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首页 > 过刊浏览>2022年第32卷第9期 >1-9. DOI:10. 3969 / j.issn.1671-7856. 2022. 09. 001
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miR-488-3p 靶向调控 RAP1A 在同型半胱氨酸介导肝细胞自噬的作用研究
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作者单位:

1.宁夏医科大学临床医学院,银川 750004;2.宁夏医科大学基础医学院,银川 750004;3.国家卫生健康委员会代谢性心血管疾病研究重点实验室(宁夏医科大学),银川 750004;4.宁夏医科大学总医院,银川 750004

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R-33


Role of miR-488-3p targeting regulation of RAP1A in homocysteine-mediated hepatocyte autophagy
Author:
Affiliation:

1. School of Clinical Medicine, Ningxia Medical University, Yinchuan 750004, China. 2. School of Basic Medicine,Ningxia Medical University, Yinchuan 750004. 3. Key Laboratory of Metabolic Cardiovascular Disease Research,National Health Commission (Ningxia Medical University), Yinchuan 750004.4. General Hospital of Ningxia Medical University, Yinchuan 750004

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    摘要:

    目的 探讨微小 RNA(miR-488-3p)通过靶向调控 RAS 癌基因家族成员 RAP1A 在同型半胱氨酸介导肝细胞自噬中的作用。 方法 体外常规培养人正常肝细胞(HL-7702),并给予 100 μmol / L Hcy 干预 24 h,采用Western blot 检测肝细胞自噬相关蛋白 LC3、p62 和 RAP1A 的表达水平,qRT-PCR 检测肝细胞中 miR-488-3p 和RAP1A 的表达;转染 miR-488-3p inhibitor 和 mimics 后,qRT-PCR 和 Western blot 分别检测 miR-488-3p 的转染效率及其对 LC3 和 p62 蛋白表达的影响;TargetScan 预测 miR-488-3p 与 RAP1A 基因相关性,Western blot 检测 miR-488-3p 下游潜在靶蛋白(RAP1A)的表达改变;Pearson 相关系数对肝细胞中 miR-488-3p 表达水平与自噬相关蛋白水平进行相关性分析。 结果 与 Control 组相比,Hcy 组肝细胞自噬相关蛋白 LC3、RAP1A 和 miR-488-3p 的表达水平升高(P<0. 01),而 p62 表达明显降低(P<0. 01);同时,转染 miR-488-3p inhibitor 和 mimics 后,与 NC-inhibitor 组比较,miR-488-3p inhibitor 组中 LC3 和 RAP1A 蛋白的表达水平显著降低(P<0. 01),p62 表达明显升高(P<0. 01);而与NC-mimics 组相比,miR-488-3p mimics 组中 LC3 和 RAP1A 蛋白表达水平明显增加(P<0. 01),p62 表达降低(P<0. 01);进一步机制研究表明 RAP1A 是 miR-488-3p 的下游靶基因并受其正向调控。 Pearson 相关性分析发现,miR-488-3p 的表达水平与 LC3( r= 0. 9329,P= 0. 0002)蛋白表达呈正相关,而与 p62( r= -0. 8086,P= 0. 0083)表达则呈负相关。 结论 miR-488-3p 在 Hcy 介导的肝细胞中高表达,可通过靶向调控 RAP1A 的表达促进肝细胞自噬。

    Abstract:

    Objective To investigate the role of the microRNA miR-488-3p in homocysteine-mediated hepatocyte autophagy through targeted regulation of the RAS oncogene family member RAP1A. Methods Human normal hepatocytes(HL-7702) were routinely cultured in vitro and administered 100 μmol / L Hcy for 24 h. Western blot was used to detect expression levels of autophagy-related proteins LC3, p62 and RAP1A in hepatocytes, whereas qRT-PCR was used to detect the expression of miR-488-3p and RAP1A in these cells. Following transfection of miR-488-3p inhibitor and mimics, qRTPCR and western blot were used to detect the transfection efficiency of miR-488-3p and its effect on LC3 and p62 protein expression. TargetScan was used to predict the correlation between miR-488-3p and RAP1A genes, whereas western blotting was used to detect changes in the expression of RAP1A, a potential downstream target protein of miR-488-3p. Pearson’s correlation coefficient was used to evaluate potential correlations between expression levels of miR-488-3p and autophagy-related proteins in hepatocytes. Results Compared with the control group, expression levels of autophagyrelated proteins LC3, RAP1A and miR-488-3p in the Hcy group were increased (P<0. 01), whereas expression of p62 was significantly decreased (P<0. 01). Following administration of miR-488-3p inhibitor and mimics, expression levels of LC3 and RAP1A proteins in the miR-488-3p inhibitor group were significantly decreased (P<0. 01) and expression of p62 was significantly increased (P<0. 01) compared with the NC-inhibitor group. Compared with the NC-mimics group, expression levels of LC3 and RAP1A proteins in the miR-488-3p mimics group were significantly increased ( P < 0. 01 ), and expression of p62 was significantly decreased (P<0. 01). Further mechanistic studies showed that RAP1A is a downstream target gene of miR-488-3p that is positively regulated by this miRNA. Pearson’ s correlation analysis indicated that the expression level of miR-488-3p was positively correlated with protein expression of LC3 ( r= 0. 9329, P= 0. 0002), but negatively correlated with p62 protein expression ( r= - 0. 8086, P= 0. 0083). Conclusions miR-488-3p is highly expressed in Hcy-mediated hepatocytes and can promote their autophagy by targeting expression of RAP1A.

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高 源,揭育祯,马天龙,汪乐新,杨慧霞,周瑜瑾,焦 运,卢冠军,马胜超. miR-488-3p 靶向调控 RAP1A 在同型半胱氨酸介导肝细胞自噬的作用研究[J].中国比较医学杂志,2022,32(9):1~9.

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  • 收稿日期:2022-01-17
  • 在线发布日期: 2023-01-16
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