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
计量
- 文章访问数: 145
- HTML全文浏览量: 111
- PDF下载量: 3
- 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

HTML全文

图 1 Tnfrsf1a在OA软骨细胞中的表达

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 3 miR-335-5p与Tnfrsf1a的靶向结合位点

下载: 全尺寸图片幻灯片

图 4 miR-335-5p对Tnfrsf1a蛋白表达的影响

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 导出CSV

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)。

下载: 导出CSV

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

下载: 导出CSV

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。

下载: 导出CSV

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)。

下载: 导出CSV

参考文献(26)

[1]	毛建杰, 徐兵, 王庆. 全膝关节置换术对老年膝骨关节炎患者的疗效及生活质量的影响[J]. 实用临床医药杂志, 2018, 22(1): 84-87. https://www.cnki.com.cn/Article/CJFDTOTAL-XYZL201801026.htm
[2]	YANG Y T, SHEN P Y, YAO T, et al. Novel role of circRSU1 in the progression of osteoarthritis by adjusting oxidative stress[J]. Theranostics, 2021, 11(4): 1877-1900. doi: 10.7150/thno.53307
[3]	MENG Y, QIU S Q, SUN L, et al. Knockdown of exosome-mediated lnc-PVT1 alleviates lipopolysaccharide-induced osteoarthritis progression by mediating the HMGB1/TLR4/NF-κB pathway via miR-93-5p[J]. Mol Med Rep, 2020, 22(6): 5313-5325. doi: 10.3892/mmr.2020.11594
[4]	GAO S T, YU Y M, WAN L P, et al. LncRNA GAS5 induces chondrocyte apoptosis by down-regulating miR-137[J]. Eur Rev Med Pharmacol Sci, 2020, 24(21): 10984-10991. http://www.researchgate.net/publication/347935600_LncRNA_GAS5_induces_chondrocyte_apoptosis_by_down-regulating_miR-137
[5]	万强, 王孝威, 原翔, 等. miRNA-9-5p通过靶向调控notch受体2对食管癌细胞增殖, 侵袭和迁移的影响研究[J]. 癌症进展, 2020, 18(2): 138-142, 171. https://www.cnki.com.cn/Article/CJFDTOTAL-AZJZ202002008.htm
[6]	魏君, 金悦, 徐少婷, 等. miR-9和miR-210在卵巢癌SKOV-3及SKOV-3/DDP细胞株中的表达及机制探讨[J]. 中华全科医学, 2018, 16(6): 938-942. https://www.cnki.com.cn/Article/CJFDTOTAL-SYQY201806025.htm
[7]	滕伟, 郑先杰, 巩贵宏, 等. 长链非编码RNA-linc00152可能通过miR-4767影响血管内皮细胞凋亡和迁移[J]. 中国临床医学, 2019, 26(2): 209-214. https://www.cnki.com.cn/Article/CJFDTOTAL-LCYX201902012.htm
[8]	何伟珍, 尹志华, 郭佩, 等. 骨关节炎患者关节液中miR-335-5p表达及意义[J]. 新医学, 2017, 48(1): 37-39. https://www.cnki.com.cn/Article/CJFDTOTAL-XYXX201701008.htm
[9]	王洁, 卞莹莹, 张川, 等. 肿瘤坏死因子及其受体在类风湿性关节炎中的研究进展[J]. 药学实践杂志, 2017, 35(4): 289-293. doi: 10.3969/j.issn.1006-0111.2017.04.001
[10]	魏钰, 魏民. 人骨关节炎软骨细胞的体外分离与培养[J]. 中国组织工程研究, 2019, 23(25): 4056-4061. doi: 10.3969/j.issn.2095-4344.1791
[11]	XIAO P, ZHU X, SUN J P, et al. LncRNA NEAT1 regulates chondrocyte proliferation and apoptosis via targeting miR-543/PLA2G4A axis[J]. Hum Cell, 2021, 34(1): 60-75. doi: 10.1007/s13577-020-00433-8
[12]	付鑫, 何锦泉, 马信龙. 神经生长因子在骨关节炎疼痛中的研究进展[J]. 中国矫形外科杂志, 2020, 28(1): 58-61. https://www.cnki.com.cn/Article/CJFDTOTAL-ZJXS202001016.htm
[13]	LU J F, QI L G, ZHU X B, et al. LncRNA RMRP knockdown promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-206/CDK9 axis[J]. Pharmazie, 2020, 75(10): 500-504. http://www.ingentaconnect.com/contentone/govi/pharmaz/2020/00000075/00000010/art00009
