敲低膜联蛋白A5对人胃癌MGC-803和MKN-45细胞周期的影响

doi:10.16098/j.issn.0529-1356.2018.03.012

摘要/Abstract

摘要：

目的探讨敲低膜联蛋白A5（ANXA5）对人胃癌细胞系MGC-803、MKN-45细胞周期相关蛋白表达的影响。方法将细胞分为干扰组、阴性对照组及空白对照组，采用脂质体转染法将靶向膜联蛋白A5的siRNA以及阴性siRNA分别转染干扰组和阴性对照组MGC-803、MKN-45细胞，空白对照组不加任何试剂。转染后48 h采用Real-time PCR和Western bloting分别从mRNA和蛋白水平检测ANXA5的表达，确定ANXA5被敲低之后，Real-time PCR和Western bloting检测各组p21^cip1 mRNA和P21^cip1的表达，Western bloting检测各组细胞周期蛋白D1（cyclinD1）蛋白的表达，流式细胞术检测各组细胞周期的变化情况。结果敲低ANXA5后，与阴性对照组和空白对照组相比，两种细胞的p21^cip1mRNA和P21^cip1蛋白均显著降低（P<0.05），cyclinD1（P<0.05）也显著降低。结论敲低ANXA5可下调MGC-803、MKN-45细胞中p21^cip1mRNA和P21^cip1蛋白以及cyclinD1的表达，推测ANXA5可能通过作用于细胞周期相关蛋白而引发细胞周期阻滞从而影响着胃癌的发展。

关键词: 膜联蛋白A5, MGC-803细胞, MKN-45细胞, 细胞周期蛋白D1, 实时定量聚合酶链反应, 免疫印迹法, 流式细胞术, 人

Abstract:

Objective To investigate the effects of annexin A5 (ANXA5) konckdown on cell cycle of human gastric cancer cell lines MGC-803, MKN-45 cells. Methods Cells were diveded into interference group, negative control group and blank control group. The siRNA targeted to ANXA5 was transfected into MGC-803, MKN-45 cells by lipofectamine 2000 respectively. At the same time, negative siRNA was also transfected into negative control group and in blank control group nothing was added in. Real-time PCR and Western blotting was used to identify whether the siRNA targeted to ANXA5 can highly suppress annexin A5 expression 48 hours after transfection. When the effect of the siRNA was assured, Real-time PCR and Western blotting were applied to observe the expression of p21^cip1 mRNA and P21^cip1 protein, Western blotting was used to detect the expression of cyclinD1, flow cytometry(FCM) was applied to observe the cell cycle progression. Results The expression of p21^cip1 mRNA, P21^cip1 protein and cyclinD1 decreased significantly when compared with the negative control group and the blank control group(P<0.05). FCM analysis showed that the percentage of cells in G1 phase increased in interference group(P<0.05). Conclusion ANXA5 knockdown can down-regulate the expression of p21^cip1 mRNA, P21^cip1 protein and cyclinD1 in human gastric cancer MGC-803 and MKN-45 cells, it is speculated that ANXA5 may affect the development of gastric cancer by inducing cell cycle arrest by acting on cell cycle related proteins.

Key words: Annexin A5, MGC-803, MKN-45, CyclinD1, Real-time PCR, Western blotting, Flow cytometry, Human

王小杰赵依纳李欣. 敲低膜联蛋白A5对人胃癌MGC-803和MKN-45细胞周期的影响[J]. 解剖学报, 2018, 49(3): 342-346.

WANG Xiao-jie ZHAO Yi-na LI Xin. Effects of annexin A5 knockdown on cell cycle of human gastric cancer MGC-803 and MKN-45 cells [J]. Acta Anatomica Sinica, 2018, 49(3): 342-346.

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