下调miR-106a-5p对过氧化氢诱导的心肌细胞损伤及JAK1/STAT3通路的影响

doi:10.11958/20220744

摘要/Abstract

摘要：

目的探讨下调miR-106a-5p对过氧化氢（H₂O₂）诱导的心肌细胞损伤及Janus激酶1（JAK1）/信号转导和转录激活因子3（STAT3）通路的影响。方法不同浓度梯度H₂O₂（0、50、100、200、400 μmol/L）处理大鼠心肌细胞H9c2。H9c2细胞分为对照组、H₂O₂组（100 μmol/L H₂O₂）、miR-106a-5p NC组（100 μmol/L H₂O₂+转染miR-106a-5p NC）和miR-106a-5p siRNA组（100 μmol/L H₂O₂+转染miR-106a-5p siRNA）。实时荧光定量PCR（qPCR）法检测miR-106a-5p表达；CCK-8法检测细胞增殖；流式细胞仪检测细胞凋亡；试剂盒检测细胞培养液中乳酸脱氢酶（LDH）含量、谷胱甘肽过氧化物酶（GSH-Px）活性及细胞中丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性；蛋白免疫印迹法检测细胞中JAK1/STAT3通路相关蛋白表达。结果根据H₂O₂浓度梯度实验，选择100 μmol/L H₂O₂用于后续实验。与对照组相比，H₂O₂组H9c2细胞增殖抑制率、凋亡率、细胞中miR-106a-5p水平、MDA含量、p-JAK1/JAK1、p-STAT3/STAT3及细胞培养液中LDH含量显著升高（P<0.05），细胞培养液GSH-Px活性及细胞中SOD活性显著降低（P<0.05）；上述指标在H₂O₂组与miR-106a-5p NC组间的差异无统计学意义（P>0.05）；与miR-106a-5p NC组相比，miR-106a-5p siRNA组H9c2细胞增殖抑制率、凋亡率、细胞中miR-106a-5p水平、MDA含量、p-JAK1/JAK1、p-STAT3/STAT3及细胞培养液中LDH含量显著降低（P<0.05），细胞培养液GSH-Px活性及细胞中SOD活性显著升高（P<0.05）。结论下调miR-106a-5p可抑制JAK1/STAT3通路激活，提高抗氧化应激水平，减轻H₂O₂诱导的心肌细胞损伤。

关键词: 肌细胞，心脏, 过氧化氢, 微RNAs, Janus激酶1, STAT3转录因子, 氧化性应激

Abstract:

Objective To investigate the effects of down-regulation of miR-106a-5p on hydrogen peroxide (H₂O₂)-induced cardiomyocyte damage and Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) pathway. Methods Different concentration gradients of H₂O₂ (0, 50, 100, 200 and 400 μmol/L H₂O₂) were used to treat rat cardiomyocytes H9c2. Rat cardiomyocytes H9c2 were divided into the control group, the H₂O₂ group (100 μmol/L H₂O₂), the miR-106a-5p NC group (100 μmol/L H₂O₂+ transfected with miR-106a-5p NC) and the miR-106a-5p siRNA group (100 μmol/L H₂O₂+ transfected with miR-106a-5p siRNA). Real-time fluorescent quantitative PCR (qPCR) method was used to detect the expression of miR-106a-5p. CCK-8 method was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. LDH content, GSH-Px activity in cell culture fluid, MDA content and SOD activity in cells were detected. Western blotting (WB) was used to detect the expression of JAK1/STAT3 pathway related proteins in cells. Results According to the H₂O₂ concentration gradient experiment, 100 μmol/L H₂O₂ was selected for subsequent experiments. Compared with the control group, the proliferation inhibition rate, apoptosis rate, miR-106a-5p level, MDA content, p-JAK1/JAK1, p-STAT3/STAT3 protein levels in H9c2 cells and LDH content in cell culture fluid were significantly increased in the H₂O₂ group (P<0.05). The GSH-Px activity in cell culture fluid and SOD activity in cells significantly reduced (P<0.05). There were no significant differences in above indicators between the H₂O₂ group and the miR-106a-5p NC group (P>0.05). Compared with the miR-106a-5p NC group, the proliferation inhibition rate, apoptosis rate, miR-106a-5p level, MDA content, p-JAK1/JAK1, p-STAT3/STAT3 protein levels in H9c2 cells and LDH content in cell culture fluid were reduced significantly in the miR-106a-5p siRNA group (P<0.05), and the GSH-Px activity in cell culture fluid and SOD activity in cells increased significantly (P<0.05). Conclusion Down-regulation of miR-106a-5p can inhibit the activation of JAK1/STAT3 pathway, increase the level of anti-oxidative stress and reduce H₂O₂-induced cardiomyocyte damage.

