Effects of miR-20a-5p targeting KDM6B on the proliferation, migration and invasion of osteosarcoma cells

doi:10.3760/cma.j.cn371439-20201105-00013

Abstract

Abstract:

Objective To investigate the expressions of miR-20a-5p and lysine (K) demethylase 6B (KDM6B) in osteosarcoma tissues and the effects of miR-20a-5p targeting KDM6B on the proliferation, migration and invasion of osteosarcoma cells and tumor growth. Methods The clinicopathological and paracancerous tissues of 20 patients with osteosarcoma admitted to the First Affiliated Hospital of Chinese Medical University from January 2017 to March 2019 were collected. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miR-20a-5p and KDM6B mRNA in tissues. The osteosarcoma MG63 cells were divided into control group, mimic NC group, miR-20a-5p mimic group, and NC+empty vector group, miR-20a-5p+empty vector group, miR-20a-5p+KDM6B group. The expression levels of miR-20a-5p and KDM6B mRNA of all groups were detected by qRT-PCR. Western blotting was used to detect the expression level of KDM6B. CCK-8 assay, cell scratch test and Transwell test were used to detect cell proliferation, migration and invasion ability. According to the random number table method, nude mice were divided into NC+empty vector group, miR-20a-5p+empty vector group and miR-20a-5p+KDM6B group, with 5 mice in each group. Tumor growth ability was detected by tumor xenograft nude mouse models. Results The relative expression level of miR-20a-5p mRNA in osteosarcoma tissues was 0.55±0.27, and that in paracancerous tissues was 1.22±0.28, with a statistically significant difference (t=7.701, P<0.001). The relative expression level of KDM6B mRNA in osteosarcoma tissues was 1.66±0.19, and that in paracancerous tissues was 1.00±0.15, with a statistically significant difference (t=12.219, P<0.001). After transfection of miR-20a-5p, KDM6B mRNA and protein expression levels decreased with the increase of miR-20a-5p expression level. After miR-20a-5p transfection for 48 h, the cell proliferation abilities of the blank control group, mimic NC group and miR-20a-5p mimic group were 0.83±0.04, 0.81±0.03 and 0.52±0.01 (F=89.655, P<0.001), compared with the blank control group and mimic NC group, the cell proliferation ability was significantly inhibited in the miR-20a-5p mimic group (both P<0.001). The cell proliferation abilities of NC+empty vector group, miR-20a-5p+empty vector group and miR-20a-5p+KDM6B group were 0.83±0.05, 0.52±0.01 and 0.67±0.05 (F=43.919, P<0.001), compared with the NC+empty vector group, the cell proliferation ability was significantly inhibited in the miR-20a-5p+empty vector group (P<0.001); compared with the miR-20a-5p+empty vector group, the cell proliferation ability of miR-20a-5p+KDM6B group increased significantly (P<0.001). The scratch healing rates of the blank control group, mimic NC group and miR-20a-5p mimic group were (32.51±2.73)%, (30.26±3.22)% and (13.52±1.77)% (F=46.314, P<0.001), compared with the control group and the mimic NC group, the scratch healing rate of the miR-20a-5p mimic group was significantly decreased (both P<0.001). The scratch healing rates of NC+empty vector group, miR-20a-5p+empty vector group and miR-20a-5p+KDM6B group were (31.34±3.11)%, (12.15±1.64)% and (28.93±2.89)% (F=47.511, P<0.001), compared with the NC+empty vector group, the scratch healing rate of the miR-20a-5p+empty vector group was significantly decreased (P<0.001); compared with the miR-20a-5p+empty vector group, the scratch healing rate of miR-20a-5p+KDM6B group was significantly increased (P=0.001). The numbers of transmembrane cells in the blank control group, mimic NC group and miR-20a-5p mimic group were 114±16, 108±11 and 42±6 (F=36.282, P<0.001), compared with the control group and mimic NC group, the number of transmembrane cells of the miR-20a-5p mimic group was significantly decreased (both P<0.001). The numbers of transmembrane cells in the NC+empty vector group, miR-20a-5p+empty vector group and miR-20a-5p+KDM6B group was 143±11, 39±4 and 139±12 (F=112.120, P<0.001), compared with the NC+empty vector group, the number of transmembrane cells of the miR-20a-5p+empty vector group was significantly decreased (P<0.001); compared with the miR-20a-5p+empty vector group, the number of transmembrane cells of the miR-20a-5p+KDM6B group was increased significantly (P<0.001). The tumor volumes of mice for 21 d in the NC+empty vector group, miR-20a-5p+empty vector group and miR-20a-5p+KDM6B group were (1 667.50±250.40) mm³, (129.20±21.00) mm³ and (775.41±77.51) mm³ respectively, with a statistically significant difference (F=77.651, P<0.001). The tumor weights of the 3 groups were (1.35±0.18) g, (0.12±0.01) g and (0.61±0.03) g respectively, with a statistically significant difference (F=104.191, P<0.001). Conclusion The expression of miR-20a-5p is significantly decreased in osteosarcoma tissues, and the expression of KDM6B is significantly increased in osteosarcoma tissues. Overexpression of miR-20a-5p may inhibit the proliferation, migration and invasion of osteosarcoma cells and tumor growth by targeting to reduce the expression of KDM6B.

