miR21介导Ang(1-7)对肺成纤维细胞AngⅡ诱导的NLR3炎性体激活的抑制作用

doi:10.3877/cma.j.issn.1674-6902.2019.05.012

目的

探讨miR21对肺成纤维细胞增殖和凋亡的影响，及其可能的作用机制。

方法

将miR21 NC、inhibitor和mimic转染至原代肺成纤维细胞，按照细胞处理方式分为空白对照组、miR21 NC组、inhibitor组和mimic组。利用CCK-8细胞增殖法检测miR21对肺成纤维细胞增殖的影响；流式细胞仪检测miR21对肺成纤维细胞凋亡的影响；通过RT-PCR法检测转染前后肺成纤维细胞miR21、Ang(1-7)基因水平的表达；应用免疫印迹Western blot检测转染前后miR21、NLRP3蛋白表达水平变化。

结果

CCK8及流式细胞仪检测显示，miR21具有显著促进肺成纤维细胞增殖、抑制肺成纤维细胞凋亡的作用；RT-PCR分析显示，细胞转染成功后，miR21 mimic/inhibitor组分别抑制/促进了Ang(1-7)表达，促进/抑制了AngⅡ表达(P<0.05)，即miR21具有抑制Ang(1-7)，促进AngⅡ表达的效果。Western Blot分析表明，miR21 mimic/inhibitor具有上调/下调AngⅡ、NLRP3蛋白表达水平的作用，与NC对照组相比，差异具有统计学意义(P<0.05)。

结论

miR21具有促进肺成纤维细胞增殖、抑制肺成纤维细胞凋亡的作用，其机制可能与miR21介导Ang(1-7)对肺成纤维细胞AngⅡ诱导的NLR3炎性体激活相关。

关键词: miR21, 肺纤维化, 增殖, 细胞凋亡, 作用机制

Objective

To investigate the effects of miR21 on the proliferation and apoptosis of pulmonary fibroblasts and the possible mechanisms.

Methods

MiR21 NC (miR21 NC group), the inhibitor (inhibitor group), the mimics (mimics group), and nothing (blank control group) were transfected into the primary lung fibroblasts, respectively. The effects of miR21 on the proliferation of lung fibroblasts were detected by CCK-8 cell proliferation. The effects of miR21 on the apoptosis of pulmonary fibroblasts were detected by flow cytometry. The expression levels of miR21 and Ang (1-7) in the pulmonary fibroblasts were detected by reverse transcription polymerase chain reaction (RT-PCR) before and after transfection. The expression levels of miR21 and NLRP3 were detected before and after transfection by Western blotting method.

Results

According to the results of CCK8 and flow cytometry detection, miR21 can significantly promote the proliferation of pulmonary fibroblasts and inhibit the apoptosis of pulmonary fibroblasts. RT-PCR analysis showed that after the success of cell transfection, the miR21 mimics group and the inhibitor group respectively inhibited/promoted Ang (1-7) expression and promoted/inhibited Ang Ⅱ expression (P<0.05). That is to say, miR21 could inhibit miR21 Ang (1-7) and promote Ang Ⅱ expression. Western blot analysis showed that the miR21 mimic/inhibitor could raise/lower the expression levels of Ang Ⅱ and the expression levels of NLRP3 protein, which had statistical significant difference compared with the NC control groups (P<0.05).

Conclusion

MiR21 can promote the proliferation and inhibit the apoptosis of pulmonary fibroblasts, and its mechanism may be related to the effect of miR21-mediated Ang (1-7) on the activation of AngⅡ-induced inflammatory body in pulmonary fibroblasts.

Key words: MiR21, Pulmonary fibrosis, Proliferation, Apoptosis, Mechanism of action

表1 波形蛋白在肺成纤维细胞中的表达

指　标	Vimentin(灰度值)
正常肺泡细胞	0.74±0.06
肺成纤维细胞	3.31±0.72
F值	4.06
P值	<0.05

表1 波形蛋白在肺成纤维细胞中的表达

指　标	Vimentin(灰度值)
正常肺泡细胞	0.74±0.06
肺成纤维细胞	3.31±0.72
F值	4.06
P值	<0.05

表2 miR21对肺成纤维细胞增殖的影响

组　别	肺成纤维细胞OD值
组　别	24 h	48 h	72 h
空白对照组	0.82±0.05	0.86±0.06	0.91±0.01
miR21 NC组	0.82±0.03^a	0.85±0.01^a	0.91±0.03^a
inhibitor组	0.81±0.04^a	0.83±0.06^a	0.86±0.07^a
mimic组	0.82±0.03^a	0.91±0.02^a	0.96±0.02^a
F值	8.252	13.706	11.485
P值	>0.05	<0.05	<0.05

表2 miR21对肺成纤维细胞增殖的影响

组　别	肺成纤维细胞OD值
组　别	24 h	48 h	72 h
空白对照组	0.82±0.05	0.86±0.06	0.91±0.01
miR21 NC组	0.82±0.03^a	0.85±0.01^a	0.91±0.03^a
inhibitor组	0.81±0.04^a	0.83±0.06^a	0.86±0.07^a
mimic组	0.82±0.03^a	0.91±0.02^a	0.96±0.02^a
F值	8.252	13.706	11.485
P值	>0.05	<0.05	<0.05