Key words: myocytes, cardiac, hydrogen peroxide, microRNAs, Janus kinase 1, STAT3 transcription factor, oxidative stress

中图分类号:

R349.5

图/表 5

表1 各组H9c2细胞中miR-106a-5p表达水平、增殖抑制率及凋亡率比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of miR-106a-5p expression level, proliferation inhibition rate and apoptosis rate between the four groups of H9c2 cells

组别	miR-106a-5p	增殖抑制率（%）	凋亡率（%）
对照组	1.00±0.00	0.00±0.00	5.62±0.52
H₂O₂组	1.64±0.12^a	32.95±1.58^a	35.08±1.63^a
miR-106a-5p NC组	1.66±0.12^a	33.54±1.43^a	34.55±1.56^a
miR-106a-5p siRNA组	0.85±0.10^bc	14.30±1.05^abc	17.26±1.04^abc
F	110.737^＊＊	1 110.412^＊＊	762.780^＊＊

图1 各组H9c2细胞凋亡情况

Fig. 1 Apoptosis of H9c2 cells in each group

表2 各组H9c2细胞培养液LDH含量、GSH-Px活性及细胞中MDA含量、SOD活性比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of LDH content, GSH-Px activity, MDA content and SOD activity in H9c2 cell culture medium between the four groups

组别	LDH（U/L）	GSH-Px（μmol/L）	MDA（nmol/mg prot）	SOD（U/mg prot）
对照组	245.50±8.79	224.68±16.15	0.40±0.06	60.94±2.82
H₂O₂组	326.36±10.22^a	133.58±12.48^a	0.76±0.09^a	48.65±2.27^a
miR-106a-5p NC组	330.20±9.86^a	135.70±13.02^a	0.75±0.09^a	48.04±2.34^a
miR-106a-5p siRNA组	282.54±9.11^abc	187.39±13.25^abc	0.57±0.07^abc	54.96±2.57^abc
F	106.526^＊＊	60.865^＊＊	28.308^＊＊	35.043^＊＊

图2 各组H9c2细胞中p-JAK1、JAK1、p-STAT3、STAT3蛋白表达情况 A：对照组；B：H2O2组；C：miR-106a-5p NC组；D：miR-106a-5p siRNA组。

Fig.2 Protein expressions of p-JAK1, JAK1, p-STAT3 and STAT3 in H9c2 cells of each group

表3 各组H9c2细胞中p-JAK1/JAK1、p-STAT3/STAT3蛋白表达水平比较（n=6，$\bar{x}±s$）

Tab.3 Comparison of protein expression levels of p-JAK1/JAK1 and p-STAT3 /STAT3 between the four groups of H9c2 cells

组别	p-JAK1/JAK1	p-STAT3/STAT3
对照组	0.30±0.04	0.34±0.05
H₂O₂组	0.84±0.07^a	0.89±0.08^a
miR-106a-5p NC组	0.81±0.07^a	0.87±0.07^a
miR-106a-5p siRNA组	0.52±0.06^abc	0.50±0.07^abc
F	105.000^＊＊	96.086^＊＊

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