miR-652-3p靶向同源异型核基因1对血管紧张素Ⅱ诱导的心肌细胞凋亡的影响

doi:10.3877/cmj.j.issn.2095-1221.2020.01.002

目的

探讨miR-652-3p靶向同源异型核基因1（PRRX1）对血管紧张素Ⅱ（AngⅡ）诱导的心肌细胞凋亡的影响。

方法

大鼠心肌细胞H9c2细胞采用正常培养基培养为对照组细胞，用含1 μmol/L AngⅡ的培养基培养为AngⅡ组细胞；分别转染miR-652-3p阳性对照序列（NC）和转染miR-652-3p mimics后用含1 μmol/L AngⅡ的培养基培养为AngⅡ+NC组和AngⅡ+miR-652-3p组细胞；将miR-652-3p mimics分别与PRRX1阳性对照质粒和PRRX1过表达质粒转染至H9c2细胞中用含1 μmol/L AngⅡ的培养基培养，分别为AngⅡ+miR-652-3p+ Vctor组和AngⅡ+miR-652-3p+PRRX1组细胞。实时荧光定量PCR （RT-qPCR）检测H9c2细胞中miR-652-3p表达水平，流式细胞术检测细胞凋亡，用Western blot检测细胞中PRRX1、Bax和Bcl-2蛋白表达水平。双荧光素酶报告基因实验验证H9c2细胞中miR-652-3p与PRRX1调控关系。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用SNK-q检验。

结果

与对照组比较，AngⅡ组H9c2细胞中miR-652-3p水平（1.00±0.08比0.21±0.05）、Bcl-2蛋白水平（0.83±0.08比0.40±0.04）均较低，而PRRX1蛋白水平（0.06±0.01比0.41±0.04）、凋亡率（5.02﹪±1.41﹪比25.33﹪±3.75﹪）、Bax蛋白水平（0.46±0.05比0.96±0.10）均较高，差异具有统计学意义（P均< 0.05）。与AngⅡ+NC组比较，AngⅡ+miR-652-3p组H9c2细胞中miR-652-3p的表达水平（0.24±0.06比0.98±0.07）、Bcl-2蛋白水平（0.38±0.04比0.72±0.07）均较高，而PRRX1蛋白水平（0.39±0.04比0.13±0.01）、凋亡率（27.02﹪±4.11﹪比12.19﹪±1.63﹪）、Bax蛋白水平（0.95±0.09比0.53±0.05）均较低，差异具有统计学意义（P均< 0.05）。与AngⅡ+miR-652-3p+Vctor组比较，AngⅡ+miR-652-3p+PRRX1组H9c2细胞凋亡率（12.88﹪±1.84﹪比25.45﹪±3.58﹪）、PRRX1蛋白水平（0.13±0.01比0.35±0.04）和Bax蛋白水平（0.54±0.05比0.82±0.08）均较高，差异具有统计学意义（P均< 0.05），而Bcl-2蛋白表达水平（0.72±0.07比0.46±0.05）降低，差异具有统计学意义（P < 0.05）。

结论

AngⅡ能够下调心肌细胞中miR-652-3p的表达，上调miR-652-3p可通过靶向抑制PRRX1的表达减少AngⅡ诱导的H9c2细胞凋亡。

关键词: miR-652-3p, 同源异型核基因1, 血管紧张素Ⅱ, 心肌细胞凋亡

Objective

To investigate the effect of miR-652-3p targeting autonomous heterologous nuclear gene 1 (PRRX1) in the regulation of angiotensinⅡ (AngII) -induced cardiomyocyte apoptosis.

Methods

Rat cardiac cell H9c2 cultured under normal conditions were used as the control group, while AngⅡgroup were established in medium containing 1 μmol/L AngⅡ. These cells were transfected with miR-652-3p+Vctor and miR-652-3p mimics respectively. In addition AngⅡ-H9c2 cells transfected with miR-652-3p mimics were transfected with pc-PRRX1 and overexpressed-PRRX1. The expression of miR-652-3p in H9c2 was determined by quantitative real-time PCR (qRT-qPCR) . Detection of cell apoptosis was evaluated by using flow cytometry. In addition the protein expression levels of Bax and Bcl-2 were assessed by Western blot analysis. Gene regulatory relations between miR-652-3p and PRRX1 were confirmed by Dual-Luciferase reporter gene system. The t-test was applied for comparison between two groups and single factor variance analysis (one-way ANOVA) was used for comparison among several groups. The SNK-q test method was used to compare between groups.

