miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制

doi:10.11958/20240086

天津医药 ›› 2024, Vol. 52 ›› Issue (8): 785-790.doi: 10.11958/20240086

• 细胞与分子生物学 • 下一篇

miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制

方杰¹(), 黄芮¹, 郑红慧¹^,², 贾倩倩¹, 鲍静²^,^△()

1 中国科学院合肥肿瘤医院血液科（邮编230031）
2 安徽医科大学第一附属医院血液内科

收稿日期:2024-01-12 修回日期:2024-04-11 出版日期:2024-08-15 发布日期:2024-08-16
通讯作者: △E-mail：baojing@ahmu.edu.cn
作者简介:方杰（1986），男，主治医师，主要从事血液肿瘤方面研究。E-mail：fangjieajie@163.com
基金资助:
安徽省自然科学基金资助项目(2008085MH296);安徽省教育厅自然科学研究项目(2022AH051178)

miR-9-5p-induced autophagy and apoptosis in multiple myeloma cells by targeting TIMP2

FANG Jie¹(), HUANG Rui¹, ZHENG Honghui¹^,², JIA Qianqian¹, BAO Jing²^,^△()

1 Department of Hematology, Hefei Cancer Hospital, Chinese Academy of Sciences, Hefei 230031, China
2 Department of Hematology, the First Affiliated Hospital of Anhui Medical University

Received:2024-01-12 Revised:2024-04-11 Published:2024-08-15 Online:2024-08-16
Contact: △E-mail：baojing@ahmu.edu.cn

方杰, 黄芮, 郑红慧, 贾倩倩, 鲍静. miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制[J]. 天津医药, 2024, 52(8): 785-790.

FANG Jie, HUANG Rui, ZHENG Honghui, JIA Qianqian, BAO Jing. miR-9-5p-induced autophagy and apoptosis in multiple myeloma cells by targeting TIMP2[J]. Tianjin Medical Journal, 2024, 52(8): 785-790.

摘要/Abstract

摘要：

目的探究miR-9-5p和组织金属蛋白酶抑制因子2（TIMP2）相互作用对多发性骨髓瘤（MM）细胞自噬和凋亡的影响机制。方法采用实时荧光定量PCR（qRT-PCR）检测初诊MM和复发MM各9例患者骨髓样本中miR-9-5p和TIMP2的表达水平，分析两者表达水平的相关性。U266细胞分为miR-对照组、miR-9-5p组、pcDNA3.1组、pcDNA3.1-TIMP2组、miR-9-5p+pcDNA3.1组、miR-9-5p+pcDNA3.1-TIMP2组。采用流式细胞术、免疫荧光染色、蛋白质印迹实验检测过表达miR-9-5p和TIMP2对U266细胞自噬和凋亡的影响；双萤光素酶报告实验验证miR-9-5p和TIMP2的靶向关系。结果与初诊MM患者相比，复发MM患者miR-9-5p表达水平升高，TIMP2表达降低；miR-9-5p和TIMP2表达水平呈负相关（P<0.05）。与miR-对照组相比，miR-9-5p组MAP1LC3B-Ⅱ的表达水平降低，MAP1LC3B-Ⅰ和SQSTM1的表达水平增加，细胞凋亡率降低（P<0.05）。与pcDNA3.1组相比，pcDNA3.1-TIMP2组MAP1LC3B-Ⅱ的表达水平升高，MAP1LC3B-Ⅰ和SQSTM1的表达水平降低，细胞凋亡率增加（P<0.05）。生物信息学和双萤光素酶报告实验证实TIMP2是miR-9-5p的靶基因。结论 miR-9-5p靶向TIMP2抑制MM细胞的自噬和凋亡，从而促进MM的发生发展。

关键词: 多发性骨髓瘤, 自噬, 细胞凋亡, 金属蛋白酶2组织抑制剂, miR-9-5p

Abstract:

