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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (02) : 106 -112. doi: 10.3877/cma.j.issn.2095-1221.2021.02.006

所属专题： 文献；

论著

miR-27a-3p通过靶向Sema7A调控病毒性心肌炎心肌细胞损伤的机制研究
鄢文娟1,†(), 代悉尼1, 谢达1, 魏燕1   
  1. 1. 433100　潜江，湖北省潜江市湖北江汉油田总医院心内科
  • 收稿日期:2020-02-26 出版日期:2021-04-01
  • 通信作者: 鄢文娟

Overexpression of miR-27a-3p inhibits myocardial cell injury in viral myocarditis by targeting Sema7A

Wenjuan Yan1,†(), Xini Dai1, Da Xie1, Yan Wei1   

  1. 1. Department of Cardiology, Jianghan Oilfield General Hospital, Qianjiang 433100, China
  • Received:2020-02-26 Published:2021-04-01
  • Corresponding author: Wenjuan Yan
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引用本文：

鄢文娟, 代悉尼, 谢达, 魏燕. miR-27a-3p通过靶向Sema7A调控病毒性心肌炎心肌细胞损伤的机制研究[J]. 中华细胞与干细胞杂志(电子版), 2021, 11(02): 106-112.

Wenjuan Yan, Xini Dai, Da Xie, Yan Wei. Overexpression of miR-27a-3p inhibits myocardial cell injury in viral myocarditis by targeting Sema7A[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(02): 106-112.

目的

探讨微小RNA-27a-3p （miR-27a-3p）过表达对病毒性心肌炎（VMC）细胞损伤的影响及其可能的作用机制。

方法

原代培养大鼠心肌细胞，柯萨奇B3病毒（CVB3）感染心肌细胞建立VMC模型（CVB3组）。正常心肌细胞作为对照组，分别将miR-NC、miR- 27a- 3p mimics、si-NC、si-Sema7A分别转染至CVB3感染的心肌细胞，记为CVB3+miR- NC组、CVB3+miR-27a-3p组、CVB3+si-NC组、CVB3+si-Sema7A组。分别将miR- 27a-3p mimics与pcDNA，miR-27a-3p mimics与pcDNA-Sema7A共转染至CVB3感染的心肌细胞，记为CVB3+miR-27a-3p+pcDNA组、CVB3+miR-27a-3p+pcDNA-Sema7A组。采用实时荧光定量聚合酶链反应（qRT-PCR）与Western blot法分别检测细胞中miR-27a-3p、信号蛋白7A （Sema7A）的表达水平；流式细胞术检测miR-27a-3p过表达或干扰Sema7A表达后心肌细胞的凋亡率；酶联免疫吸附（ELISA）法检测细胞中肿瘤坏死因子-α （TNF-α）、白细胞介素-1 （IL-1）的含量；双荧光素酶报告基因验证miR-27a-3p与Sema7A的靶向关系；Western blot检测B淋巴细胞瘤-2相关蛋白（Bax）、活化的含半胱氨酸的天冬氨酸蛋白水解酶3 （cleaved caspase-3）、B淋巴细胞瘤-2 （Bcl-2）蛋白表达。两组间比较采用独立样本t检验。

结果

与对照组比较，CVB3组miR-27a-3p的表达水平（1.01±0.09比0.42±0.04）降低，Sema7A的mRNA和蛋白表达水平（1.02±0.09比2.15±0.21、0.43±0.04比0.94±0.09）升高，TNF-α、IL-1水平[（403.56± 38.16）pg/mL比（1156.48±63.59）pg/mL、（29.45±3.54）pg/mL比（136.57±12.47）pg/mL]升高，细胞凋亡率[（5.36±0.54）﹪比（24.58±2.14）﹪]升高，Bax、cleaved caspase-3的表达水平升高，Bcl-2的表达水平降低（P均< 0.05）；与CVB3+miR-NC组比较，CVB3+miR-27a-3p组TNF-α、IL-1水平[（1187.69±71.42）pg/mL比（516.28±48.31）pg/mL、（147.25±14.05）pg/ mL比（43.68±5.02）pg/mL]和细胞凋亡率[（26.38±2.55）﹪比10.24±1.15）﹪]降低，Bax、cleaved caspase-3表达水平降低，Bcl-2表达水平升高（P均< 0.05）；双荧光素酶报告实验证实miR- 27a-3p可靶向调控Sema7A的表达；Sema7A过表达可逆转miR-27a-3p过表达对CVB3诱导的VMC细胞损伤的作用。

结论

miR-27a-3p过表达可降低VMC细胞中炎症因子的表达及抑制细胞凋亡从而减轻心肌损伤，其作用机制可能与靶向调控Sema7A的表达有关。

关键词: 病毒性心肌炎, miR-27a-3p, Sema7A, 细胞损伤
Objective

To investigate the effect of overexpressed microRNA-27a-3p (miR-27a-3p) on viral myocarditis (VMC) cell injury and its mechanisms.