Results

Compared with control group, the level of miR-652-3p (1.00±0.08 vs 0.21±0.05) and Bcl-2 protein (0.83±0.08 vs 0.40±0.04) in H9c2 cells were lower in AngⅡgroup, while PRRX1 protein level (0.06±0.01 vs 0.41±0.04) , apoptosis rate (5.02﹪±1.41﹪vs 25.33﹪±3.75﹪) , Bax protein level (0.46±0.05 vs 0.96±0.10) were higher (all P< 0.05) . Compared with AngⅡ+NC group, the expression level of miR-652-3p (0.24±0.06 vs 0.98±0.07) and Bcl-2 protein level in the AngⅡ+miR-652-3p group (0.38±0.04 vs 0.72±0.07) were higher while the PRRX1 protein level (0.39±0.04 vs 0.13±0.01) , and apoptosis rate (27.02﹪±4.11﹪ vs 12.19﹪±1.63﹪) , Bax protein level (0.95±0.09 vs 0.53±0.05) were lower (all P< 0.05) . Compared with the AngⅡ+miR-652-3p+Vctor group, the apoptosis rate of H9c2 cells (12.88﹪±1.84﹪vs 25.45﹪±3.58﹪) as well as expression of PRRX1 protein (0.13±0.01 vs 0.35±0.04) and Bax protein (0.54±0.05 vs 0.82±0.08) in the AngⅡ+ miR-652-3p+PRRX1 group were higher (all P< 0.05) , whereas Bcl-2 protein level (0.72±0.07 vs 0.46±0.05) was lower (all P< 0.05) .

Conclusion

AngⅡcould down-regulatethe expression of miR-652-3p in cardiomyocytes. Additionally, Up-regulation of miR-652-3p could reduce the apoptosis of H9c2 cells induced by AngⅡ by targeting the inhibition of PRRX1 expression.

Key words: miR-652-3p, Autonomous heterologous nuclear gene 1, AngiotensinⅡ, Cardiomyocyte apoptosis

图1 上调miR-652-3p对AngⅡ诱导H9c2细胞中PRRX1蛋白表达的影响

表1 不同组H9c2细胞中miR-652-3p和PRRX1蛋白表达水平比较（±s， n = 3）

分组	miR-652-3p	PRRX1蛋白
对照组	1.00±0.08	0.06±0.01
AngⅡ组	0.21±0.05^a	0.41±0.04^a
AngⅡ+NC组	0.24±0.06^a	0.39±0.04^a
AngⅡ+miR-652-3p组	0.98±0.07^b	0.13±0.01^b
F值	404.052	337.676
P值	< 0.001	< 0.001

表1 不同组H9c2细胞中miR-652-3p和PRRX1蛋白表达水平比较（±s， n = 3）

分组	miR-652-3p	PRRX1蛋白
对照组	1.00±0.08	0.06±0.01
AngⅡ组	0.21±0.05^a	0.41±0.04^a
AngⅡ+NC组	0.24±0.06^a	0.39±0.04^a
AngⅡ+miR-652-3p组	0.98±0.07^b	0.13±0.01^b
F值	404.052	337.676
P值	< 0.001	< 0.001

图2 miR-652-3p与PRRX13'UTR的结合位点

表2 不同组H9c2细胞相对荧光素酶活性比较（±s，n = 3）

分组	相对荧光素酶活性
NC+PRRX1-Wt组	1.00±0.10
miR-652-3p+PRRX1-Wt组	0.20±0.02^a
NC+PRRX1-Mut组	0.98±0.09
miR-652-3p+PRRX1-Mut组	0.98±0.10^b
F值	195.537
P值	< 0.001