表3 miR21对肺成纤维细胞凋亡的影响

组　别	肺成纤维细胞凋亡率(%)
组　别	24 h	48 h	72 h
空白对照组	26.24±4.20	35.27±4.37	39. 25±4.61
miR21 NC组	28.20±5.27^a	35.23±4.32^a	38.34±5.57^a
inhibitor组	42.91±3.76^a	51.27±4.21^a	68.94±4.21^a
mimic组	15.23±3.06^a	18.72±4.24^a	23.63±4.04^a
F值	10.814	12.922	10.723
P值	<0.05	<0.05	<0.05

表3 miR21对肺成纤维细胞凋亡的影响

组　别	肺成纤维细胞凋亡率(%)
组　别	24 h	48 h	72 h
空白对照组	26.24±4.20	35.27±4.37	39. 25±4.61
miR21 NC组	28.20±5.27^a	35.23±4.32^a	38.34±5.57^a
inhibitor组	42.91±3.76^a	51.27±4.21^a	68.94±4.21^a
mimic组	15.23±3.06^a	18.72±4.24^a	23.63±4.04^a
F值	10.814	12.922	10.723
P值	<0.05	<0.05	<0.05

表4 PCR过程中引物序列设计

引物名称	前引	后引	长度(bp)
miR21	5′-GGAAGAGCTTCGACCAGCTG-3′	5′-GAGCCTTGTTAGTTTACAG-3′	124
Ang(1-7)	5′-AACCGAGCTTAGGCCAGCTG-3′	5′-AAGCCTTGACGCGTTTACAG-3′	132
AngⅡ	5′-TCTGGATCAATGAAGCCATG-3′	5′-TCTGCGTTGCAGTTTACAG-3′	138
GAPDH	5′-CAGCATTGGAAGTGCTATGG-3′	5′-TAGAGCGAGTATGCATGACA-3′	132

表4 PCR过程中引物序列设计

引物名称	前引	后引	长度(bp)
miR21	5′-GGAAGAGCTTCGACCAGCTG-3′	5′-GAGCCTTGTTAGTTTACAG-3′	124
Ang(1-7)	5′-AACCGAGCTTAGGCCAGCTG-3′	5′-AAGCCTTGACGCGTTTACAG-3′	132
AngⅡ	5′-TCTGGATCAATGAAGCCATG-3′	5′-TCTGCGTTGCAGTTTACAG-3′	138
GAPDH	5′-CAGCATTGGAAGTGCTATGG-3′	5′-TAGAGCGAGTATGCATGACA-3′	132

表5 RT-PCR检测miR21、Ang(1-7)、AngⅡ基因表达情况

组　别	miR21(灰度值)	Ang(1-7)(灰度值)	AngⅡ(灰度值)
空白对照组	2.24±0.40	2.74±0.32	2.34±0.61
miR21 NC组	2.36±0.41	2.75±0.72	2.33±0.58
inhibitor组	1.06±0.06	4.79±0.52	1.08±0.04
mimic组	5.23±0.21	1.10±0.08	5.03±0.22
F值	8.375	12.726	13.215
P值	<0.05	<0.05	<0.05

表5 RT-PCR检测miR21、Ang(1-7)、AngⅡ基因表达情况

组　别	miR21(灰度值)	Ang(1-7)(灰度值)	AngⅡ(灰度值)
空白对照组	2.24±0.40	2.74±0.32	2.34±0.61
miR21 NC组	2.36±0.41	2.75±0.72	2.33±0.58
inhibitor组	1.06±0.06	4.79±0.52	1.08±0.04
mimic组	5.23±0.21	1.10±0.08	5.03±0.22
F值	8.375	12.726	13.215
P值	<0.05	<0.05	<0.05

图1 RT-PCR检测miR21、Ang(1-7)基因表达情况

表6 miR21对肺成纤维细胞对AngⅡ、NLRP3蛋白表达水平的影响

组　别	AngⅡ(灰度值)	NLRP3(灰度值)
空白对照组	2.04±0.29	2.25±028
miR21 NC组	2.06±0.31	2.23±0.21
inhibitor组	1.08±0.04	1.0±0.04
mimic组	5.03±0.22	4.63±0.50
F值	8.212	12.323
P值	<0.05	<0.05

表6 miR21对肺成纤维细胞对AngⅡ、NLRP3蛋白表达水平的影响

组　别	AngⅡ(灰度值)	NLRP3(灰度值)
空白对照组	2.04±0.29	2.25±028
miR21 NC组	2.06±0.31	2.23±0.21
inhibitor组	1.08±0.04	1.0±0.04
mimic组	5.03±0.22	4.63±0.50
F值	8.212	12.323
P值	<0.05	<0.05

图2 Western Blot检测AngⅡ、NLRP3蛋白表达情况

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Inhibition of miR21-mediated Ang (1-7) on activation of AngⅡ-induced inflammatory body in pulmonary fibroblasts

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Inhibition of miR21-mediated Ang (1-7) on activation of AngⅡ-induced inflammatory body in pulmonary fibroblasts