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

doi:10.11958/20240086

Abstract

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

CLC Number:

R733.3

Figures/Tables 13

References 17

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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

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

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^＊＊

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^＊＊

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^＊