Methods

Primary cardiomyocytes of rats were infected with Coxsackie B3 virus (CVB3) to establish VMC cell model (CVB3 group) . Normal cardiomyocytes as control (con) , and the miR-NC, miR-27a-3p mimics, si-NC, and si-Sema7A were transfected into CVB3-infected cardiomyocytes respectively, which were named as CVB3+miR-NC, CVB3+miR-27a-3p, CVB3+si-NC, and CVB3+si-Sema7A group. The miR-27a-3p mimics and pcDNA, miR-27a-3p mimics and pcDNA-Sema7A were co-transfected into CVB3-infected cardiomyocytes, respectively, and named as CVB3+miR-27a-3p+pcDNA and CVB3+miR-27a-3p+pcDNA-Sema7A group. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression levels of miR-27a-3p and Sema7A in cells. Flow cytometry was used to detect the apoptotic rate of cardiomyocytes after overexpression of miR-27a-3p or knock-down Sema7A. The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL) -1 in the cells were detected by enzyme-linked immunosorbent assay (ELISA) . The targeting relationship between miR-27a-3p and Sema7A was verified by dual luciferase reporter gene assay. The expression of Bax, cleaved caspase-3 and Bcl-2 protein was detected by Western blot.

Results

Compared with Con group, the expression level of miR-27a-3p in CVB3 group was significantly decreased (1.01±0.09 vs 0.42±0.04, P < 0.05) , and the mRNA and protein levels of Sema7A were significantly increased (1.02±0.09 vs 2.15±0.21, P < 0.05, 0.43±0.04 vs 0.94±0.09, P < 0.05) , the levels of TNF-α and IL-1 were significantly increased [ (403.56±38.16) pg/mL vs (1156.48±63.59) pg/mL, (29.45±3.54) pg/mL vs (136.57±12.47) pg/mL, P < 0.05], the apoptosis rate was increased significantly (5.36±0.54 vs 24.58±2.14, P < 0.05) , the expression levels of Bax and cleaved caspase-3 were increased significantly (P < 0.05) , and the expression level of Bcl-2 was decreased significantly (P < 0.05) . Compared with the CVB3+miR-NC group, the levels of TNF-α and IL-1 in the CVB3+miR-27a-3p group were significantly reduced [ (1187.69± 71.42) pg/mL vs (516.28±48.31) pg/mL, (147.25±14.05) pg/mL vs (43.68±5.02) pg/mL, P < 0.05], the apoptosis rate was decreased [ (26.38±2.55) ﹪ vs (10.24± 1.15) ﹪, P < 0.05], the expression levels of Bax and cleaved caspase-3 were decreased (P < 0.05) , and the expression level of Bcl-2 was increased (P < 0.05) . Double luciferase reporting experiments confirmed that miR-27a-3p could target Sema7A expression. Sema7A overexpression could reverse the effect of miR-27a-3p overexpression on CVB3- induced VMC cell injury.

Conclusion

Overexpression of miR-27a-3p can reduce the expression of inflammatory factors and inhibit apoptosis in VMC cells, thereby reducing myocardial injury. The mechanism may be related to the targeted regulation of the expression of Sema7A.

