miR-335-5p对骨关节炎软骨细胞增殖和凋亡的分子机制研究

吴洁; 范文强; 许振丹; 梁舒; 秦艺璐; 王培山

doi:10.16766/j.cnki.issn.1674-4152.001906

miR-335-5p对骨关节炎软骨细胞增殖和凋亡的分子机制研究

doi: 10.16766/j.cnki.issn.1674-4152.001906

1.
新乡市中心医院风湿免疫科，河南新乡 453000
2.
新乡市中心医院门诊手术部，河南新乡 453000

基金项目:

河南省医学科技攻关计划联合共建项目 LHGJ20200941

2019年度新乡市科技攻关计划项目 GG2019026

详细信息

通讯作者:
王培山，E-mail：ccff-218000@163.com

中图分类号: R684.3 R329.25
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- 文章访问数: 216
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- 被引次数: 0
出版历程
- 收稿日期: 2020-08-04
- 网络出版日期: 2022-02-16

Study on the molecular mechanism of miR-335-5p on proliferation and apoptosis of osteoarthritis chondrocytes

1.
Department of Rheumatology and Immunology, Xinxiang Central Hospital, Xinxiang, Henan 453000, China

摘要

摘要: 目的研究miR-335-5p对骨关节炎(OA)软骨细胞增殖和凋亡的影响及其可能机制。方法体外分离培养OA软骨细胞及正常软骨细胞，qRT-PCR法检测细胞中miR-335-5p表达，蛋白印迹法检测Tnfrsf1a蛋白表达。将OA软骨细胞分为anti-miR-NC组、anti-miR-335-5p组、anti-miR-335-5p+si-NC组和anti-miR-335-5p+si-Tnfrsf1a组，MTT法、流式细胞术及蛋白印迹法分别检测细胞增殖、凋亡及p53和Survivin蛋白表达。双荧光素酶报告基因检测实验miR-335-5p与Tnfrsf1a靶向关系。2组间比较行成组t检验；多组间比较行单因素方差分析，组间两两比较采用LSD-t检验。结果与正常软骨细胞比较，OA软骨细胞中miR-335-5p表达降低(0.31±0.08 vs. 1.00±0.00，t=25.875，P < 0.05)，Tnfrsf1a蛋白表达升高(0.95±0.18 vs. 0.18±0.03，t=12.659，P < 0.05)。与anti-miR-NC组比较，anti-miR-335-5p组OA软骨细胞OD值降低(0.27±0.01 vs. 0.34±0.01，t=14.849，P < 0.05)，凋亡率升高[(19.18±0.88)% vs. (6.89±0.33)%，t=39.230，P < 0.05], p53蛋白表达显著升高(P < 0.05)，Survivin蛋白表达显著降低(P < 0.05)。miR-335-5p靶向负调控Tnfrsf1a表达。与miR-335-5p+si-NC组比较，miR-335-5p+si-Tnfrsf1a组OA软骨细胞OD值升高，凋亡率降低，p53蛋白表达显著降低，Survivin蛋白表达显著升高(P < 0.05)。结论敲减miR-335-5p可靶向Tnfrsf1a抑制OA软骨细胞增殖并促进其凋亡，miR-335-5p有可能成为OA治疗的新靶点。
- miR-335-5p /
- Tnfrsf1a /
- 骨关节炎 /
- 增殖 /
- 凋亡
Abstract: Objective To explore the effect of miR-335-5p on the proliferation and apoptosis of osteoarthritis (OA) chondrocytes. Methods OA chondrocytes and normal chondrocytes were isolated and cultured in vitro. The expression of miR-335-5p in the cells was detected by qRT-PCR, and the expression of Tnfrsf1a protein was detected by western blotting. OA chondrocytes were divided into anti-miR-NC group, anti-miR-335-5p group, anti-miR-335-5p+pcDNA group, and anti-miR-335-5p+pcDNA-Tnfrsf1a group. MTT, flow cytometry and western blotting were used to detect proliferation, apoptosis and the protein expression of p53 and Survivin, respectively. Dual luciferase reporter assay verified the targeted regulation relationship between miR-335-5p and Tnfrsf1a. The comparison between the two groups was performed by independent sample t test, the comparison between multiple groups was performed by one-way analysis of variance, and the further comparison between the two groups was performed by LSD-t test. Results Compared with normal chondrocyte, the expression of miR-335-5p in OA chondrocytes reduced (0.31±0.08 vs. 1.00±0.00, t=25.875, P < 0.05), while the expression of Tnfrsf1a miRNA (6.54±0.30 vs. 1.00±0.00, t=55.400, P < 0.05) and protein (0.95±0.18 vs. 0.18±0.03, t=12.659, P < 0.05) increased. Compared with the anti-miR-NC group, the OD value of OA chondrocytes in the anti-miR-335-5p group decreased (0.27±0.01 vs. 0.34±0.01, t=14.849, P < 0.05), while the apoptosis rate increased [(19.18±0.88) % vs. (6.89±0.33) %, t=39.230, P < 0.05], and the expression of p53 protein increased (P < 0.05), but the expression of Survivin protein decreased (P < 0.05). miR-335-5p targeted and negatively regulated the expression of Tnfrsf1a. Compared with the miR-335-5p + si-NC group, the OD value of OA chondrocytes in the miR-335-5p + si-Tnfrsf1a group increased, while the apoptosis rate was decreased, and the expression of p53 protein decreased (P < 0.05), but the expression of Survivin protein increased (P < 0.05). Conclusion Knockdown of miR-335-5p could inhibit the proliferation and promote apoptosis of OA chondrocyte cells by negatively regulating Tnfrsf1a, and it may become a new target for OA treatment.
- MiR-335-5p /
- Tnfrsf1a /
- Osteoarthritis /
- Proliferation /
- Apoptosis

