin and snail significantly lower at the same time,prompt TRPC3 in epithelial ovarian cancer cell migration,invasive malignant biological behavior plays an important role.In EOC cells,the reduction of TPRC3 can significantly raise BMPR2 protein expression,TRPC3 may through related signaling pathways mediating BMPR2,intensified its downstream related proteins to promote EOC malignant biological behavior.The next step is to further verify the results of BMPR2-related signaling pathways and in vivo experiments in nude mice.This research mainly through the cell level in vitro experiment of TRPC3 discussed influence on ovarian cancer invasion and metastasis,to further clarify back ovarian cancer invasion and metastasis mechanisms and potential molecular targeted therapy provides a preliminary theoretical basis.