miR-181a-5p通过HMGB1/NF-κB信号通路调控狼疮性肾炎小鼠肾小球系膜细胞增殖和凋亡

doi:10.11958/20253169

摘要/Abstract

摘要：

目的探究微小RNA（miR）-181a-5p通过高迁移率族蛋白B1（HMGB1）/核因子（NF）-κB信号通路对狼疮性肾炎（LN）小鼠肾小球系膜细胞增殖和凋亡的调控机制。方法分离并体外培养LN小鼠肾小球系膜细胞，分为空白组、mimics-NC组、miR-181a-5p mimics组、Si-NC组（转染Si-NC）、Si-HMGB1组，mimics-NC+pcDNA-NC组、miR-181a-5p mimics+pcDNA-NC组、mimics-NC+pcDNA-HMGB1组、miR-181a-5p mimics+pcDNA-HMGB1组，分离并体外培养MRL/MPJ小鼠肾小球系膜细胞作为对照组。qRT-PCR法检测细胞中miR-181a-5p、HMGB1 mRNA表达水平；双萤光素酶实验验证miR-181a-5p与HMGB1的靶向关系；CCK-8法、流式细胞仪分别检测细胞增殖、凋亡；Western blot检测细胞中HMGB1、人核因子κB抑制蛋白-α（IκB-α）、NF-κB p65蛋白表达水平。结果 miR-181a-5p与HMGB1存在靶向关系；与对照组相比，空白组的细胞增殖率、HMGB1、NF-κB p65蛋白表达均显著增加，miR-181a-5p、IκB-α表达均显著降低（P<0.05）；与mimics-NC组相比，miR-181a-5p mimics组的细胞增殖率、HMGB1、NF-κB p65蛋白表达均显著降低，miR-181a-5p、IκB-α表达均显著增加（P<0.05）；与Si-NC组相比，Si-HMGB1组的细胞增殖率、HMGB1、NF-κB p65蛋白表达均显著降低，IκB-α表达显著增加（P<0.05），且过表达HMGB1可以逆转上调miR-181a-5p对LN小鼠肾小球系膜细胞过度增殖的抑制作用（P<0.05）。结论过表达miR-181a-5p可抑制LN小鼠肾小球系膜细胞过度增殖，可能与抑制HMGB1/NF-κB信号通路有关。

关键词: 狼疮肾炎, 微RNAs, HMGB1蛋白质, NF-κB, 肾小球系膜细胞, 细胞增殖, 细胞凋亡

Abstract:

Objective To explore the regulatory mechanism of microRNA (miR)-181a-5p on the proliferation and apoptosis of glomerular mesangial cells in mice with lupus nephritis (LN) through the high mobility group protein box 1 (HMGB1)/nuclear factor kappa B (NF-κB) signaling pathway. Methods LN mouse mesangial cells were isolated and cultured in vitro, and divided into the blank group, the mimics-NC group, the miR-181a-5p mimics group, the Si-NC group, the Si-HMGB1 group (transfected with Si-HMGB1), the mimics-NC+pcDNA-NC group, the miR-181a-5p mimics+pcDNA-NC group, the mimics-NC+pcDNA-HMGB1 group and the miR-181a-5p mimics+pcDNA-HMGB1 group. MRL/MPJ mouse mesangial cells were isolated and cultured in vitro as the control group. qRT-PCR method was implemented to measure the mRNA expression levels of miR-181a-5p and HMGB1 in cells. Dual-luciferase experiments were performed to verify the targeting relationship of miR-181a-5p to HMGB1. CCK-8 method and flow cytometry were implemented to measure cell proliferation and apoptosis, respectively. Western blot assay was performed to measure the protein expression levels of HMGB1, human inhibitor of nuclear factor κB-α (IκB-α) and NF-κB p65 in cells. Results There was a targeting relationship between miR-181a-5p and HMGB1. Compared with the control group, the cell proliferation rate, HMGB1 and NF-κB p65 protein expression were obviously increased in the blank group, and the miR-181a-5p and IκB-α expression were obviously decreased (P<0.05). Compared with the mimics-NC group, the cell proliferation rate, HMGB1 and NF-κB p65 protein expression were obviously decreased in the miR-181a-5p mimics group, and the miR-181a-5p and IκB-α expression were obviously increased (P<0.05). Compared with the Si-NC group, the cell proliferation rate, HMGB1 and NF-κB p65 protein expression were obviously decreased in the Si-HMGB1 group, and the IκB-α expression was obviously increased (P<0.05). The overexpression of HMGB1 was able to reverse the inhibitory effect of up-regulated miR-181a-5p on the excessive proliferation of glomerular mesangial cells in LN mice (P<0.05). Conclusion Overexpression of miR-181a-5p can inhibit the excessive proliferation of glomerular mesangial cells in LN mice, which may be related to the inhibition of HMGB1/NF-κB signaling pathway.