Key words: Osteosarcoma, Cell movement, Neoplasm invasiveness, MicroRNAs, Lysine (K) demethylase 6B

Li Bingliang, Yang Ya, Huang Yingli, Si Wen, Li Xingwei, Zhang Yuanmin, Bian Jichao, Chen Yu. Effects of miR-20a-5p targeting KDM6B on the proliferation, migration and invasion of osteosarcoma cells[J]. Journal of International Oncology, 2021, 48(2): 65-73.

Figures/Tables 14

References 12

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基因名称	引物序列
miR-20a-5p	正向引物:5'-TAAAGTGCTTATAGTGCAGGTAG-3'
	反向引物:5'-CTACCTGCACTATAAGCACTTTA-3'
U6	正向引物:5'-GCTTCGGCAGCACATATACTAAAAT-3'
	反向引物:5'-CGCTTCACGAATTTGCGTGTCAT-3'
KDM6B	正向引物:5'-CTGGAGAGCAAACGGGATG-3'
	反向引物:5'-AGGGTCTTGGTGGAGAAGAGG-3'
GAPDH	正向引物:5'-CTCCTCTGACTTCAACAGCGACAC-3'
	反向引物:5'-TGCTGTAGCCAAATTCGTTGTCAT-3'

组别	miR-20a-5p	KDM6B
空白对照组	1.00±0.01	0.99±0.01
mimic NC组	0.97±0.05	0.98±0.03
miR-20a-5p mimic组	3.12±0.20^ab	0.25±0.06^ab
F值	325.708	380.471
P值	<0.001	<0.001

组别	0 h	24 h	48 h	72 h
空白对照组	0.21±0.02	0.49±0.02	0.83±0.04	1.42±0.08
mimic NC组	0.22±0.01	0.49±0.01	0.81±0.03	1.46±0.06
miR-20a-5p mimic组	0.22±0.01^ab	0.32±0.02^ab	0.52±0.01^ab	0.75±0.05^ab
F值	0.207	63.044	89.655	111.843
P值	0.818	<0.001	<0.001	<0.001

组别	0 h	24 h	48 h	72 h
NC+空载体组	0.21±0.01	0.49±0.01	0.83±0.05	1.44±0.09
miR-20a-5p+空载体组	0.22±0.01	0.33±0.02^a	0.52±0.01^a	0.75±0.05^a
miR-20a-5p+KDM6B组	0.22±0.01	0.45±0.03^b	0.67±0.05^b	1.05±0.12^b
F值	0.233	50.107	43.919	45.280
P值	0.799	<0.001	<0.001	<0.001

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