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图 1 Tnfrsf1a在OA软骨细胞中的表达

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图 2 敲减miR-335-5p对OA软骨细胞中p53、Survivin蛋白表达的影响

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图 3 miR-335-5p与Tnfrsf1a的靶向结合位点

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图 4 miR-335-5p对Tnfrsf1a蛋白表达的影响

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图 5 敲减Tnfrsf1a的软骨细胞中Tnfrsf1a、p53、Survivin的蛋白表达

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表 1 miR-335-5p、Tnfrsf1a在OA软骨细胞中的表达(x ±s)

组别	例数	miR-335-5p	Tnfrsf1a蛋白
正常软骨细胞	5	1.00±0.00	0.18±0.03
OA软骨细胞	10	0.31±0.08	0.95±0.18
t值		18.926	9.329
P值		< 0.001	< 0.001

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表 2 敲减miR-335-5p对OA软骨细胞增殖、凋亡的影响(x ±s)

组别	次数	增殖(OD₄₉₀)			凋亡率(%)	p53蛋白	Survivin蛋白
组别	次数	24 h	48 h	72 h	凋亡率(%)	p53蛋白	Survivin蛋白
ant-miR-NC	9	0.21±0.02	0.34±0.01	0.52±0.01	6.89±0.33	0.28±0.02	0.98±0.11
ant-miR-335-5p	9	0.22±0.02	0.27±0.01	0.36±0.03	19.18±0.88	0.95±0.09	0.21±0.02
t值		1.061	14.849	15.179	39.230	21.802	20.661
P值		0.305	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
注：增殖(OD490)组别与时间存在交互作用(F_时间=81.584, F_组别=36.832，F_交互=38.449，均P < 0.001)。

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表 3 双荧光素酶报告基因检测实验结果(x ±s)

组别	次数	WT-Tnfrsf1a	MUT-Tnfrsf1a
miR-NC	9	1.00±0.01	1.00±0.10
miR-335-5p	9	0.42±0.03	0.98±0.09
t值		55.024	0.446
P值		< 0.001	0.662

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表 4 miR-335-5p对Tnfrsf1a蛋白表达的影响(x ±s)

组别	次数	Tnfrsf1a蛋白
anti-miR-NC	9	1.11±0.01
anti-miR-335-5p	9	2.30±0.43^a
miR-NC	9	1.01±0.01
miR-335-5p	9	0.31±0.02^b
F值		132.296
P值		< 0.001
注：与anti-miR-NC组比较，^aP < 0.05；与miR-NC组比较，^bP < 0.05。

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表 5 敲减Tnfrsf1a逆转敲减miR-335-5p对OA软骨细胞增殖、凋亡的影响(x ±s)

组别	次数	Tnfrsf1a	增殖(OD₄₉₀)			凋亡率(%)	p53蛋白	Survivin蛋白
组别	次数	Tnfrsf1a	24 h	48 h	72 h	凋亡率(%)	p53蛋白	Survivin蛋白
anti-miR-335-5p	9	2.35±0.14	0.25±0.03	0.36±0.04	0.55±0.06	21.01±2.10	0.93±0.11	0.20±0.02
anti-miR-335-5p+si-NC	9	2.33±0.13	0.24±0.02	0.35±0.04	0.54±0.05	20.11±2.01	0.92±0.13	0.22±0.02
anti-miR-335-5p+si-Tnfrsf1a	9	1.18±0.11^ab	0.27±0.03	0.48±0.05^ab	0.72±0.07^ab	8.94±0.89^ab	0.35±0.04^ab	1.18±0.12^ab
F值		249.241	2.864	24.790	12.846	132.078	97.265	557.289
P值		< 0.001	0.077	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
注：与anti-miR-335-5p组比较，^aP < 0.05；anti-miR-335-5p+si-NC组比较，^bP < 0.05。增殖(OD490)组别与时间存在交互作用(F_时间=108.297, F_组别=26.667，F_交互=33.875，均P < 0.001)。

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