Objective To investigate the mechanism of the interaction between miR-9-5p and tissue metalloproteinase inhibitor 2 (TIMP2) on autophagy and apoptosis in multiple myeloma (MM) cells. Methods Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect expression levels of miR-9-5p and TIMP2 in bone marrow samples of 9 patients with newly diagnosed MM and 9 patients with recurrent MM. The correlation of expression levels between the two were analyzed. U266 cells were divided into the miR-control group, the miR-9-5p group, the pcDNA3.1 group, the pcDNA3.1-TIMP2 group, the miR-9-5p+pcDNA3.1 group, and the miR-9-5p+pcDNA3.1-TIMP2 group. The effects of overexpressed miR-9-5p and TIMP2 on autophagy and apoptosis in U266 cells were detected by flow cytometry, immunofluorescence staining and Western blot experiments. The dual luciferase report experiment verified the interaction between miR-9-5p and TIMP2. Results Compared with newly diagnosed MM patients, the expression level of miR-9-5p was increased and the expression level of TIMP2 was decreased in patients with recurrent MM. The expression levels of miR-9-5p and TIMP2 were negatively correlated (P<0.05). Compared with the miR-control group, the miR-9-5p group showed a decrease in the expression level of MAP1LC3B-Ⅱ, an increase in expression levels of MAP1LC3B-Ⅰ and SQSTM1, and a decrease in cell apoptosis rate (P<0.05). Compared with the pcDNA3.1 group, the expression level of MAP1LC3B-Ⅱwas increased in the pcDNA3.1-TIMP2 group, while the expression levels of MAP1LC3B-Ⅰand SQSTM1 were decreased, and the apoptosis rate of cells increased (P<0.05). Bioinformatics and dual luciferase reporter experiments confirmed that TIMP2 was the target gene of miR-9-5p. Conclusion miR-9-5p inhibits autophagy and apoptosis in MM cells by targeting TIMP2, thereby promoting the occurrence and development of MM.

Key words: multiple myeloma, autophagy, apoptosis, tissue inhibitor of metalloproteinase-2, miR-9-5p

中图分类号:

R733.3

图/表 13

表1 引物序列

Tab.1 Primer sequence

基因名称	引物序列（5'→3'）	产物大小/bp
miR-9-5p	上游：GCCCGCTCTTTGGTTATCTAG	95
miR-9-5p	下游：CCAGTGCAGGGTCCGAGGT	95
TIMP2	上游：AGCACCACCCAGAAGAAGAG	183
TIMP2	下游：GTGACCCAGTCCATCCAGAG	183
U6	上游：GCTTCGGCAGCACATATACTAAAAT	113
U6	下游：CGCTTCACGAATTTGCGTGTCAT	113
GAPDH	上游：TGGAGAAACCTGCCAAGTATG	120
GAPDH	下游：GGAGACAACCTGGTCCTCAG	120

表2 miR-9-5p和TIMP2在多发性骨髓瘤不同细胞系中的表达水平比较（$\bar{x} \pm s$）

Tab.2 Comparison of expression levels of miR-9-5p and TIMP 2 between different cell lines of multiple myeloma

细胞系	n	miR-9-5p	TIMP2
GM50113	3	1.00±0.12	1.00±0.03
U266	3	3.58±0.24^a	0.50±0.05^a
NCI-H929	3	2.67±0.24^a	0.80±0.03^a
RPMI-8226	3	2.25±0.31^a	0.65±0.06^a
F		61.060^＊＊	69.304^＊＊

图1 过表达miR-9-5p对U266细胞自噬相关蛋白表达的影响 1：miR-对照组；2：miR-9-5p组。

Fig.1 Effects of overexpressed miR-9-5p on the expression of autophagy-related proteins in U266 cells

图2 过表达miR-9-5p对U266细胞凋亡的影响

Fig.2 Effect of overexpressed miR-9-5p on apoptosis in U266 cells

表3 U266细胞miR-对照组和miR-9-5p组中3种自噬相关蛋白的表达水平及凋亡率比较（n=6, $\bar{x} \pm s$）

Tab.3 Comparison of protein expression levels of three autophagy-related proteins and apoptosis ratio between the miR-control group and the miR-9-5p group of U266 cells

组别	MAP1LC3B-Ⅰ	MAP1LC3B-Ⅱ	SQSTM1	细胞凋亡率/%
miR-对照组	0.66±0.01	0.42±0.02	0.50±0.01	8.95±1.33
miR-9-5p组	0.86±0.03	0.35±0.03	0.67±0.01	5.91±0.33
t	10.955^＊＊	3.363^＊	20.821^＊＊	3.843^＊

图3 miR-9-5p与TIMP2之间的靶向结合位点及调控关系 1：miR-对照组；2：miR-9-5p组。

Fig.3 Targeted binding sites and regulatory relationships between miR-9-5p and TIMP2

