miR-20a-5p靶向KDM6B对骨肉瘤细胞增殖、迁移和侵袭能力的影响

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

摘要/Abstract

摘要：

目的探讨miR-20a-5p、赖氨酸脱甲基酶6B(KDM6B)在骨肉瘤组织中的表达及miR-20a-5p靶向KDM6B对骨肉瘤细胞增殖、迁移、侵袭及肿瘤生长的影响。方法收集2017年1月至2019年3月在中国医科大学附属第一医院接受治疗的20例骨肉瘤患者的临床病理标本和癌旁组织,采用实时定量PCR(qRT-PCR)检测组织中miR-20a-5p及KDM6B mRNA表达水平。将骨肉瘤MG63细胞分为空白对照组、mimic NC组、miR-20a-5p mimic组和NC+空载体组、miR-20a-5p+空载体组、miR-20a-5p+KDM6B组,采用qRT-PCR检测各组细胞miR-20a-5p和KDM6B mRNA表达水平,蛋白质印迹法检测KDM6B蛋白的表达水平,CCK-8法、细胞划痕实验和Transwell侵袭实验检测细胞增殖、迁移和侵袭能力。按照随机数字表法将裸鼠分为NC+空载体组、miR-20a-5p+空载体组和miR-20a-5p+KDM6B组,每组各5只,采用裸鼠移植瘤模型检测肿瘤细胞的生长能力。结果骨肉瘤组织中miR-20a-5p mRNA相对表达量为0.55±0.27,癌旁组织为1.22±0.28,差异具有统计学意义(t=7.701,P<0.001);骨肉瘤组织中KDM6B mRNA相对表达量为1.66±0.19,癌旁组织为1.00±0.15,差异具有统计学意义(t=12.219,P<0.001)。转染miR-20a-5p后,KDM6B mRNA和蛋白表达水平均随miR-20a-5p表达水平的升高而降低。转染miR-20a-5p 48 h后,空白对照组、mimic NC组、miR-20a-5p mimic组细胞增殖能力分别为0.83±0.04、0.81±0.03、0.52±0.01(F=89.655,P<0.001),与空白对照组、mimic NC组相比,miR-20a-5p mimic组均受到明显抑制(均P<0.001);NC+空载体组、miR-20a-5p+空载体组、miR-20a-5p+KDM6B组细胞增殖能力分别为0.83±0.05、0.52±0.01、0.67±0.05(F=43.919,P<0.001),与NC+空载体组相比,miR-20a-5p+空载体组受到明显抑制(P<0.001),与miR-20a-5p+空载体组相比,miR-20a-5p+KDM6B组显著增强(P<0.001)。空白对照组、mimic NC组、miR-20a-5p mimic组划痕愈合率分别为(32.51±2.73)%、(30.26±3.22)%、(13.52±1.77)%(F=46.314,P<0.001),与空白对照组、mimic NC组相比,miR-20a-5p mimic组均显著降低(均P<0.001);NC+空载体组、miR-20a-5p+空载体组、miR-20a-5p+KDM6B组划痕愈合率分别为(31.34±3.11)%、(12.15±1.64)%、(28.93±2.89)%(F=47.511,P<0.001),与NC+空载体组相比,miR-20a-5p+空载体组显著降低(P<0.001),与miR-20a-5p+空载体组相比,miR-20a-5p+KDM6B组显著增强(P=0.001)。空白对照组、mimic NC组、miR-20a-5p mimic组穿膜细胞数分别为114±16、108±11和42±6(F=36.282,P<0.001),与空白对照组、mimic NC组相比,miR-20a-5p mimic组均显著减少(均P<0.001);NC+空载体组、miR-20a-5p+空载体组、miR-20a-5p+KDM6B组穿膜细胞数分别为143±11、39±4、139±12(F=112.120,P<0.001),与NC+空载体组相比,miR-20a-5p+空载体组显著减少(P<0.001),与miR-20a-5p+空载体组相比,miR-20a-5p+KDM6B组显著增多(P<0.001)。NC+空载体组、miR-20a-5p+空载体组、miR-20a-5p+KDM6B组小鼠第21天肿瘤体积分别为(1 667.50±250.40)mm³、(129.20±21.00)mm³、(775.41±77.51)mm³,差异具有统计学意义(F=77.651,P<0.001);3组小鼠肿瘤重量分别为(1.35±0.18)g、(0.12±0.01)g、(0.61±0.03)g,差异具有统计学意义(F=104.191,P<0.001)。结论 miR-20a-5p在骨肉瘤组织中表达显著下降,KDM6B在骨肉瘤组织中表达显著升高,过表达miR-20a-5p可能通过靶向降低KDM6B的表达来抑制骨肉瘤细胞的增殖、迁移、侵袭及肿瘤生长能力。

