Preliminary Exploration On Signal Transduction Pathway Of Kiss-1 Gene In Human Colorectal Cancer Cells

Objective: 1、To observe the effect of Kiss-1 gene on the cell proliferation, migration and invasion ability of human colorectal cancer cells. 2、To explore the potential relevant signal transduction pathways of Kiss-1 gene in human colorectal cancer cells.Methods: 1、q RT- PCR method was used to screen the lowest Kiss-1 gene m RNA expression level of 5 kinds of human colorectal carcinoma cells — Lo Vo, SW480, HT-29, HCT116, SW620. 2、Built the lentiviral vectors of Kiss-1 gene and non-carrier, then transfected HCT116 cells. q RT-PCR and Western blot methods were used to detect Kiss-1 gene product m RNA and metastin level of primary HCT116 human colorectal carcinoma cells, non-carrier transfected cells and lentiviral vector transfected cells. Then, divided them respectively into: the blank control group(CON group), the non-carrier contral group(NC group) and the over-expression group(OE). 3、Counting Kit- 8(CCK8) and Transwell Chambers methods were used to detect the changes of cell proliferation, invasion and migration ability of the three groups. 4、Western blot was used to detect the expression of key molecular of PKC signal pathway—PKCα、PKCβII and downstream effector—E cadherin of the three groups. 5、 Western blot was used to detect the expression of Rho A, the key molecular Rho/Rho Kinase signaling pathway.Results: 1、q RT-PCR showed that Kiss-1 gene m RNA expressions of of Lo Vo, SW480, HT29, HCT116 and SW620 human colorectal carcinoma cells were all medium, the lowest expression was HCT116 cells(P < 0.001) and the highest was SW620 cells, both with statistical significances(P < 0.005). 2、After lentiviral transfection, HCT-116 cells could stably express EGFP report gene, and the fluorescence rate was over 80%. q RT-PCR and Western blot showed that the Kiss-1 gene product m RNA and metastin level of the over-expression group were significantly increased, there were statistically significant differences(P < 0.05). 3、CCK-8 showed that cell proliferation ability of the over-expression group decreased obviously, there was statistically significant difference(P < 0.05); Transwell chambers experiments showed that cell invasion, migration ability of the over-expression group also significantly decreased with statistical significance(P < 0.05). 4、Western blot showed that PKCα expression level of the over-expression group had little change compared to that of the control group and the non-carrier group(P>0.05), PKCβII expression level of the over-expression group decreased obviously compared to that of the control group and the non-carrier group, there was a clear statistical difference(P <0.05) and E-cadherin expression level rose highly compared to that of the other two groups with statistical significance(P < 0.05). 5、Western blot showed that Rho A expression level of the over-expression group had little change compared to those of the control group and the non-carrier group(P>0.05).Conclusion:1、In Lo Vo, SW480, HT29, HCT116, SW620 these five kinds of human colorectal carcinoma cells, Kiss-1 gene expression of HCT116 cell was the lowest. 2、After lentiviral transfection, HCT-116 cells could stably over-expressed kiss-1 gene.3、Kiss-1 gene can suppress cell proliferation, cell invasion and cell migration ability of HCT116 human colorectal carcinoma cells. 4、PKC signal pathway could be the possible signal transduction pathway of Kiss-1 gene in suppression of human colorectal carcinoma metastasis, maybe work by up-regulating expression level of downstream protein E-cadherin. 5、Rho/Rho kinase signal pathway is irrelevant to Kiss-1 gene in suppression of human colorectal carcinoma metastasis, or other key moleculars may be relevant.