微小RNA-378抑制高血压心肌纤维化的作用和机制研究

The mechanisms of microRNA-378 on suppressing cardiac fibrosis induced by hypertension

目的 研究微小RNA-378(miR-378)在高血压心肌纤维化中的作用和机制.方法 通过对体外细胞施加机械应力刺激模拟高血压压力负荷对心肌细胞的作用.试验分为四部分:(1)对心肌细胞进行机械牵张刺激或不干预,分别提取心肌细胞上清中的外泌体,用电镜观察外泌体数目和形态、实时定量聚合酶链式反应(RT-PCR)检测外泌体中微小RNA(miRNA)的表达、免疫蛋白印迹法(Western blot)检测CD81蛋白表达水平.(2)用诺瑟杂交(Northern blot)检测体外心肌细胞和心肌成纤维细胞中内源性miR-378的表达水平;用RT-PCR检测不同培养基中成纤维细胞的miR-378的表达水平.(3)用经机械牵张刺激和未经刺激的心肌细胞上清分别培养成纤维细胞后,再对成纤维细胞进行机械牵张刺激,用RT-PCR检测成纤维细胞的miR-378和胶原mRNA表达水平、Western blot检测基质金属蛋白酶9(MMP9)和基质金属蛋白酶3(MMP3)的蛋白表达水平.(4)用miR-378模拟物转染心肌成纤维细胞,并分别加入p38 MAPK抑制剂SB203580和STAT3抑制剂S3I-201,再经机械牵张刺激后,用RT-PCR检测成纤维细胞中胶原mRNA、Western blot检测p38 MAPK、磷酸化p38 MAPK、STAT3、MMP9的表达.结果 (1)对比未干预的心肌细胞上清,机械牵张刺激后的心肌细胞上清中外泌体数量和miR-378含量显著增多(P<0.01).(2)miR-378在心肌细胞中高表达,而在心肌成纤维细胞中不表达;而经牵张刺激的心肌细胞上清培养的成纤维细胞中miR-378水平较未牵张组显著升高(P<0.01).(3)成纤维细胞经牵张刺激的心肌细胞上清培养后,其Col1a1、Col3a1 mRNA和MMP9、MMP3蛋白在机械应力刺激下的表达水平显著下降(P<0.01).(4)成纤维细胞经miR-378类似物转染后,其Col1a1和Col3a1 mRNA(P<0.05)、MMP9和磷酸化p38 MAPK(P<0.01)在机械应力刺激下的表达水平显著下降,而STAT3表达升高(P<0.01).在miR-378的基础上,阻断p38 MARK磷酸化,成纤维细胞STAT3在机械应力刺激下的表达水平进一步上升;而阻断STAT3,成纤维细胞磷酸化p38 MARK在机械应力下的表达水平升高.结论 机械应力促进心肌细胞中的miR-378以外泌体途径进入心肌成纤维细胞中.miR-378通过调节p38 MAPK和STAT3信号通路从而抑制心肌纤维化.

Objective To investigate the effects and mechanisms of microRNA-378 ( miR-378 ) in cardiac fibrosis induced by hypertension .Methods Effect of pressure overload on cardiac cells induced by hypertension was simulated by exposure to mechanical stress (MS) in vitro.The study was divided into four parts.( 1 ) In exomes purified from cardiomyocyte culture supernatant with or without MS , electron microscopy , quantitative real-time polymerase chain reaction ( RT-PCR ) , Western blot was performed to evaluate quantity and morphology of exosomes , expression of microRNA, expression of CD81, respectively. (2) Northern blot was used to evaluate endogenous miR-378 expression in cardiomyocyte and cardiac fibroblasts(CFs).Western blot was used to evaluate miR-378 in CFs after cultured in different mediums . (3) After cultured in the cardiomyocyte supernatant with or without MS , CFs were exposed to MS or no MS invention.Then RT-PCR was used to evaluate expression of miR-378 and collagen mRNA , Western blot was used to detect MMP9 and MMP3 expression level in CFs.(4) After transfection of miR-378 analogue, SB203580 and S3I-201 were used to inhibit p38 MAPK and STAT3 in CFs, respectively , then CFs were exposed to MS or no MS.RT-PCR was used to evaluate collagen mRNA , Western blot was used to evaluate expression of p38 MARK, phosphorylated p38 MAPK, STAT3, MMP9 in CFs.Results (1) Compared with cardiomyocyte culture supernatant without MS , exomes and miR-378 increased significantly in cardiomyocyte culture supernatant with MS ( P<0.01 ) . ( 2 ) miR-378 was highly expressed in cardiomyocytes , but not expressed in CFs .However , miR-378 increased significantly after cultured in cardiomyocyte supernatant with MS .( 3 ) After cultured in the cardiomyocyte supernatant with MS , expression of Col1a1, Col3a1 mRNA and MMP9,MMP3 protein significantly decreased in CFs under MS (P<0.01).(4)After transfection of miR-378 analogue, expression of Col1a1 and Col3a1 mRNA(P<0.05), MMP9 and phosphorylated p38 MARK(P<0.01) also decreased significantly in CFs under MS , but STAT3 expression increased .Combination with miR-378 analogue , block of p38 MARK phosphorylation could further increase STAT3 expression, in turn, block of STAT3 expression could increase p38 MARK phosphorylation expression under MS .Conclusion Mechanical stress promotes the secretion of miR-378 from cardiomyocytes in an exosome-dependent manner to CFs .MiR-378 can suppress cardiac fibrosis by regulating p38 MAPK and STAT3 pathways.