Key words: lupus nephritis, microRNAs, HMGB1 protein, NF-kappa B, mesangial cells, cell proliferation, apoptosis

中图分类号:

R593.242

图/表 9

参考文献 18

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引物名称		引物序列（5′→3′）	产物大小/bp
miR-181a-5p	上游：	TCGACCGTTGTACCCT	113
	下游：	TCGACTGCATAGCTAGCTCA	113
HMGB1	上游：	AGCAATCTGAACGTCTGTCC	107
	下游：	GTTCTTGTGATAGCCTTCGC	107
U6	上游：	CTCGCTTCGGCAGCACATA	134
	下游：	AACGATTCACGAATTTGCGT	134
β-actin	上游：	AAGACCTCTATGCCAACACAG	107
	下游：	GGAGGAGCAATGATCTTGATC	107

引物名称		引物序列（5′→3′）	产物大小/bp
miR-181a-5p	上游：	TCGACCGTTGTACCCT	113
	下游：	TCGACTGCATAGCTAGCTCA	113
HMGB1	上游：	AGCAATCTGAACGTCTGTCC	107
	下游：	GTTCTTGTGATAGCCTTCGC	107
U6	上游：	CTCGCTTCGGCAGCACATA	134
	下游：	AACGATTCACGAATTTGCGT	134
β-actin	上游：	AAGACCTCTATGCCAACACAG	107
	下游：	GGAGGAGCAATGATCTTGATC	107

组别	增殖率/ %		凋亡率/ %		miR-181a -5p		HMGB1 mRNA
对照组	100.00±0.00		25.38±2.54		1.00±0.00		1.00±0.00
空白组	156.68±15.65^a		24.34±2.43		0.63±0.07^a		2.12±0.22^a
mimics-NC组	149.34±14.50^a		24.42±2.44		0.62±0.07^a		2.18±0.22^a
miR-181a-5p mimics组	99.03±9.91^b		25.43±2.54		0.89±0.09^b		1.42±0.15^b
Si-NC组	149.64±14.51		24.28±2.26		0.64±0.07		2.16±0.22
Si-HMGB1组	98.83±9.89^c		25.44±2.54		0.67±0.07		1.51±0.16^c
F	34.970^＊＊		0.343		34.176^＊＊		45.326^＊＊
组别		HMGB1/ β-actin		IκB-α/ β-actin		NF-κB p65/ β-actin
对照组		0.35±0.03		1.05±0.10		0.29±0.03
空白组		0.98±0.09^a		0.41±0.04^a		1.01±0.10^a
mimics-NC组		0.96±0.09^a		0.43±0.04^a		0.98±0.09^a
miR-181a-5pmimics组		0.62±0.06^b		0.68±0.07^b		0.61±0.06^b
Si-NC组		0.97±0.09		0.39±0.04		0.95±0.09
Si-HMGB1组		0.58±0.06^c		0.65±0.07^c		0.63±0.06^c
F		77.985^＊＊		93.800^＊＊		85.214^＊＊

组别	增殖率/ %		凋亡率/ %		miR-181a -5p		HMGB1 mRNA
对照组	100.00±0.00		25.38±2.54		1.00±0.00		1.00±0.00
空白组	156.68±15.65^a		24.34±2.43		0.63±0.07^a		2.12±0.22^a
mimics-NC组	149.34±14.50^a		24.42±2.44		0.62±0.07^a		2.18±0.22^a
miR-181a-5p mimics组	99.03±9.91^b		25.43±2.54		0.89±0.09^b		1.42±0.15^b
Si-NC组	149.64±14.51		24.28±2.26		0.64±0.07		2.16±0.22
Si-HMGB1组	98.83±9.89^c		25.44±2.54		0.67±0.07		1.51±0.16^c
F	34.970^＊＊		0.343		34.176^＊＊		45.326^＊＊
组别		HMGB1/ β-actin		IκB-α/ β-actin		NF-κB p65/ β-actin
对照组		0.35±0.03		1.05±0.10		0.29±0.03
空白组		0.98±0.09^a		0.41±0.04^a		1.01±0.10^a
mimics-NC组		0.96±0.09^a		0.43±0.04^a		0.98±0.09^a
miR-181a-5pmimics组		0.62±0.06^b		0.68±0.07^b		0.61±0.06^b
Si-NC组		0.97±0.09		0.39±0.04		0.95±0.09
Si-HMGB1组		0.58±0.06^c		0.65±0.07^c		0.63±0.06^c
F		77.985^＊＊		93.800^＊＊		85.214^＊＊

组别	增殖率/ %		凋亡率/ %		HMGB1/ β-actin
mimics-NC+pcDNA-NC组	156.36±15.64		24.34±2.43		0.86±0.08
miR-181a-5p mimics+ pcDNA-NC组	95.32±9.53^a		25.52±2.52		0.38±0.04^a
mimics-NC+pcDNA-HMGB1组	192.43±19.24^a		24.42±2.39		1.08±0.11^a
miR-181a-5p mimics+ pcDNA-HMGB1组	159.35±16.04^bc		24.37±2.44		0.88±0.08^bc
F	40.836^＊＊		0.329		79.940^＊＊
组别		IκB-α/ β-actin		NF-κB p65/ β-actin
mimics-NC+pcDNA-NC组		0.43±0.04		0.82±0.08
miR-181a-5p mimics+pcDNA-NC组		0.98±0.09^a		0.32±0.03^a
mimics-NC+pcDNA-HMGB1组		0.21±0.02^a		0.98±0.09^a
miR-181a-5p mimics+pcDNA-HMGB1组		0.41±0.04^bc		0.78±0.08^bc
F		223.778^＊＊		88.477^＊＊