miR-149-5p靶向FGF21对大鼠心肌细胞的缺氧/复氧损伤的促进作用

MiR-149-5p targeting FGF21 promotes hypoxia/reoxygenation injury of rat's cardiomyocytes

目的 探究miR-149-5p与成纤维细胞生长因子21(FGF21)的靶向关系,及其对大鼠心肌细胞缺氧/复氧(H/R)损伤的影响。方法 将10只C56BL/6J雄性小鼠构建缺血再灌注(I/R)模型,qRT-PCR法检测I/R小鼠心脏组织中miR-149-5p表达水平的变化。将培养至生长对数期的H9c2大鼠心肌细胞分为对照组、H/R组、H/R+miR-149-5pinhibitor组、H/R+miR-149-5p NC组、H/R+miR-149-5p mimics组、H/R+miR-149-5p mimics+FGF21组,并诱导H/R模型及转染处理。ELISA法检测H9c2细胞中乳酸脱氢酶(LDH)和肌酸激酶(CK)的活性;MTT法检测细胞活力;流式细胞术检测细胞凋亡情况;qRT-PCR检测细胞中miR-149-5p的表达水平;Western blotting检测Bcl-2、Bax、cleaved caspase-3、FGF21蛋白的表达水平;双荧光素酶测定miR-149-5p与FGF21的靶向关系。结果 与假手术组比较,I/R小鼠心脏组织中miR-149-5p表达水平明显升高(P<0.05)。与对照组比较,H/R组H9c2细胞凋亡率、LDH和CK活性及Bax、cleaved caspase-3蛋白表达水平均明显升高(P<0.05),细胞活力及FGF21、Bcl-2蛋白表达水平明显降低(P<0.05);与H/R组和H/R+miR-149-5p NC组比较,H/R+miR-149-5pinhibitor组细胞凋亡率、LDH和CK活性及Bax、cleavedcaspase-3蛋白表达水平均明显降低(P<0.05),细胞活力及FGF21、Bcl-2蛋白表达水平均明显升高(P<0.05);与H/R组比较,H/R+miR-149-5pmimics组细胞凋亡率、LDH和CK活性及Bax、cleaved caspase-3蛋白表达水平均明显升高(P<0.05),细胞活力及FGF21、Bcl-2蛋白表达水平均明显降低(P<0.05)。与H/R+miR-149-5p mimics组比较,H/R+miR-149-5p mimics+FGF21组细胞凋亡率及LDH和CK活性均明显降低(P<0.05),细胞活力明显升高(P<0.05)。双荧光素酶检测结果显示miR-149-5p与FGF21基因有靶向调控关系。结论 miR-149-5p在I/R小鼠心肌组织中明显上调,可负向调控FGF21水平,导致H/R处理的大鼠心肌细胞活力下降、凋亡增加。

Objective To investigate the targeting relationship of miR-149-5p and fibroblast growth factor 21(FGF21),and the effect of miR-149-5p on hypoxia/reoxygenation(H/R)injury of rat`s cardiomyocytes.Methods Ten male C56BL/6J mice were used to construct the cardiac ischemia-reperfusion(I/R)models,and qRT-PCR was performed to detect the changes of miR-149-5p levels in the heart tissues of I/R mice.H9c2 cells cultured to logarithmic phase were divided into control group,H/R group,H/R+miR-149-5p inhibitor group,H/R+miR-149-5p NC group,H/R+miR-149-5p mimics group and H/R+miR-149-5p mimics+FGF21 group, and induced H/R model and transfection treatment. The activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in H9c2 cardiomyocytes were detected by ELISA;Cell viability was detected by MTT assay;apoptosis was detected by flow cytometry;the level of miR-149-5p was detected by qRT-PCR;the expression levels of Bcl-2, Bax, cleaved caspase-3 and FGF21 proteins were detected by Western blotting;the targeting relationship between miR-149-5p and FGF21 were determined by dual luciferase assay. Results Compared with the sham operation group, the level of miR-149-5p in the heart tissue of I/R mice was significantly increased (P<0.05). Compared with those in control group, the apoptosis rate, LDH and CK activities and Bax, cleaved caspase-3 protein expression levels of H9c2 cells in H/R group were significantly increased (P<0.05), cell viability and FGF21, Bcl-2 protein expression levels decreased significantly (P<0.05). Compared with those in H/R group and H/R+miR-149-5p NC group, the apoptosis rate, LDH and CK activities and Bax, cleaved caspase-3 protein expression levels of H9c2 cells in H/R+ miR-149-5p inhibitor group were significantly decreased (P<0.05), cell viability and FGF21, Bcl-2 protein expression levels were significantly increased (P<0.05). Compared with those in H/R group, the apoptosis rate, LDH and CK activities and Bax, cleaved caspase-3 protein expression levels of H9c2 cells in H/R+miR-149-5p mimics group were significantly increased (P<0.05), cell viability and FGF21, Bcl-2 protein expression levels decreased significantly (P<0.05). Compared with those in H/R+miR-149-5p mimics group, the apoptosis rate, and LDH and CK activities of H9c2 cells in miR-149-5p mimics+FGF21 group were significantly decreased (P<0.05), cell viability was significantly increased (P<0.05). The result of dual luciferase assay indicated the targeted regulatory relationship between miR-149-5p and FGF21. Conclusions MiR-149-5p is up-regulated significantly in the myocardial tissue of I/R mice, and negatively regulate the level of FGF21, leads to decreased cell viability and increased apoptosis of H/R-treated rat's cardiomyocytes.