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

doi:10.13481/j.1671-587x.20200613

摘要/Abstract

摘要： 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。

关键词: B7-H4, 结直肠肿瘤, 细胞增殖, 细胞迁移, 细胞凋亡

Abstract:

Objebtive： 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.

Key words: B7-H4, colorectal carcinoma, proliferation, cell migration, apoptosis

中图分类号:

R735.3

马绪哲,湛玉东,王丹,历春,盖晓东. 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[J]. 吉林大学学报(医学版), 2020, 46(6): 1187-1193.

Xuzhe MA,Yudong ZHAN,Dan WANG,Chun LI,Xiaodong GAI. Effect of inhibiting B7-H4 expression on proliferation, apoptosis，and migration of colorectal cancer HT-29 cells and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(6): 1187-1193.

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