表4 U266细胞miR-对照组和miR-9-5p组中TIMP2的mRNA和蛋白表达水平比较（$\bar{x} \pm s$）

Tab.4 Comparison of protein expression levels of TIMP2 between the miR-control group and the miR-9-5p group in U266 cells

组别	n	TIMP2 mRNA	TIMP2 蛋白
miR-对照组	3	1.01±0.01	1.01±0.03
miR-9-5p组	3	0.46±0.02	0.58±0.01
t		46.603^＊＊	23.552^＊＊

图4 过表达TIMP2后GFP的荧光表达情况绿色为GFP染色，蓝色为DAPI染色。

Fig.4 The fluorescence expression of GFP after overexpressed TIMP2

图5 U266细胞中过表达TIMP2后，MAP1LC3B-Ⅰ、MAP1LC3B-Ⅱ、SQSTM1蛋白表达电泳图 1：pcDNA3.1组；2：pcDNA3.1-TIMP2组。

Fig.5 Protein electrophoretogram of MAP1LC3B-I, MAP1LC3B-II and SQSTM1 proteins after overexpressed TIMP2 in U266 cells

表5 U266细胞中过表达TIMP2后，3种自噬相关蛋白的表达水平以及凋亡率比较（n=6，$\bar{x} \pm s$）

Tab.5 Comparison of protein expression levels of three autophagy-related proteins, and apoptosis ratio after overexpressed TIMP2 in U266 cells

组别	MAP1LC3B-Ⅰ	MAP1LC3B-Ⅱ
pcDNA3.1组	0.51±0.01	0.44±0.02
pcDNA3.1-TIMP2组	0.31±0.02	0.65±0.01
t	15.492^＊＊	16.267^＊＊
组别	SQSTM1	细胞凋亡率/%
pcDNA3.1组	0.33±0.03	4.97±0.52
pcDNA3.1-TIMP2组	0.12±0.03	19.61±3.23
t	8.573＊＊	7.751＊＊

图6 过表达TIMP2细胞凋亡的流式图

Fig.6 Flow diagram of cell apoptosis after overexpressed TIMP2

图7 U266细胞中过表达miR-9-5p和TIMP2，MAP1LC3B-Ⅰ、MAP1LC3B-Ⅱ、SQSTM1蛋白表达电泳图 1：miR-对照组；2：miR-9-5p组；3：pcDNA3.1-TIMP2组；4：pcDNA3.1组；5：miR-9-5p+pcDNA3.1-TIMP2组；6：miR-9-5p+pcDNA3.1组。

Fig.7 Protein electrophoretogram of MAP1LC3B-I, MAP1LC3B-II and SQSTM1 after overexpressed miR-9-5p and TIMP2 in U266 cells

表6 U266细胞中过表达miR-9-5p和TIMP2后3种自噬相关蛋白的表达水平以及凋亡率比较（n=3，$\bar{x} \pm s$）

Tab.6 Comparison of protein expression levels of three autophagy-related proteins and apoptosis ratio after overexpressed miR-9-5p and TIMP2

组别	MAP1LC3B-Ⅰ			MAP1LC3B-Ⅱ
miR-对照组	0.32±0.02			0.56±0.04
miR-9-5p组	0.53±0.01^a			0.39±0.02^a
pcDNA3.1组	0.59±0.01			0.36±0.03
pcDNA3.1-TIMP2组	0.35±0.03^b			0.60±0.05^b
miR-9-5p+pcDNA3.1组	0.58±0.04			0.39±0.02
miR-9-5p+pcDNA3.1-TIMP2组	0.36±0.04^c			0.60±0.04^c
F	59.550^＊＊			31.978^＊＊
组别		SQSTM1	细胞凋亡率/%
miR-对照组		0.22±0.02	8.40±0.98
miR-9-5p组		0.37±0.03^a	5.78±0.55^a
pcDNA3.1组		0.41±0.05	5.82±0.45
pcDNA3.1-TIMP2组		0.28±0.01^b	24.03±1.59^b
miR-9-5p+pcDNA3.1组		0.45±0.05	5.39±0.48
miR-9-5p+pcDNA3.1-TIMP2组		0.30±0.02^c	20.07±1.35^c
F		19.844^＊＊	203.821^＊＊

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miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制

miR-9-5p-induced autophagy and apoptosis in multiple myeloma cells by targeting TIMP2

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