Key words: Viral myocarditis, miR-27a-3p, Sema7A, Cell injury
表1 引物序列信息
基因 序列（5'→3'） 预期扩增产物长度（bp）
miR-27a-3p 上游TGCGGTTCACAGTGGCTAAG 292
下游CTCAACTGGTGTCGTGGA
Sema7A 上游GGCGGAAGCTCTATGTGA 272
下游GACTGCAGCACTGATCGTTGG
U6 上游CTCGCTTCGGCAGCACA 285
下游AACGCTTCACGAATTTGCGT
GAPDH 上游GGCAAGTTCAACGGCACAG 494
下游CGCCAGTAGACTCCACGACAT
表2 miR-27a-3p和Sema7A在CVB3诱导的病毒性心肌炎细胞中的表达（±s）
分组 实验次数 miR-27a-3p Sema7A mRNA Sema7A蛋白
对照 3 1.01±0.09 1.02±0.09 0.43±0.04
CVB3 3 0.42±0.04 2.15±0.21 0.94±0.09
t值   10.376 8.567 8.969
P值   < 0.001 0.001 0.001
图1 Sema7A蛋白在柯萨奇B3病毒诱导的病毒性心肌炎细胞中的表达
表3 miR-27a-3p过表达对CVB3诱导的病毒性心肌炎细胞炎症因子表达的影响（±s）
分组 实验次数 miR-27a-3p TNF-α （pg/mL） IL-1（pg/mL）
对照 3 1.01±0.09 403.56±38.16 29.45±3.54
CVB3 3 0.44±0.04 1156.48±63.59 136.57±12.47
t值   10.024 17.585 14.313
P值   0.001 <0.001 <0.001
CVB3+miR-NC 3 0.41±0.04 1187.69±71.42 147.25±14.05
CVB3+miR-27a-3p 3 0.82±0.08 516.28±48.31 43.68±5.02
t值   7.940 13.487 12.023
P值   0.001 < 0.001 < 0.001
表4 miR-27a-3p过表达对CVB3诱导的病毒性心肌炎细胞凋亡的影响（±s
分组 实验次数 凋亡率（﹪） Bcl-2蛋白 Bax蛋白 cleaved caspase-3蛋白
对照 3 5.36±0.54 0.75±0.07 0.24±0.03 0.29±0.03
CVB3 3 24.58±2.14 0.32±0.03 0.58±0.05 0.67±0.06
t值   15.083 9.779 10.100 9.812
P值   < 0.001 0.001 0.001 0.001
CVB3+miR-NC 3 26.38±2.55 0.31±0.03 0.62±0.06 0.68±0.07
CVB3+miR-27a-3p 3 10.24±1.15 0.64±0.06 0.35±0.03 0.41±0.04
t值   9.994 8.521 6.971 5.801
P值   0.001 0.001 0.002 0.004
图2 miR-27a-3p过表达对CVB3诱导的病毒性心肌炎细胞凋亡相关蛋白表达的影响
图3 miR-27a-3p过表达对CVB3诱导的病毒性心肌炎细胞凋亡的影响
表5 干扰Sema7A表达对CVB3诱导的病毒性心肌炎细胞损伤的影响（±s
分组 实验次数 Sema7A蛋白 TNF-α （pg/mL） IL-1 （pg/mL） 凋亡率（﹪） Bcl-2蛋白 Bax蛋白 cleaved caspase-3蛋白
对照 3 0.42±0.04 387.14±36.24 34.58±3.65 6.75±0.67 0.74±0.07 0.23±0.03 0.28±0.03
CVB3 3 0.93±0.08a 1087.54±73.15a 148.25±13.44a 25.13±2.47a 0.33±0.03a 0.59±0.05a 0.68±0.06a
t值   9.876 14.860 14.137 12.439 9.325 10.694 10.328
P值   0.001 < 0.001 < 0.001 < 0.001 0.001 < 0.001 < 0.001
CVB3+si-NC 3 0.94±0.07 1116.58±84.32 156.37±15.24 26.18±2.64 0.32±0.03 0.61±0.06 0.69±0.07
CVB3+si-Sema7A 3 0.53±0.05b 587.12±51.23b 57.21±5.36b 13.48±1.44b 0.61±0.06b 0.39±0.04b 0.46±0.04b
t值   8.255 9.295 10.631 7.315 7.488 5.284 4.941
P值   0.001 0.001 <0.001 0.002 0.002 0.006 0.008
图4 干扰Sema7A表达对CVB3诱导的病毒性心肌炎细胞Sema7A和凋亡相关蛋白表达的影响
表6 荧光素酶报告实验检测荧光素酶相对活性（±s
分组 实验次数 WT-Sema7A MUT-Sema7A
miR-NC 3 1.02±0.08 0.98±0.09
miR-27a-3p 3 0.36±0.04 1.01±0.08
t值   12.781 0.432
P值   < 0.001 0.688
表7 miR-27a-3p调控Sema7A蛋白的表达（±s）
分组 实验次数 Sema7A蛋白
miR-NC 3 0.46±0.04
miR-27a-3p 3 0.23±0.03a
t值   7.967
P值   0.001
anti-miR-NC 3 0.44±0.05
anti-miR-27a-3p 3 0.89±0.08b
t值   8.262
P值   0.001
图5 Sema7A的3'UTR中含有与miR-27a-3p互补的核苷酸序列
图6 miR-27a-3p异常表达对Sema7A表达的影响
表8 Sema7A过表达逆转miR-27a-3p过表达对CVB3诱导的病毒性心肌炎细胞损伤的作用（±s
分组 实验次数 Sema7A蛋白 TNF-α （pg/mL） IL-1 （pg/mL） 凋亡率（﹪） Bcl-2蛋白 Bax蛋白 cleaved caspase-3蛋白
CVB3+miR-NC 3 0.86±0.08 1245.36±86.32 142.65±13.54 23.45±2.14 0.29±0.03 0.61±0.06 0.72±0.07
CVB3+miR-27a-3p 3 0.39±0.03 526.18±49.32 47.58± 4.26 12.65±1.24 0.65±0.06 0.33±0.03 0.38±0.03
t值   9.528 12.530 11.601 7.563 9.295 7.230 7.733
P值   0.001 < 0.001 < 0.001 0.002 0.001 0.002 0.002
CVB3+miR-27a-3p+pcDNA 3 0.37±0.04 514.26±43.87 43.87±5.03 10.42±1.04 0.66±0.05 0.31±0.03 0.36±0.04
CVB3+miR-27a-3p+pcDNA-Sema7A 3 0.74±0.07 987.36±65.48 120.45±12.35 19.57±1.64 0.41±0.04 0.49±0.04 0.61±0.05
t值   7.949 10.397 9.947 8.161 6.763 6.235 6.763
P值   0.001 < 0.001 0.001 0.001 0.002 0.003 0.002
图7 Sema7A过表达逆转miR-27a-3p过表达对CVB3诱导的病毒性心肌炎细胞Sema7A和凋亡相关蛋白表达的作用
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