MiR-374a通过PTEN/AKT信号通路调节乳腺癌细胞的增殖和凋亡

MicroRNA-374a Regulates Proliferation and Apoptosis of Breast Cancer Cells via PTEN/AKT Signaling Pathway

[目的]探讨miR-374a在乳腺癌细胞增殖和凋亡中的作用,并进一步探讨其机制。[方法]采用实时荧光定量PCR (qRT-PCR)检测人乳腺癌细胞MCF-7及正常人乳腺细胞Hs 578Bst的miR-374a及PTEN mRNA转录水平,Western blot法测定MCF-7、Hs 578Bst组细胞及对照、 miR-374a抑制剂组细胞的PTEN、p-AKT蛋白表达变化,用CCK-8法及Tunel法分别观察对照组和miR-374a抑制剂组细胞的增殖和凋亡,并用Western blot法检测两组细胞的凋亡相关蛋白Bcl-2、Cleaved caspase-3、Cleaved caspase-9表达。[结果 ]与人正常乳腺Hs 578Bst细胞相比,乳腺癌MCF-7细胞的miR-374a表达水平显著性增高(P<0.001),PTEN mRNA及蛋白的表达水平显著性降低(P<0.001),而p-AKT蛋白表达水平明显升高(P<0.001)。miR-374a抑制剂可抑制乳腺癌细胞的增殖(P<0.001),并促进其凋亡的发生(P<0.001),降低抗凋亡蛋白Bcl-2表达水平(P<0.001)的同时活化凋亡相关蛋白Caspase-3和Caspase-9(P<0.001)。miR-374a通过靶点PTEN影响AKT通路,miR-374a组细胞PTEN mRNA表达水平显著性降低(P<0.001),而miR-374a抑制剂处理后,PTEN蛋白水平显著性升高(P<0.001),p-AKT表达降低(P<0.001)。[结论] miR-374a在乳腺癌细胞高表达,并通过调节PTEN/AKT信号通路抑制乳腺癌细胞的增殖,促进凋亡的发生。miR-374a/PTEN/AKT信号通路有望成为乳腺癌的新治疗靶点。

[Objective] To investigate the effects of micro RNA-374 a(miR-374 a) on the proliferation and apoptosis of breast cancer cells,and its mechanism. [Methods] Real-time fluorescence quantitative polymerase chain reaction(q RT-PCR) was used to detect the transcript levels of miR-374 a and phosphatase and tensin homolog deleted on chromosome ten(PTEN) in human breast cancer MCF-7 cells and normal human breast Hs 578 Bst cells. The expression of miR-374 a was inhibited by transfection with miR-374 a inhibitor. Western blot method was used to detect the changes of PTEN,p-AKT,AKT protein expression in MCF-7 cells after transfection. CCK-8 and TUNEL methods were used to measure the cell proliferation and apoptosis,and the expressions of apoptosis-related proteins Bcl-2,cleaved caspase-3 and cleaved caspase-9 were detected by Western blot. [Results] Compared with Hs 578 Bst cells,the expression of miR-374 a in breast cancer MCF-7 cells significantly increased(P<0.001),the expression of PTEN gene and protein significantly decreased(P<0.001),and the protein expression level of p-AKT was significantly increased(P<0.001). The miR-374 a inhibitor inhibited the proliferation of breast cancer cells(P<0.001) and promoted the cell apoptotic(P<0.001),and decreased the expression of antiapoptotic protein Bcl-2(P<0.001),meanwhile activated apoptosis-related protein cleaved caspase-3 and cleaved caspase-9(P <0.001). Compared with control group,PTEN m RNA levels in MCF-7 cells were significantly down-regulated(P <0.001),but when treated with miR-374 a inhibitor,the protein level of PTEN was significantly up-regulated(P<0.001),and p-AKT protein expression was significantly downregulated(P<0.001). [Conclusion] The miR-374 a is highly expressed in breast cancer cells,which promotes the proliferation of breast cancer cells and inhibits cell apoptosis by regulating PTEN/AKT signaling pathway. miR-374 a/PTEN/AKT signaling pathway is expected to be a new therapeutic target for breast cancer.