miR-195靶向MYB激活PI3K-Akt通路调控皮肤鳞癌细胞增殖与恶性转移

miR-195 targeting MYB activates the PI3K-Akt pathway to regulate the proliferation and malignant metastasis of squamous cell carcinoma of the skin

目的:探讨微小RNA 195(miR-195)对皮肤鳞癌细胞增殖与恶性转移的影响及其机制。方法:通过实时荧光定量PCR(RT-qPCR)检测miR-195和MYB基因在皮肤鳞癌细胞(A431细胞)和人正常皮肤细胞(HaCaT细胞)中的表达。CCK-8实验检测过表达及下调miR-195对A431细胞增殖能力的影响;Western blot检测过表达及下调miR-195对A431细胞凋亡相关蛋白Bcl-2相关蛋白X(Bax)、B淋巴细胞瘤-2(Bcl-2)表达的影响;Western blot分析过表达及下调miR-195对A431细胞上皮细胞-间充质转化(EMT)的影响。TargetScan和双荧光素酶报告基因实验分析miR-195和MYB之间的关系,分析下调MYB对A431细胞增殖和EMT的影响。Western blot检测过表达MYB后对PI3K-Akt通路的影响。LY294002作用细胞后,检测过表达MYB对A431细胞增殖和EMT的影响。结果:与HaCaT细胞相比,A431细胞中miR-195表达明显下调(P<0.01),MYB表达明显上调(P<0.01)。过表达miR-195抑制A431细胞增殖与EMT,促进细胞凋亡,下调miR-195则起到相反作用;miR-195和MYB具有靶向和负调控关系;下调MYB的表达抑制A431细胞增殖与EMT;过表达MYB激活细胞内PI3K-Akt通路,且通过该通路促进A431细胞增殖与EMT。结论:miR-195通过靶向MYB激活PI3K-Akt通路调控A431细胞的增殖和转移。

Objective:To investigate the effect and mechanism of miR-195 on the proliferation and malignant metastasis of squamous cell carcinoma of the skin.Methods:The expressions of miR-195 and MYB gene in the epidermoid carcinoma cell line(A431 cells)and the human epidermal keratinocyte line(HaCaT cells)were detected by RT-qPCR.The effects of up-regulation and down-regulation of miR-195 on the proliferation ability of A431 cells were detected by CCK-8 experiment.The effects of up-regulation and down-regulation of miR-195 on the expression of apoptosis related proteins Bax and Bcl-2 in A431 cells were detected by Western blot.The effects of up-regulation and down-regulation of miR-195 on EMT of A431 cells were detected by Western blot.The relationship between miR-195 and MYB was analyzed by TargetScan,and the effects of down-regulation of MYB on A431 cell proliferation and EMT were analyzed by dual luciferase reporter gene assay.Western blot was used to detect the effects of MYB on PI3K-Akt pathway.Results:Compared with the expressions of miR-195 and MYB in HaCaT cells,the expressions of miR-195 in A431 cells were significantly down-regulated(P<0.01),the expressions of MYB were significantly up-regulated(P<0.01).Up-regulation of miR-195 inhibited the proliferation and EMT of A431 cells and promoted apoptosis,while down-regulation of miR-195 produced an opposite effect.miR-195 and MYB had a targeted and negative regulatory relationship.Down-regulation of MYB inhibited A431 cell proliferation and EMT.Overexpression of MYB activated the PI3K-Akt pathway and promoted proliferation and EMT of A431 cells.Conclusion:miR-195 regulates the proliferation and metastasis of A431 cells by targeting the MYB to activate the PI3K-Akt pathway.