消岩汤抑制WISP2/CCN5基因敲减的MCF-7细胞增殖的分子机制

The research on mechanism of Xiaoyan decoction inhibiting the proliferation of MCF-7 with knockdown of WISP2/CCN5 gene

目的观察消岩汤对WISP2/CCN5基因敲减的MCF-7细胞增殖的抑制作用,以及WISP2/CCN5、Skp2和p27Kip1 mRNA和蛋白水平变化,探索消岩汤抑制WISP2/CCN5基因敲降的MCF-7细胞增殖的分子机制。方法WISP2/CCN5 shRNA慢病毒质粒转染MCF-7细胞,构建WISP2/CCN5基因敲减的MCF-7细胞系,采用高、中、低浓度的消岩汤孵育WISP2/CCN5基因敲减的MCF-7细胞24、36及48 h后,检测细胞增殖抑制率、WISP2/CCN5、Skp2及p27Kip1的mRNA和蛋白水平。结果WISP2/CCN5基因敲减的MCF-7细胞增殖抑制率低于空白组和空载组(P<0.05),消岩汤干预组细胞增殖抑制率高于WISP2/CCN5基因敲减组(P<0.05);高浓度消岩汤孵育WISP2/CCN5基因敲减的MCF-7细胞48 h组细胞增殖抑制率高于相同浓度孵育24 h组(P<0.05)。WISP2/CCN5基因敲减组Skp2 mRNA和蛋白水平均高于空白组和空载组(P<0.05,P<0.05),而p27Kip1 mRNA和蛋白水平则低于空白组和空载组(P<0.05,P<0.05)。消岩汤干预组的Skp2 mRNA和蛋白水平低于WISP2/CCN5基因敲减组(P<0.05,P<0.05),而p27Kip1 mRNA和蛋白水平则高于WISP2/CCN5基因敲减组(P<0.05,P<0.05)。结论消岩汤干预WISP2/CCN5基因敲减的MCF-7细胞,在mRNA和蛋白水平下调Skp2表达,上调p27Kip1表达,抑制MCF-7细胞增殖;2.高浓度消岩汤对细胞增殖活性的抑制较显著,孵育时间较长组对细胞增殖活性的抑制较显著。

Objective To investigate the inhibition of Xiaoyan decoction on proliferation of MCF-7 cells with WISP2/CCN5 gene knockdown,and both mRNA and protein levels of WISP2/CCN5,Skp2 and p27Kipl,so the mechanism of Xiaoyan decoction inhibiting proliferation of MCF-7 cells.Methods WISP2/CCN5 shRNA lentivirus plasmid was used to establish the WISP2/CCN5 gene knockdown MCF-7 cells.Xiaoyan decoction with different concentration were used to incubate the MCF-7 cells with W1SP2/CCN5 gene knockdown for 24 h,36 h and 48 h.RT-PCR and Western blot were used to evaluate the effect of Xiaoyan decoction on both mRNA and protein levels of WISP2/CCN5,Skp2 and p27Kipl.Results The inhibition on proliferation of MCF-7 cells with WISP2/CCN5 gene knockdown was significantly higher than that of control group(P<0.05),while the proliferation inhibition rate of MCF-7 cells with WISP2/CCN5 gene knockdown incubated with higher concentration of Xiaoyan decoction for 48 h was significantly higher than that of 24 h with same concentration(P<0.05).Compared to control group,Skp2 mRNA and protein levels of WISP2/CCN5 gene knockdown group was significantly increased(P<0.05),while p27Kipl mRNA and protein levels was significantly reduced(P<0.05).Compared to WISP2/CCN5 gene knockdown group,Skp2 mRNA and protein levels of MCF-7 cells with WISP2/CCN5 gene knockdown incubated by Xiaoyan decoction were significantly reduced(P<0.05),while p27Kipl mRNA and protein levels were significantly increased(P<0.05).Conclusion Xiaoyan decoction inhibits the proliferation of MCF-7 cells with WISP2/CCN5 gene knockdown by up-regulation of Skp2 and down-regulation of p27Kipl at both mRNA and protein levels.Higher concentration and longer incubation of Xiaoyan decoction presents more significant inhibition effects on MCF-7 cells with WISP2/CCN5 gene knockdown.