MiR-129-5p靶向HMGB1抑制骨肉瘤细胞增殖和迁移

MiRNA-129-5p Targeting HMGB1 Inhibits Proliferation and Migration of Osteosarcoma Cells

目的探讨miR-129-5p对骨肉瘤(OS)细胞增殖和迁移的影响以及对HMGB1的调控作用。方法 RT-PCR和Western blot法分别检测骨肉瘤细胞株MG-63、Saos-2和成骨细胞hFOB1.19中miR-129-5p和HMGB1的表达。生物信息学预测miR-129-5p与HMGB1基因是否存在结合位点,双荧光素酶报告基因检测验证miR-129-5p和HMGB1的相互作用。将miR-129-5p模拟物和抑制物分别转染到miR-129-5p表达量较低和较高的骨肉瘤细胞株中,RT-PCR检测转染效率。转染成功后分别用CCK-8法、划痕实验和Transwell迁移实验检测OS细胞株的增殖和迁移能力;Western blot检测转染后OS细胞株中HMGB1的表达。结果 miR-129-5p在OS细胞中的表达低于正常成骨细胞(P<0.05),HMGB1在OS细胞株中的表达高于正常成骨细胞(P<0.05)。miR-129-5p和HMGB1基因存在结合位点,miR-129-5p mimic转染能下调HMGB1-WT质粒组的荧光素酶活性(P<0.05)。转染miR-129-5p模拟物可显著提高MG-63细胞中miR-129-5p的表达量(P<0.05),且可抑制MG-63细胞的增殖和迁移(P<0.05),同时降低HMGB1的表达水平;转染miR-129-5p抑制物后可显著降低Saos-2细胞中miR-129-5p的表达量(P<0.05),且可增强Saos-2细胞增殖和迁移能力(P<0.05),同时提高HMGB1表达水平。结论 miR-129-5p可能通过HMGB1基因抑制骨肉瘤细胞增殖和迁移。

Objective To investigate the effects of miR-129-5p on the proliferation and migration of osteosarcoma cells and the regulation of HMGB1 gene.Methods The expression of miR-129-5p and HMGB1 in osteosarcoma cell line MG-63,Saos-2 and osteoblast hFOB1.19 were detected by RT-PCR and Western blot.Bioinformatics methods were used to predict whether there were binding sites between mir-129-5p and HMGB1 gene.Double luciferase reporter gene system was used to verify the interaction between miR-129-5p and the target gene HMGB1.miR-129-5p mimic and inhibitor were transfected into osteosarcoma cell lines with low and high miR-129-5p expression,respectively,and the transfection efficiency was detected by RT-PCR.After successful transfection,the proliferation and migration of osteosarcoma cell lines were detected by CCK-8 assay,scratch assay and Transwell migration assay,respectively,and Western blot was used to detect the expression of HMGB1 in the transfected osteosarcoma cell lines.Results Expression of miR-129-5p in osteosarcoma cells was lower than that in normal osteoblasts (P<0.05),and the expression of HMGB1 in osteosarcoma cell lines was higher than that in normal osteoblasts (P<0.05).There were binding sites between miR-129-5p and HMGB1 genes,and the luciferase activity of HMGB1-WT plasmid group was down-regulated after transfection with miR-129-5p mimic (P<0.05).Transfection of miR-129-5p mimic significantly increased the expression of miR-129-5p in MG-63 cells (P<0.05),inhibited the proliferation and migration of MG-63 cells (P<0.05),and decreased the expression level of HMGB1.After transfection with miR-129-5p inhibitor,the expression of miR-129-5p in Saos-2 cells was significantly decreased (P<0.05),the proliferation and migration abilities of Saos-2 cells were enhanced (P<0.05),and the expression level of HMGB1 was also increased.Conclusion miR-129-5p may inhibit the proliferation and migration of osteosarcoma cells through HMGB1 gene.