关键词: 骨肉瘤, 细胞运动, 肿瘤浸润, 微RNAs, 赖氨酸脱甲基酶6B

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

李炳亮, 杨娅, 黄英丽, 司文, 李兴伟, 张元民, 卞继超, 陈语. miR-20a-5p靶向KDM6B对骨肉瘤细胞增殖、迁移和侵袭能力的影响[J]. 国际肿瘤学杂志, 2021, 48(2): 65-73.

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.

图/表 14

表1

表2

表3

图1

图2

图3

图4

表4

表5

图5

图6

图7

图8

图9

参考文献 12

[1]	Kobayashi E, Hornicek FJ, Duan Z. MicroRNA involvement in osteosarcoma[J]. Sarcoma, 2012,2012:359739. DOI: 10.1155/2012/359739. doi: 10.1155/2012/359739 pmid: 22550419
[2]	Liao SA, Guan J, Mo H, et al. lncRNA LSINCT5 regulates miR-20a-5p/XIAP to inhibit the growth and metastasis of osteosarcoma cells[J]. Onco Targets Ther, 2020,13:8209-8221. DOI: 10.2147/OTT.S251843. doi: 10.2147/OTT.S251843 pmid: 32884299
[3]	Lan F, Bayliss PE, Rinn JL, et al. A histone H3 lysine 27 demethylase regulates animal posterior development[J]. Nature, 2007,449(7163):689-694. DOI: 10.1038/nature06192. doi: 10.1038/nature06192 pmid: 17851529
[4]	Kruidenier L, Chung CW, Cheng Z, et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response[J]. Nature, 2012,488(7411):404-408. DOI: 10.1038/nature11262. doi: 10.1038/nature11262
[5]	Wang DY, Wu YN, Huang JQ, et al. Hippo/YAP signaling pathway is involved in osteosarcoma chemoresistance[J]. Chin J Cancer, 2016,35:47. DOI: 10.1186/s40880-016-0109-z. doi: 10.1186/s40880-016-0109-z pmid: 27206784
[6]	Wang J, Liu S, Shi J, et al. The role of miRNA in the diagnosis, prognosis, and treatment of osteosarcoma[J]. Cancer Biother Radio-pharm, 2019,34(10):605-613. DOI: 10.1089/cbr.2019.2939.
[7]	Yu Y, Zhang J, Jin Y, et al. MiR-20a-5p suppresses tumor proliferation by targeting autophagy-related gene 7 in neuroblastoma[J]. Cancer Cell Int, 2018,18:5. DOI: 10.1186/s12935-017-0499-2. doi: 10.1186/s12935-017-0499-2 pmid: 29311760
[8]	Huang Y, Yang N. MicroRNA-20a-5p inhibits epithelial to mesenchymal transition and invasion of endometrial cancer cells by targeting STAT3[J]. Int J Clin Exp Pathol, 2018,11(12):5715-5724. pmid: 31949657
[9]	Han J, Hu J, Sun F, et al. MicroRNA-20a-5p suppresses tumor angiogenesis of non-small cell lung cancer through RRM2-mediated PI3K/Akt signaling pathway[J]. Mol Cell Biochem, 2020. DOI: 10.1007/s11010-020-03936-y. doi: 10.1007/s11010-021-04098-1 pmid: 33611674
[10]	Li H, Zhang K, Liu LH, et al. MicroRNA screening identifies circulating microRNAs as potential biomarkers for osteosarcoma[J]. Oncol Lett, 2015,10(3):1662-1668. DOI: 10.3892/ol.2015.3378. doi: 10.3892/ol.2015.3378 pmid: 26622728
[11]	Liang S, Yao Q, Wei D, et al. KDM6B promotes ovarian cancer cell migration and invasion by induced transforming growth factor-beta1 expression[J]. J Cell Biochem, 2019,120(1):493-506. DOI: 10.1002/jcb.27405. doi: 10.1002/jcb.27405 pmid: 30277596
[12]	Sui A, Xu Y, Yang J, et al. The histone H3 Lys 27 demethylase KDM6B promotes migration and invasion of glioma cells partly by regulating the expression of SNAI1[J]. Neurochem Int, 2019,124:123-129. DOI: 10.1016/j.neuint.2019.01.006. doi: 10.1016/j.neuint.2019.01.006 pmid: 30633952

