miR-106a负调控PTEN促进骨肉瘤细胞增殖并抑制凋亡

目的:研究miR-106a在骨肉瘤组织和MG-63细胞中的表达水平及其对MG-63细胞增殖和凋亡的影响及机制。方法:用荧光实时定量PCR法检测20对骨肉瘤和相邻正常组织及MG-63和成骨细胞hFOB 1.19中miR-106a的表达。用miR-106a mimics、miR-106a antagomir 及两者相应的对照物转染MG-63细胞，然后分别用CCK-8法检测四组细胞增殖活性和FCM法检测细胞凋亡率。miR-106a mimics和mimics control与野生型或突变型PTEN 3'-UTR 重组载体共转染后，应用荧光素酶基因报告系统检测miR-106a是否与PTEN基因3'-UTR 结合。利用Western blot技术检测PTEN蛋白在上述四组转染MG-63细胞和骨肉瘤标本中的表达水平。结果:与相邻正常组织（1.19±0.15）相比，肿瘤组织miR-106a的表达水平（2.60±0.86）显著升高；同时，miR-106a在MG-63中的表达水平（2.60±0.92）明显高于hFOB 1.19（1.19±0.39），以上差异均有统计学意义（P＜0.05）。CCK-8和FCM检测结果显示，与mimics control组相比，miR-106a mimics组的增殖率明显增加，而细胞凋亡率下降；反之，miR-106a antagomir组与antagomir control组相比，增殖率前者低于后者，而凋亡率前者高于后者，上述差异均有统计学意义（P＜0.05）。荧光素酶报告实验显示，miR-106a mimics和wt PTEN 3'-UTR共转染组的荧光强度值明显低于mimics control和wt PTEN 3'-UTR组（P＜0.05）。Western blot发现，与对照组相比，miR-106a mimics组PTEN表达下调，而miR-106a antagomir表达上调；临床标本，肿瘤组织PTEN表达明显低于正常组织，差异均有统计学意义（P＜0.05）。结论:miR-106a在骨肉瘤组织及细胞中过表达，并靶向负调控PTEN表达，促进骨肉瘤细胞增殖并抑制其凋亡，从而发挥促癌作用。因此，miR-106a可为骨肉瘤的诊治提供新的潜在分子靶点。

miR-106a promoted osteosarcoma cell proliferation and inhibited apoptosis via negatively regulating the expression of PTEN

Objective:To study the expression level of miR-106a in osteosarcoma and MG-63 cells and its effect on proliferation and apoptosis of MG-63 cells and the underlying mechanism.Methods:The expression level of miR-106a in 20 pairs of osteosarcoma and adjacent normal tissues,MG-63 and osteoblasts hFOB 1.19 was detected by real-time quantitative PCR.MG-63 cells were transfected with miR-106a mimics,mimics control,miR-106a antagomir and antagomir control,respectively.Then the cell viability of four groups was detected by CCK-8 and their apoptosis rate was detected by FCM.After co-transfection of miR-106a mimics and mimics control with wild or mutant PTEN 3'-UTR recombinant vectors,the luciferase gene report system was used to detect whether miR-106a was combined with the 3'-UTR of gene PTEN.The expression level of PTEN protein in the four groups of MG-63 cells transfected and osteosarcoma samples was detected by Western blot.Results:Compared with the adjacent normal tissues （1.19±0.15）,the relative expression level of miR-106a in tumor tissues （2.60±0.86）was significantly higher.Meanwhile,the expression level of miR-106a in MG-63（2.60±0.92） was significantly higher than that of hFOB 1.19 （1.19±0.39）,and the above differences were statistically significant (P＜0.05).The results of CCK-8 and FCM assay showed that compared with the mimics control group,the proliferation rate of miR-106a mimics group increased significantly,while its apoptosis rate decreased.On the contrary,the proliferation rate in miR-106a antagomir groupwas obviously lower than that of antagomir control group,and its apoptosis rate was higher than that of the latter,and the all difference was statistically significant (P＜0.05).The fluorescence intensity of miR-106a mimics and wt PTEN 3'-UTR co-transfection group was significantly lower than that of mimics control and wt PTEN 3'-UTR group (P＜0.05).Western blot found that compared with the corresponding control group,the protein expression of PTEN in the miR-106a mimics group was significantly down-regulated,but the expression of PTEN in miR-106a antagomir was up-regulated,and the expression of PTEN in the tumor tissue was significantly lower than that of the adjacent normal tissue,and all difference was statistically significant (P＜0.05).Conclusion:miR-106a was overexpressed in the osteosarcoma tissues and MG-63 cells and negatively regulated the expression of PTEN,which promotes the proliferation of osteosarcoma cells and inhibits its apoptosis,and thus may play an important role in promotingtumorigenesis.Therefore,miR-106a likely becomes a new potential molecular target for the diagnosis and treatment of osteosarcoma.