Song Yanling, Mai Huade, Lin Yunyun, Wang Yachun, Wang Xiaoxi, Gu Shenhong
Department of General Practice, The First Affiliated Hospital of Hainan Medical University Haikou, Hainan Province, China.
Int J Clin Exp Pathol. 2020 Feb 1;13(2):142-152. eCollection 2020.
Diabetic cardiomyopathy (DCM) is a common complication of diabetes and can lead to heart failure, arrhythmia, and sudden death. microRNAs (miRNAs) are reportedly involved in many human disease, including DCM. However, little is known about the biologic functions of miR-144 in DCM progression.
The expression levels of miR-144 and C1q/TNF-related protein-3 (CTRP3) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used to determine the protein levels of CTRP3, phosphorylated c-Jun amino-terminal kinase (p-JNK), JNK, Bax, Bcl-2, and cleaved-caspase-3. Cell proliferation and apoptosis were detected by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The potential binding sites between miR-144 and CTRP3 were predicted by microRNA.org databases and further determined using a dual-luciferase assay. AC16 cardiomyocytes were cultured in high glucose (HG, 30 mmol/L) to mimic hyperglycemia.
MiR-144 expression level was enhanced, while CTRP3 expression was reduced in HG-induced AC16 cardiomyocytes. Knockdown of miR-144 or overexpression of CTRP3 dramatically promoted cell proliferation and reduced apoptosis of AC16 cardiomyocytes treated with HG. Inhibition of miR-144 evidently decreased the protein levels of Bax and p-JNK, but elevated Bcl-2 expression in HG-induced AC16 cardiomyocytes. Moreover, CTRP3 was a direct target of miR-144, and its abrogation reversed the effects of miR-144 knockdown on proliferation and apoptosis in HG-induced AC16 cardiomyocytes. SP600125 (a JNK inhibitor, 10 μmol/L) attenuated the si-CTRP3-mediated inhibition of proliferation and promotion of apoptosis in AC16 cardiomyocytes transfected with anti-miR-144 and stimulated with HG.
MiR-144 regulates proliferation and apoptosis of HG-induced AC16 cardiomyocytes through targeting the CTRP3/JNK signaling pathway, providing a novel avenue for treatment of DCM.
糖尿病性心肌病（DCM）是糖尿病常见的并发症，可导致心力衰竭、心律失常和猝死。据报道，微小RNA（miRNA）参与包括DCM在内的多种人类疾病。然而，关于miR-144在DCM进展中的生物学功能知之甚少。
采用定量实时聚合酶链反应（qRT-PCR）检测miR-144和C1q/TNF相关蛋白-3（CTRP3）的表达水平。蛋白质印迹法用于测定CTRP3、磷酸化c-Jun氨基末端激酶（p-JNK）、JNK、Bax、Bcl-2和裂解的半胱天冬酶-3的蛋白质水平。分别通过细胞计数试剂盒-8（CCK-8）检测法和流式细胞术检测细胞增殖和凋亡。通过microRNA.org数据库预测miR-144与CTRP3之间的潜在结合位点，并使用双荧光素酶检测法进一步确定。将AC16心肌细胞培养于高糖（HG，30 mmol/L）环境中以模拟高血糖状态。
在HG诱导的AC16心肌细胞中，miR-144表达水平升高，而CTRP3表达降低。敲低miR-144或过表达CTRP3可显著促进HG处理的AC16心肌细胞的增殖并减少其凋亡。抑制miR-144明显降低HG诱导的AC16心肌细胞中Bax和p-JNK的蛋白质水平，但提高Bcl-2的表达。此外，CTRP3是miR-144的直接靶点，其缺失逆转了miR-144敲低对HG诱导的AC16心肌细胞增殖和凋亡的影响。SP600125（一种JNK抑制剂，10 μmol/L）减弱了si-CTRP3介导的对转染了抗miR-144并经HG刺激的AC16心肌细胞增殖的抑制和凋亡的促进作用。
miR-144通过靶向CTRP3/JNK信号通路调节HG诱导的AC16心肌细胞的增殖和凋亡，为DCM的治疗提供了一条新途径。