表2 不同组H9c2细胞相对荧光素酶活性比较（±s，n = 3）

分组	相对荧光素酶活性
NC+PRRX1-Wt组	1.00±0.10
miR-652-3p+PRRX1-Wt组	0.20±0.02^a
NC+PRRX1-Mut组	0.98±0.09
miR-652-3p+PRRX1-Mut组	0.98±0.10^b
F值	195.537
P值	< 0.001

图3 流式细胞术检测上调miR-652-3p后AngⅡ诱导的H9c2细胞凋亡

图4 Western blot检测上调miR-652-3p后AngⅡ诱导H9c2细胞Bax和Bcl-2蛋白表达

表3 上调miR-652-3p后AngⅡ诱导的H9c2细胞凋亡率及Bax和Bcl-2表达水平（±s，n = 3）

分组	细胞凋亡率（﹪）	Bax	Bcl-2
对照组	5.02±1.41	0.46±0.05	0.83±0.08
AngⅡ组	25.33±3.75^a	0.96±0.10^a	0.40±0.04^a
AngⅡ+NC组	27.02±4.11^a	0.95±0.09^a	0.38±0.04^a
AngⅡ+miR-652-3p组	12.19±1.63^b	0.53±0.05^b	0.72±0.07^b
F值	113.205	111.221	127.841
P值	< 0.001	< 0.001	< 0.001

表3 上调miR-652-3p后AngⅡ诱导的H9c2细胞凋亡率及Bax和Bcl-2表达水平（±s，n = 3）

分组	细胞凋亡率（﹪）	Bax	Bcl-2
对照组	5.02±1.41	0.46±0.05	0.83±0.08
AngⅡ组	25.33±3.75^a	0.96±0.10^a	0.40±0.04^a
AngⅡ+NC组	27.02±4.11^a	0.95±0.09^a	0.38±0.04^a
AngⅡ+miR-652-3p组	12.19±1.63^b	0.53±0.05^b	0.72±0.07^b
F值	113.205	111.221	127.841
P值	< 0.001	< 0.001	< 0.001

图5 流式细胞术检测上调miR-652-3p和过表达PRRX1后AngⅡ诱导H9c2细胞凋亡

图6 Western blot检测上调miR-652-3p和过表达PRRX1后AngⅡ诱导的H9c2细胞Bax和Bcl-2蛋白表达

表4 上调miR-652-3p和过表达PRRX1后AngⅡ诱导H9c2细胞凋亡率及PRRX1、Bax和Bcl-2蛋白水平比较（±s，n = 3）

分组	PRRX1	细胞凋亡率（﹪）	Bax	Bcl-2
AngⅡ+miR-652-3p组	0.14±0.01	13.02±2.12	0.52±0.05	0.73±0.07
AngⅡ+miR-652-3p+Vctor组	0.13±0.01	12.88±1.84	0.54±0.05	0.72±0.07
AngⅡ+miR-652-3p+PRRX1组	0.35±0.04^a	25.45±3.58^a	0.82±0.08^a	0.46±0.05^a
F值	231.500	67.953	66.632	31.439
P值	< 0.001	< 0.001	< 0.001	< 0.001

表4 上调miR-652-3p和过表达PRRX1后AngⅡ诱导H9c2细胞凋亡率及PRRX1、Bax和Bcl-2蛋白水平比较（±s，n = 3）

分组	PRRX1	细胞凋亡率（﹪）	Bax	Bcl-2
AngⅡ+miR-652-3p组	0.14±0.01	13.02±2.12	0.52±0.05	0.73±0.07
AngⅡ+miR-652-3p+Vctor组	0.13±0.01	12.88±1.84	0.54±0.05	0.72±0.07
AngⅡ+miR-652-3p+PRRX1组	0.35±0.04^a	25.45±3.58^a	0.82±0.08^a	0.46±0.05^a
F值	231.500	67.953	66.632	31.439
P值	< 0.001	< 0.001	< 0.001	< 0.001

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Effects of miR-652-3p targeting homeotype nuclear gene 1 in the regulation of angiotensin Ⅱ-induced cardiomyocyte apoptosis

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