MiR-17-5p调控STAT3信号通路在心肌慢性缺氧适应中意义的研究

Significance of miR-17-5p in adaptation of chronic hypoxia in myocardium via modulation of STAT3 signaling pathway

目的探讨心肌慢性缺氧环境下,miR-17-5p的表达变化以及miR-17-5p对STAT3信号通路的调控对于缺氧心肌细胞的影响。方法 1选取新桥医院心外科2014年8月至2015年4月期间先心病患儿20例,其中紫绀型(年龄61.5±46.5月,术后诊断为法洛氏四联症)与非紫绀型(年龄43.2±32.1月,术后诊断为室缺伴右心室流出道狭窄)各10例。术中取右室流出道心肌组织作为标本,采用RT-PCR检测两组先心病患儿心肌组织miR-17-5p的表达情况。2建立大鼠源H9c2心肌细胞慢性缺氧适应模型,分为缺氧0、12、24、48、72 h组(n=5),RT-PCR分别检测各组miR-17-5p的表达。3脂质体转染antagomir miR-17-5p和agomir miR-17-5p至H9c2心肌细胞,建立过表达miR-17-5p和沉默miR-17-5p的H9c2心肌细胞系,慢性缺氧培养48 h,CCK8实验检测细胞增殖情况,TUNNEL实验检测细胞凋亡情况,Western blot检测STAT3,p-Tyr-705-STAT3及其下游调控蛋白c-myc蛋白表达。结果 1与非紫绀组(0.482±0.163)相比,紫绀组(1.450±0.705)患儿miR-17-5p表达明显升高(P<0.01)。2慢性缺氧细胞模型研究结果显示慢性缺氧时H9c2细胞miR-17-5p表达逐渐增高(P<0.01)。3缺氧环境下miR-17-5p过表达明显抑制p-Tyr-705-STAT3、STAT3、c-myc表达,引起心肌心肌细胞增殖受抑制,凋亡增加(P<0.01);而抑制miR-17-5p对p-Tyr-705-STAT3、STAT3、c-myc表达改变不明显,且对于心肌细胞的增殖和凋亡改变也不明显(P>0.05)。结论在心肌慢性缺氧过程中,miR-17-5p可能参与调控STAT3-c-myc信号通路,对于心肌细胞的存活和功能调节有重要意义。

Objective To explore the expression of miR-17-5p and its effect on STAT3 signaling pathway in the myocardium under chronic hypoxic condition. Methods Twenty children with congenital heart disease, who were prepared to undergo surgical treatment from August 2014 to April 2015, were recruited and divided into 10 cases of cyanotic type （average age 61.5±46.5 months, post-operation diagnosis as Tetralogy of Fallot） and 10 cases of acyanotic type （average age 43.2±32.1 months, post-operation diagnosis as interventricular septal defect and right ventricular outflow tract stenosis）. The right myocardial tissue of ventricular outflow tract was taken as the specimen to detect the expression of miR-17-5p by RT-PCR. Chronic hypoxia models of H9c2 cardiomyocytes were established and divided into 5 groups including hypoxia 0-h group, hypoxia 12-h group, hypoxia 24-h group, hypoxia 48-h group and hypoxia 72-h group （n=5）, and RT-PCR was employed to detect the expression of miR-17-5p in each group. H9c2 cardiomyocytes were transfected with antagomir miR-17-5p and agomir miR-17-5p by liposomes to build miR-17-5p overexpression and silence models of H9c2 cardiomyocytes, and then the cells were cultured in hypoxia incubator for 48 h. CCK8 assay was used to detect cell proliferation, TUNEL method was adopted to detect cell apoptosis, and Western blotting was employed to detect the expressions of STAT3, p-Tyr-705-STAT3 and downstream c-myc. Results Compared with the acyanosis group （0.482±0.163）, the miR-17-5p expression of the cyanotic group （1.45±0.705） was significantly higher （P〈0.01）. The chronic hypoxia cell model showed that the expression of miR-17-5p was increased gradually along with chronic hypoxia time （P〈0.01）. Overexpression of miR-17-5p inhibited cardiomyocyte proliferation and promoted apoptosis significantly through inhibiting the expression of STAT3, p-Tyr-705-STAT3 and c-myc （P〈0.01）. The inhibition of miR-17-5p had no effect on the expression of p-Tyr-705-STAT3, STAT3 and c-myc, and the changes of proliferation and apoptosis of cardiomyocytes （P〉0.05）. Conclusion During chronic hypoxia, miR-17-5p may be involved in the regulation of STAT3-c-myc signal pathway, and has important significance in survival and function regulation of cardiomyocytes.