Objective: To investigate if microRNA (miR) -23a knockdown could attenuate angiotensin Ⅱ(AngⅡ) induced cardiac hypertrophy by activating phosphatase and tensin homolog deleted on chromosome ten(PTEN) and AMP-activated protein kinase(AMPK) pathway. Methods: Rat H9c2 cells were cultured in DMEM high glucose medium and put in 5% CO(2) incubator at 37 ℃(normal group). After 48 hours of culture, H9c2 cells were stimulated with 10 nmol/L AngⅡ to establish cell hypertrophy model (AngⅡgroup). The H9c2 cells were inoculated in a 6-well cell culture plate and cultured in an incubator at 37 ℃. When the confluence degree of cell growth was about 70%, the cells were transfected with different reagents, and 24 hours after transfection, 10 nmol/L AngⅡ was used to interfere with the cells. The H9c2 cells were divided into different groups according to the reagents, namely AngⅡ+anti-miR group(transfected with miR-23a inhibitor), Ang Ⅱ+NC group(transfected with miR-23a inhibitor negative control), Ang Ⅱ+anti-miR+si-PTEN group(cotransfected with miR-23a inhibitor and PTEN small interference RNA(siRNA)), and AngⅡ+anti-miR+si-NC group(cotransfected with miR-23a inhibitor and PTEN siRNA negative control). The surface area of single cell was measured by Image J software.The mRNA expression levels of α-actin 1 (ACTA1) and β-myosin heavy chain (β-MHC) and miR-23a were detected by quantitative real-time PCR(qRT-PCR). The expression levels of PTEN and AMPK signal pathway related proteins were detected by Western blot. In order to verify whether miR-23a targets PTEN gene, double luciferase reporter gene experiment was performed. The luciferase reporter gene vector recombinant plasmids of wild type pGL-WT-PTEN and mutant pGL-MUT-PTEN were constructed and prepared after normal sequencing. H9c2 cells was inoculated into 24-well cell culture plate and cultured overnight in 37 ℃ incubator. The cells were co-transfected with miR-23a mimic or miR-23a mimic negative control and wild type or mutant reporter gene recombinant plasmid. Forty-eight hours after transfection, firefly luciferase activity and sea kidney luciferase activity were measured, and the ratio of them was recorded as relative luciferase activity. Results: Compared with the normal group, the cell surface area, the mRNA expression levels of ACTA1, β-MHC and miR-23a were significantly higher, while the protein expression levels of PTEN and p-AMPK were significantly lower in the Ang Ⅱ group(all P<0.05). The results of double luciferase reporter gene assay showed that the relative luciferase activity of cells co-transfected with miR-23a mimic and wild-type reporter gene recombinant plasmid was lower than that of miR-23a mimic negative control (P<0.05), and PTEN served as the target gene of miR-23a. In AngⅡ+anti-miR group the mRNA expression levels of miR-23a, ACTA1 and β-MHC were lower, and the cell surface area was smaller, while the protein expression levels of PTEN and p-AMPK were higher than that in AngⅡ group and AngⅡ+NC group(all P<0.05). Compared with AngⅡ+anti-miR group, the cell surface area was bigger, the expression of ACTA1 and β-MHC mRNA was up-regulated, and the protein expression levels of PTEN and p-AMPK were down-regulated in Ang Ⅱ+anti-miR+si-PTEN group(all P<0.05). Conclusion: Inhibition of miR-23a can attenuate Ang Ⅱ-induced hypertrophy in H9c2 cells through targeting PTEN and activating AMPK signaling pathway.
目的： 探讨微小RNA（miR）-23a是否通过靶向10号染色体缺失的磷酸酶及张力蛋白同源（PTEN）基因抑制腺苷酸活化蛋白激酶（AMPK）信号通路诱导的心肌细胞肥大。 方法： 将大鼠心肌细胞株H9c2细胞置于DMEM高糖培养基中，于37 ℃ 5% CO(2)培养箱中培养，作为正常组。稳定培养48 h后，采用10 nmol/L血管紧张素Ⅱ（AngⅡ）刺激H9c2细胞构建细胞肥大模型，为AngⅡ组。将H9c2细胞接种于培养板中，置于37 ℃培养箱中培养，待细胞生长汇合度约70%时，使用不同试剂转染细胞，转染24 h后以10 nmol/L AngⅡ干预细胞。其中，转染miR-23a抑制剂的细胞为AngⅡ+anti-miR组，转染miR-23a抑制剂阴性对照者为AngⅡ+NC组，共转染miR-23a抑制剂和PTEN小干扰RNA（siRNA）者为AngⅡ+anti-miR+si-PTEN组，共转染miR-23a抑制剂和PTEN siRNA阴性对照者为Ang Ⅱ+anti-miR+si-NC组。采用Image J软件测定单细胞表面积，通过实时荧光定量PCR（qRT-PCR）检测细胞中心肌肥厚标志基因α-肌动蛋白1（ACTA1）和β-肌球蛋白重链（β-MHC）基因mRNA和miR-23a的表达水平，Western blot检测细胞中PTEN蛋白和AMPK信号通路相关蛋白表达水平。为验证miR-23a是否靶向作用于PTEN基因，进行双荧光素酶报告基因实验。构建野生型pGL-WT-PTEN和突变型pGL-MUT-PTEN的荧光素酶报告基因载体重组质粒，测序正常后备用。H9c2细胞接种到24孔细胞培养板中，置37 ℃培养箱培养过夜，将miR-23a模拟物或miR-23a模拟物阴性对照与野生型或突变型报告基因重组质粒共转染细胞。转染48 h后测定萤火虫荧光素酶活性和海肾荧光素酶活性，以二者比值作为相对荧光素酶活性。 结果： AngⅡ组的细胞表面积、ACTA1和β-MHC mRNA及miR-23a的表达水平明显高于正常组，而PTEN及p-AMPK蛋白表达水平明显低于正常组（P均<0.05）。双荧光素酶报告基因实验结果显示，miR-23a模拟物与野生型报告基因重组质粒共转染细胞的相对荧光素酶活性比miR-23a模拟物阴性对照共转染者低（P<0.05），PTEN是miR-23a的靶基因。与AngⅡ组和AngⅡ+NC组比较，AngⅡ+anti-miR组细胞中miR-23a、ACTA1和β-MHC mRNA的表达水平更低，细胞表面积更小，PTEN和p-AMPK蛋白表达水平更高（P均<0.05）。与AngⅡ+anti-miR组比较，AngⅡ+anti-miR+si-PTEN组细胞表面积较大，ACTA1和β-MHC mRNA的表达水平较高，PTEN和p-AMPK蛋白表达水平较低（P均<0.05）。 结论： 抑制miR-23a能够减轻AngⅡ诱导的心肌细胞肥大，其作用机制可能与miR-23a靶向PTEN抑制AMPK信号通路有关。.
Keywords: AMPK signaling pathway; Hypertrophic; MicroRNA-23a; Myocytes, cardiac; PTEN gene.