Expression Of KISS1 Gene And Its Downstream PI3K/AKT-MMPs Pathway In Human Colorectal Cancer Cell Line LOVO Regulated By TCF21

Purpose :1.To study the effect of Lentiviral transfection overexpressing TCF21 on KISS1 gene expression in human colorectal cancer LOVO cells.2.To observe the biological changes of colorectal cancer LOVO cell proliferation,invasion and migration after transfection of TCF21 by lentivirus.3.To analyze the expression of PI3 K,AKT,p-AKT,MMP-2,MMP-9 and other key molecules in PI3K/AKT-MMPs signaling axis downstream of KISS1 after LVP overexpression of colorectal cancer LOVO cells.Methods :1.The human colorectal cancer cell line LOVO with low TCF21 expression was used as the research object.The TCF21 gene overexpression stable cell line was constructed using the lentivirus infection system.The TCF21 mRNA levels in the cell lines before and after transfection were detected by real-time fluorescence quantitative PCR and Western blotting,respectively.With the expression of protein levels,the expression stability of TCF21 in each cell line was identified for subsequent studies.2.The blank control group,negative control group(empty vector control group)and TCF21 gene overexpressing cell group were set in the experiment,and the protein expression level of KISS1 before and after transfection was detected by immunoblotting.3.MTT assays were used to analyze the changes in the ability of colorectal cancer cell proliferation in different subgroups;functional changes such as scratches and Transwell chambers were used to detect changes in cell metastasis and invasion.4.Western blot was used to examine the changes of protein expression levels of key molecules PI3 K,AKT,p-AKT,and MMPs in the PI3K/AKT-MMP9 signal axis before and after transfection.Results:1.After successfully transfecting LOVO cells with TCF21 overexpression vector packaged in Lentivirus,TCF21 mRNA and protein levels were significantly higher than those of the blank control group and the negative control group(empty vector control group),the difference was statistically significant(P<0.05)TCF21 is stably overexpressed in cells.2.Compared with the blank control group and the negative control group,the protein expression of KISS1 was increased in the TCF21 gene overexpression group,and the difference was statistically significant(P < 0.05).3.After successful overexpression of LOVO cells by Lentivirus,MTT assay showed that the cell proliferation ability was significantly weakened(p<0.05);Scratch test showed that the cell migration ability was significantly weakened(P<0.05);Transwell chamber experiments showed that the cell invasion ability was also weakened significantly.(P <0.01)4.After transfection of TCF21 gene in OE group,the expression level of PI3 K was significantly decreased compared with CON group(P<0.05).There was no significant difference between NC group and CON group(P>0.05).OE group p-The expression level of AKT was significantly lower than that of CON group(P<0.01).There was no statistical difference between NC group and CON group(P>0.05).The expression level of AKT in OE group had no significant change compared with CON group.There was no statistical difference between NC group and CON group(P>0.05).The expression level of downstream metalloproteinase MMP-2 in OE group was significantly lower than that in CON group(P<0.05).There was no significant difference between NC group and CON group(P>0.05).OE group MMP The expression level of-9 was significantly lower than that of CON group(P<0.05).There was no statistical difference between NC group and CON group(P>0.05).Conclusions:1.In colorectal cancer LOVO cell line,overexpression of TCF21 gene can up-regulate the expression of KISS1 gene and lead to the decrease of cell proliferation,invasion and migration.2.The KISS1 gene may act as a downstream functional gene during the progression of colorectal cancer lesions,undergo transcriptional regulation of the TCF21 gene,and exert its tumor metastasis suppressive effect by inhibiting the PI3K/AKT-MMPs pathway.