[14]	WU Y Z, HONG Z H, XU W B, et al. Circular RNA circPDE4D protects against osteoarthritis by binding to mir-103a-3p and regulating FGF18[J]. Mol Ther, 2021, 29(1): 308-323. doi: 10.1016/j.ymthe.2020.09.002
[15]	LI Z C, WANG J, YANG J. TUG1 knockdown promoted viability and inhibited apoptosis and cartilage ECM degradation in chondrocytes via the miR-17-5p/FUT1 pathway in osteoarthritis[J]. Exp Ther Med, 2020, 20(6): 154. doi: 10.3892/etm.2020.9283
[16]	LU J J, ZHOU Z B, SUN B, et al. MiR-520d-5p modulates chondrogenesis and chondrocyte metabolism through targeting HDAC1[J]. Aging (Albany NY), 2020, 12(18): 18545-18560. http://www.researchgate.net/publication/344332089_MiR-520d-5p_modulates_chondrogenesis_and_chondrocyte_metabolism_through_targeting_HDAC1
[17]	ZHANG Y, XIA Q M, LIN J. LncRNA H19 attenuates apoptosis in MPTP-induced Parkinson's disease through regulating miR-585-3p/PIK3R3[J]. Neurochem Res, 2020, 45(7): 1700-1710. doi: 10.1007/s11064-020-03035-w
[18]	LU X K, LI Y, CHEN H M, et al. miR-335-5P contributes to human osteoarthritis by targeting HBP1[J]. Exp Ther Med, 2021, 21(2): 109. http://www.researchgate.net/publication/347198460_miR-335-5P_contributes_to_human_osteoarthritis_by_targeting_HBP1
[19]	LU J F, QI L G, ZHU X B, et al. LncRNA RMRP knockdown promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-206/CDK9 axis[J]. Pharmazie, 2020, 75(10): 500-504. http://www.ingentaconnect.com/contentone/govi/pharmaz/2020/00000075/00000010/art00009
[20]	GAO S T, YU Y M, WAN L P, et al. LncRNA GAS5 induces chondrocyte apoptosis by down-regulating miR-137[J]. Eur Rev Med Pharmacol Sci, 2020, 24(21): 10984-10991. http://www.researchgate.net/publication/347935600_LncRNA_GAS5_induces_chondrocyte_apoptosis_by_down-regulating_miR-137
[21]	LI H W, LIU J. Circ_0009910 promotes proliferation and metastasis of hepatocellular carcinoma cells through miR-335-5p/ROCK1 axis[J]. Eur Rev Med Pharmacol Sci, 2020, 24(4): 1725-1735. http://www.ncbi.nlm.nih.gov/pubmed/32141540
[22]	霍佳莉, 任翔, 李昆鑫, 等. miR-335-5p/ADCY3诱导的再生障碍性贫血患者免疫异常的机制研究[J]. 中国实验血液学杂志, 2020, 28(3): 909-917. https://www.cnki.com.cn/Article/CJFDTOTAL-XYSY202003037.htm
[23]	何伟珍, 尹志华, 徐晓东, 等. 骨关节炎患者外周血单个核细胞miR-335-5p表达及其意义[J]. 新医学, 2017, 48(5): 323-325. doi: 10.3969/j.issn.0253-9802.2017.05.008
[24]	NAGAHAMA A, YASHIRO M, KAWASHIMA T, et al. Combination of p53 and Ki67 as a promising predictor of postoperative recurrence of meningioma[J]. Anticancer Res, 2021, 41(1): 203-210. doi: 10.21873/anticanres.14766
[25]	HAN L C, WANG H, NIU F L, et al. Effect miR-214-3p on proliferation and apoptosis of breast cancer cells by targeting survivin protein[J]. Eur Rev Med Pharmacol Sci, 2019, 23(17): 7469-7474. http://www.ncbi.nlm.nih.gov/pubmed/31539134
[26]	田佳音, 陈康勇, 张畅, 等. 斑马鱼肿瘤坏死因子(tnfα)及其受体(tnfrsf1a)应答细菌和病毒感染的表达[J]. 上海海洋大学学报, 2020, 29(2): 171-179. https://www.cnki.com.cn/Article/CJFDTOTAL-SSDB202002002.htm