目的:探讨miR-23a-3p对人肝星状细胞(LX-2)活化的影响并探索其相关机制。方法:通过实时定量聚合酶链式反应(qRT-PCR)实验和免疫蛋白印迹法(WesternBlot),检测miR-23a-3p、PTEN基因、α-SMA和collagenI等蛋白表达水平;通过转染构建过表达miR-23a-3p和PTEN的LX-2细胞系;CCK-8实验检测miR-23a-3p对LX-2细胞增殖的影响;通过多个数据库筛选miR-23a-3p的可能靶点;用双萤光素酶报告基因实验检测miR-23a-3p和目标靶点的结合情况。结果:与正常LX-2细胞相比,过表达miR-23a-3p显著促进LX-2细胞增殖和α-SMA和collagenI蛋白的表达。miR-23a-3p可以靶向PTEN并抑制其表达。过表达PTEN能够逆转miR-23a-3p对LX-2细胞增殖和α-SMA和collagenI蛋白表达的影响。miR-23a-3p促进了TGF-β1诱导的PI3K、Akt和mTOR的磷酸化,同时过表达PTEN抑制了miR-23a-3p诱导的磷酸化。结论:miR-23a-3p通过调节PTEN促进肝星状细胞活化,从而调控肝纤维化。
Objective: To investigate the effect of miR-23a-3p on the activation of human hepatic stellate cells (LX-2) and to explore its related mechanisms. Methods: The expression levels of miR-23a-3p and PTEN genes and proteins such as α-SMA and collagen I were detected by real-time quantitative polymerase chain reaction (qRT-PCR) assay and immunoprotein blotting (Western Blot); LX-2 cell lines overexpressing miR-23a-3p and PTEN were constructed by transfection; and the CCK-8 assay to detect the effect of miR-23a-3p on the proliferation of LX-2 cells; screening of possible targets of miR-23a-3p by multiple databases; and detection of the binding of miR-23a-3p and the target targets by dual luciferase reporter gene assay. Results: Overexpression of miR-23a-3p significantly promoted LX-2 cell proliferation and expression of α-SMA and collagen I proteins compared with normal LX-2 cells. miR-23a-3p could target PTEN and inhibit its expression. Overexpression of PTEN reversed the effects of miR-23a-3p on LX-2 cell proliferation and α-SMA and collagen I protein expression. miR-23a-3p promoted TGF-β1-induced phosphorylation of PI3K, Akt, and mTOR, while overexpression of PTEN inhibited miR-23a-3p-induced phosphorylation. Conclusion: miR-23a-3p promotes hepatic stellate cell activation by regulating PTEN, thereby modulating liver fibrosis.
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