摘要
目的探讨心力衰竭患者循环中微小RNA(miR)-30a的表达水平及诱导人心肌细胞凋亡的可能分子机制。方法采集心力衰竭患者和健康志愿者的血清用于检测miRNAs的表达水平。体外实验,将人心肌细胞分为3组:空白对照组(MOCK组)、空质粒组(NC组)和转染miR-30a模拟物组(miR-30a组)。用脂质体2000(Lipofectamine2000,Li2)转染miR-30a模拟物(终浓度为100 nmol/L)。采用实时逆转录聚合酶链反应(RT-PCR)检测各组的miR-30a的表达水平。用细胞计数-8试剂盒(CCK-8)检测各组细胞增殖。用流式细胞仪和Tunel法检测各组心肌细胞的凋亡并检测各组细胞的Caspase-3活性。用荧光素酶报告基因检测法检测miR-30a和接头蛋白Gab1(Gab1)的相互作用。用蛋白免疫印记法(WB)检测各组增殖细胞核抗原(PCNA)、Ki67和核转录因子Kappa B(NF-κB)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、以及Gab1、β-肌动蛋白(β-actin)的表达水平。结果心力衰竭患者血清中的miRNA-30a表达水平显著高于健康志愿者(P<0.05)。逆转录聚合酶链式反应(RT-PCR)结果显示miR-30a组的人心肌细胞miR-30a的表达显著增高相对于NC组(P<0.05)。CCK-8结果显示miR-30a组OD值低于MOCK组和NC组(P<0.05)。流式细胞仪和Tunel结果显示miR-30a过度表达的人心肌细胞凋亡高于MOCK组和NC组(P<0.05)。Caspase-3活性检测显示miR-30组Caspase-3活性显著高于MOCK组和NC组(P<0.05)。荧光素酶报告基因检测法报告miR-30a与Gab1有互补配对序列。WB结果显示miR-30a组PCNA、ki67、Bcl-2、Gab1表达下降,NF-κB、Bax表达上升(P<0.05)。结论 miR-30a通过下调Gab1抑制人心肌细胞增殖和诱导人心肌细胞凋亡。
Objective To explore the expression of RNA （miR） -30a in the serum of heart failure patients and test its mechanism by which miR - 30a induces apoptosis of human cardiomyoblast cells. Methods Serum levels of miRNAs were detected in patients with heart failure and healthy volunteers. Human eardiomyoblast cells were divided into 3grnups： blank control group （MOCK group）, negative control group（ NC group） and transfected miR -30a mimics group. Lipofectamine2000 was transfected with miR -30a mimics （final concentration of 100 nmol/L）. The levels of miR - 30a in each group were detected by real - time reverse transcription polymerase chain reaction （ RT - PCR）. CCk - 8 method was used to detect the cell proliferation. Apoptosis was detected by flow cytometry and Terminal deoxynucleotidyl transferase （TdT）dUTP Nick - End Labeling （Tunel） method, and the Caspase -3 activity of the cells in each group were detected. The interaction of miR -30a and Gabl was detected by luciferase reporter gene assay. The expression levels of PCNA, Ki67 and nuclear factor kappa B （ NF - κB ） , Bax, Bcl - 2 and Gab1, β - actin and were detected by western blot （WB）. Results The level of miRNA - 30a in the serum of patients with heart failure was significantly higher than that in healthy volunteers （ P 〈 0. 05 ）. RT - PCR results showed that the expression of miR - 30a in miR - 30a group was significantly higher than that in NC group （ P 〈 0.05 ）. CCK - 8 results showed that the OD value of miR - 30a group was lower than MOCK group and NC group （ P 〈 0. 05）. Flow cytometry and Tunel results showed that human cardiomyoblast ceils apoptosis in miR - 30a group was higher than MOCK group and NC group （ P 〈 0. 05 ）. Caspase - 3 activity detection showed that the Caspase - 3 activity of miR - 30 group was significantly higher than MOCK group and NC group （ P 〈 0.05）. Lueiferase reporter gene assay reported that miR -30a and Gabl have complementary pairing sequences. WB results showed that PCNA , Ki67, Bcl - 2, Gabl expression decreased, NF - κB , Bax expression increased （ P 〈 0.05 ）. Conclusion MiR - 30a may inhibit the proliferation and induce the apnptosis of human cardiomyoblast cells by down - regulation Gabl.
出处
《临床和实验医学杂志》
2016年第22期2180-2185,共6页
Journal of Clinical and Experimental Medicine
基金
上海市卫生局项目(SHXH201126)
