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

Objective: 1、To explore the effect of Kiss-1 gene on the cell proliferation, migration and invasion ability of human colorectal cancer cells. 2、To explore the relevant signal transduction pathways of Kiss-1 gene in human colorectal cancer cells. 3、To analyze the possible mechanism of Kiss-1 gene in suppression of human colorectal cancer cells metastasis. Methods: 1、QRT- PCR method was used to screen the lowest Kiss-1 gene mRNA expression level of 5 kinds of human colorectal cancer cells—LOVO, SW480, HT-29, HCT116, SW620. 2、Built the lentiviral vectors of Kiss-1 gene and non-carrier, then transfected HCT116 cells. Primary HCT116 human colorectal cancer cells, non-carrier transfected cells and lentiviral vector transfected cells are divided respectively into: the blank control group(CON group), the non-carrier contral group(NC group) and the over-expression group(OE). 3、Using Counting Kit- 8(CCK8) and Transwell Chambers mehtod 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 MEK of the three groups—key molecular of MAPK signal pathway, and phosphorylation level of downstream effector—MLC.5 、 Western blot was used to detect the expression of I-kB of the three groups—inhibitive protein of NF-kB signal pathway, and expression of downstream effector—MMP-9. Results: 1、QRT-PCR showed that the Kiss-1 gene mRNA expressions of LOVO, SW480, HT29, HCT116 and SW620 human colorectal cancer cells were all medium, the expression in HCT116 cells was the lowest, with statistical significance(P < 0.01).2、After lentiviral transfection, HCT-116 cells could stably express EGFP report gene, and the fluorescence rate was over 80%. QRT-PCR and Western blot showed that the Kiss-1 gene product m RNA and its protein-metastin level of the over-expression group were significantly increased, there were statistically significant differences(P < 0.05).3、After lentiviral transfection, CCK8 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 significances(P < 0.05).4、Western blot showed that expression level of MEK and phosphorylation level of MLC of the over-expression group decreased significantly compared to those of the control group and the non-carrier group with clear statistical differences(P < 0.05).5、 Western blot showed that the I-kB expression level of the over-expression group rised and the MMP-9 expression level decreased compared to those of the control group and the non-carrier group(P<0.05).Conclusion:1、After lentiviral transfection, HCT-116 human colorectal cancer cells could stably over-expressed Kiss-1 gene. And Kiss-1 gene can suppress cell proliferation, invasion and migration ability.2、MAPK signal pathway is relevant to suppression of human colorectal tumor proliferation, invasion and migration ability via Kiss-1 gene, maybe work by reducing phosphorylation level of downstream protein MLC.3、NF-?B signal pathway is relevant to suppression of human colorectal tumor proliferation, invasion and migration ability via Kiss-1 gene, maybe work by reducing expression level of downstream protein MMP-9.4、There may be many signal pathways taking parts in suppression of human colorectal tumor proliferation, invasion and migration ability via Kiss-1 gene.