抑制B7-H4表达对结直肠癌HT-29细胞增殖、凋亡和迁移的影响及其机制

Effect of inhibiting B7-H4 expression on proliferation,apoptosis,and migration of colorectal cancer HT-29 cells and its mechanism

目的:探讨抑制B7-H4表达对结直肠癌HT-29细胞增殖、凋亡和迁移的影响,阐明B7-H4在结直肠癌发生发展中的作用。方法:以脂质体法转染pSilencer4.1-B7-H4-shRNA和pSilencer4.1-scrambled shRNA载体至HT-29细胞,作为B7-H4 shRNA组和scrambled shRNA组。RT-qPCR法检测2组HT-29细胞中B7-H4 mRNA表达水平,Western blotting法检测2组HT-29细胞中B7-H4蛋白表达水平,CCK-8法检测2组HT-29细胞增殖活性,流式细胞术检测2组不同细胞周期HT-29细胞百分率和细胞凋亡率,Transwell实验检测2组迁移细胞数,ELISA法检测2组HT-29细胞上清液中基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)水平,Western blotting法检测2组HT-29细胞中Bcl-2和Caspase-3蛋白表达量,实时荧光定量PCR(RT-qPCR)法检测抑制PI3K/Akt/mTOR信号通路前后HT-29细胞中B7-H4 mRNA表达水平,Western blotting法检测抑制PI3K/Akt/mTOR信号通路前后HT-29细胞中B7-H4蛋白表达水平。结果:B7-H4 shRNA组HT-29细胞中B7-H4 mRNA和蛋白表达水平明显低于scrambled shRNA组(P<0.01)。与scrambled shRNA组比较,B7-H4 shRNA组HT-29细胞增殖活性降低(P<0.05),G0/G1期和S期细胞百分率比较差异无统计学意义(P>0.05),细胞凋亡率和细胞中Casapse-3蛋白表达水平明显升高(P<0.05),细胞中Bcl-2蛋白表达水平明显降低(P<0.05),HT-29细胞的迁移细胞数减少(P<0.01),MMP-2和MMP-9水平也明显降低(P<0.05)。与对照组比较,LY294002组和雷帕霉素组HT-29细胞中B7-H4 mRNA和蛋白表达水平均降低(P<0.05)。结论:抑制B7-H4表达能够明显抑制结直肠癌HT-29细胞的增殖、凋亡和迁移,其作用机制可能与PI3K/AktmTOR信号通路活性有关联。

Objective:To investigate the effect of inhibiting B7-H4 expression on the proliferation,apoptosis,and migration of colorectal cancer HT-29 cells,and to elucidate the role of B7-H4 in the occurrence and development of colorectal cancer.Methods:The pSilencer4.1-B7-H4-shRNA and pSilencer4.1-scrambled shRNA vectors were converted into the HT-29 cells by liposome method as B7-H4 shRNA group and scrambled shRNA group.The expression levels of B7-H4 mRNA in the HT-29 cells in two groups was detected by RT-qPCR method;the expression levels of B7-H4 protein in the HT-29 cells in two groups were detected by Western blotting method;CCK-8 method was performed to detect the proliferation activities of the HT-29 cells in two groups;flow cytometry was used to detect the percentages of HT-29 cells at different cell cycles and the apoptotic rates of the HT-29 cells in two groups;Transwell chamber assay was used to measure the number of migration cells of the HT-29 cells in two groups;the levels of matrix metalloproteinases 2(MMP-2)and matrix metalloproteinases 9(MMP-9)in liquid supernatant of the HT-29 cells in two groups were detected by ELISA assay;the expression amounts of Bcl-2 and Caspase-3 in the HT-29 cells in two groups were detected by Western blotting method;the expression levels of B7-H4 mRNA in HT-29 cells before and after inhibiting the PI3K/Akt/mTOR signal pathway were analyzed by RT-qPCR method;Western blotting method was used to detect the expression levels of B7-H4 protein in the HT-29 cells before and after inhibiting the PI3K/Akt/mTOR signal pathway.Results:The expression levels of B7-H4 mRNA and protein in the HT-29 cells in B7-H4 shRNA group were significantly lower than those in scrambled shRNA group(P<0.01).Compared with scrambled shRNA group,the proliferation ability of the HT-29 cells in B7-H4 shRNA group was decreased(P<0.05),and the percentages of cells in G0/G1 phase and S phase had were no statistically significant differences(P>0.05),the apoptotic rate and the expression level of Caspase-3 protein in the HT-29 cells were significantly increased(P<0.05),the expression level of Bcl-2 protein was significantly decreased(P<0.05),the number of migrating cells was decreased(P<0.01),and the levels of MMP-2 and MMP-9 were also significantly decreased(P<0.05).Compared with control group,the expression levels of B7-H4 mRNA and protein in the HT-29 cells in LY294002 group and rapamycin group were significantly decreased(P<0.05).Conclusion:Silencing of B7-H4 expression can significantly inhibit the proliferation,apoptosis and migration of the HT-29 cells,and its mechanism may be related to the activity of PI3K/Akt/mTOR signaling pathway.