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

[1]	王培鑫, 赵军, 徐世红, 姜朝阳, 王小强, 杨红娟. 铁死亡相关机制在骨肉瘤中的应用进展[J]. 国际肿瘤学杂志, 2024, 51(5): 308-311.
[2]	王子豪, 王宇, 杨鑫, 何艺, 莫兴奎, 袁涛. 铁死亡在骨肉瘤中的分子机制及相关治疗的研究进展[J]. 国际肿瘤学杂志, 2024, 51(4): 239-244.
[3]	龚艳, 陈洪雷. 微RNA调控卵巢癌顺铂耐药的机制研究进展[J]. 国际肿瘤学杂志, 2024, 51(3): 186-190.
[4]	向玉玲, 谭佳杰, 熊远果, 赵丽蓉, 黎晨, 张洪. 脱水淫羊藿素对肝癌细胞增殖、迁移和凋亡的影响[J]. 国际肿瘤学杂志, 2023, 50(9): 513-519.
[5]	全祯豪, 徐飞鹏, 黄哲, 黄先进, 陈日红, 孙开裕, 胡旭, 林琳. 沉默lncRNA FTX通过miR-22-3p/NLRP3炎症体通路抑制胃癌细胞增殖[J]. 国际肿瘤学杂志, 2023, 50(4): 202-207.
[6]	马小平, 常君丽, 孙星媛, 杨燕萍. 长非编码RNA调控骨肉瘤耐药机制的研究进展[J]. 国际肿瘤学杂志, 2023, 50(1): 51-54.
[7]	周仁邦, 张仲传, 许志远, 朱勋兵. miR-219a-5p通过负调控HMGA2抑制骨肉瘤U2OS细胞增殖、侵袭和迁移[J]. 国际肿瘤学杂志, 2022, 49(4): 193-198.
[8]	石雨, 陈曦, 许梦琪, 张滢滢, 冀洪海, 蒋英英. 沉默PSIP1基因对口腔鳞状细胞癌细胞迁移及侵袭的影响[J]. 国际肿瘤学杂志, 2022, 49(3): 129-133.
[9]	来比江·吾斯曼, 曹博威, 张文斌, 高华. 外源性AGR2对结肠癌细胞增殖及侵袭能力的影响[J]. 国际肿瘤学杂志, 2022, 49(2): 73-78.
[10]	金嘉会, 陈存海, 马学真. 放射相关miRNA在乳腺癌放疗中的作用[J]. 国际肿瘤学杂志, 2022, 49(12): 735-738.
[11]	井文君, 赵文文, 冯青青, 赵文飞, 赵丽丽, 张雪, 魏红梅. miR-34家族用于胃癌治疗的分子基础及临床前景[J]. 国际肿瘤学杂志, 2022, 49(11): 681-686.
[12]	罗丽云, 赖灿辉, 梁仁佩, 杨爱武, 林志敏. 晚期胃癌中miR-524-5p与SOX9表达的相关性及其对化疗疗效和预后的影响[J]. 国际肿瘤学杂志, 2022, 49(1): 45-50.
[13]	刘佩, 蒲嘉泽, 黄雯, 汪斐. 吉非替尼敏感与耐药NSCLC患者miR-200c、miR-19a、miR-155表达差异及其对患者预后的影响[J]. 国际肿瘤学杂志, 2021, 48(7): 409-414.
[14]	王阳, 刘芊, 龙辉, 吴清明. 结直肠癌标志物粪便检测的研究现状[J]. 国际肿瘤学杂志, 2021, 48(7): 441-444.
[15]	蒋梦婷, 王家淳, 宗静. 抑制NFAT5对肺腺癌细胞增殖、侵袭、迁移及凋亡能力的影响[J]. 国际肿瘤学杂志, 2021, 48(6): 321-327.