miR-146b-5p对骨肉瘤细胞增殖抑制及克隆形成的机制研究

Study on the Mechanism of miR-146b-5p on Proliferation Inhibition and Clonogenesis of Osteosarcoma Cells

目的检测骨肉瘤组织中miR-146b-5p表达,探究其对骨肉瘤(osteosarcoma,OS)细胞增殖、克隆形成的影响及潜在分子作用机制。方法选取2018年1月~2020年12月内蒙古自治区巴彦淖尔市医院病理科30组临床骨肉瘤组织及邻近正常组织标本,采用实时荧光定量PCR实验(qRT-PCR)法检测组织中miR-146b-5p表达水平;通过介导转染miR-146b-5p mimics和miR-146b-5p ASO分别过表达和敲低miR-146b-5p表达,探究其对骨肉瘤细胞增殖及克隆形成的影响。通过生物学信息数据库预测miR-146b-5p的潜在靶基因,荧光素酶实验验证两者结合关系,分析miR-146b-5p对靶基因的调控作用。分析靶基因在骨肉瘤中的表达特征及与miR-146b-5p的相关性,通过细胞增殖实验验证两者的调控作用,以其探究在骨肉瘤中发挥功能的潜在分子机制。结果骨肉瘤组织中miR-146b-5p表达(4.096±1.237)显著低于邻近正常组织(5.895±0.834),差异有统计学意义(t=6.605,P<0.001)。miR-146b-5p过表达抑制了骨肉瘤细胞的增殖能力(t=13.233~34.599,均P<0.01)。miR-146b-5p过表达组克隆形成数目(48.912±2.032)较对照组(160.834±9.031)明显降低,差异有统计学意义(t=20.942,P<0.001)。DUSP16是miR-146b-5p的潜在靶基因,miR-146b-5p负向调控双特异性磷酸酶(dual specificity phosphatase 16,DUSP16)表达。骨肉瘤组织中DUSP16表达(5.683±0.457)较邻近正常组织(4.665±0.531)显著升高,差异有统计学意义(t=7.959,P<0.001),与miR-146b-5p表达呈负相关(r=-0.667,P<0.05)。共转染过表达DUSP16逆转了miR-146b-5p对骨肉瘤细胞增殖的抑制作用。miR-146b-5p过表达组P21水平(4.995±0.214)较对照组(1.001±0.002)显著升高,差异有统计学意义(t=32.325,P<0.001);当共转染DUSP16过表达后,P21表达(0.986±0.012)较单独过表达miR-146b-5p组(4.995±0.214)显著降低,差异有统计学意义(t=32.398,P<0.001)。结论骨肉瘤组织中miR-146b-5p显著低表达,其过表达抑制了骨肉瘤细胞的增殖及克隆形成;miR-146b-5p通过靶向负调控DUSP16表达参与P53信号通路从而在骨肉瘤发生发展起重要作用。

Objective The expression of miR-146b-5p in osteosarcoma tissues was detected to explore the effect of miR-146b-5p on proliferation and clonogenesis of osteosarcoma cells and its potential molecular mechanism.Methods Thirty clinical osteosarcoma tissue samples and adjacent normal tissue samples were selected in the Department of Pathology,Bayannur City Hospital,Inner Mongolia Autonomous Region from January 2018 to December 2020.The expression level of miR-146b-5p in the tissues was detected by qRT-PCR.The effects of miR-146b-5p mimics and miR-146b-5p ASO on the proliferation and clonal formation of osteosarcoma cells were investigated by mediating the transfection of miR-146b-5p mimics and miR-146b-5p ASO overexpression and knockdown,respectively.The potential target genes of miR-146b-5p were predicted through biological information database,and the binding relationship between the two was verified by luciferase assay,and the regulatory effect of miR-146b-5p on target genes was analyzed.The expression characteristics of target genes in osteosarcoma and their correlation with miR-146b-5p were analyzed.The expression characteristics of target genes in osteosarcoma and their correlation with miR-146b-5p were analyzed,and the regulatory effects of the two genes were verified by cell proliferation assay,so as to explore the underlying molecular mechanisms of function in osteosarcoma.Results The expression of miR-146b-5p in osteosarcoma tissues(4.096±1.237)was significantly lower than that in adjacent normal tissues(5.895±0.834),the difference was statistically significant(t=6.605,P<0.001).The overexpression of miR-146b-5p inhibited the proliferation of osteosarcoma cells(t=13.233~34.599,all P<0.01).The number of clone formation in miR-146b-5p overexpression group(48.912±2.032)was significantly lower than that in control group(160.834±9.031),the difference was statistically significant(t=20.942,P<0.001).DUSP16 was a potential target gene of miR-146b-5p,and miR-146b-5p negatively regulates the expression of DUSP16.The expression of DUSP16 in osteosarcoma tissues(5.683±0.457)was significantly higher than that in adjacent normal tissues(4.665±0.531),the difference was statistically significant(t=7.959,P<0.001),and was negatively correlated with the expression of miR-146b-5p(r=-0.667,P<0.05).The co-transfection and overexpression of DUSP16 reversed the inhibitory effect of miR-146b-5p on the proliferation of osteosarcoma cells.The P21 level of miR-146b-5p overexpression group(4.995±0.214)was significantly higher than that of the control group(1.001±0.002),the difference was statistically significant(t=32.325,P<0.001).After co-transfection with DUSP16 overexpression,the expression of P21(0.986±0.012)was significantly lower than that of miR-146b-5p alone(4.995±0.214),the difference was statistically significant(t=32.398,P<0.001).Conclusion The expression of miR-146b-5p was significantly lower in osteosarcoma tissues,and its overexpression inhibited the proliferation and clonal formation of osteosarcoma cells.MiR-146b-5p plays an important role in the development of osteosarcoma by negatively regulating the expression of DUSP16 and participating in the P53 signaling pathway.