微小RNA-130b靶向调控PTEN表达及对骨肉瘤细胞上皮间质转化的影响

Targeted regulation of PTEN expression by microRNA-130b and its effect on epithelial mesenchymal transition of osteosarcoma cells

目的探讨微小RNA-130b(miR-130b)对第10号染色体缺失的磷酸酶及张力蛋白同源物基因(PTEN)的靶向调控及骨肉瘤(OS)MG-63细胞上皮间质转化(EMT)的影响。方法从美国国家生物技术信息中心的GEO数据库中下载基因表达谱OS样本数据集GSE39058、GSE79181的series matrix数据分别用于分析OS组织的miR-130b水平及与预后的关系。采用脂质体法将miR-130b抑制剂转染至MG-63(抑制组),同时设置未转染组和空白对照组,活细胞计数CCK-8和Annexin V-FITC/PI双染流式细胞术检测增殖活力和凋亡率,实时定量PCR和Western blotting检测PTEN及上皮表型标志物E-钙黏蛋白(E-cadherin)和间质表型标志物波形蛋白(Vimentin)和α-平滑肌肌动蛋白(α-SMA)的水平,在线预测miR-130b与PTEN的靶向结合序列并利用双荧光素酶报告基因实验验证两者的靶向关系。结果GSE39058数据集显示,65例OS患者的组织miR-130b水平为11.040±1.008,高于26例化疗后切除组织的9.614±1.154(P<0.05);GSE79181数据集显示,miR-130b低水平组的中位生存期为2823天,优于高水平组的1463天(P<0.05)。抑制组的miR-130b水平为0.038±0.006,低于未转染组的1.160±0.137和空白对照组的1.049±0.089;抑制组的凋亡率为(17.672±1.664)%,高于未转染组的(7.975±0.364)%和空白对照组的(7.438±0.560)%,差异有统计学意义(P<0.05)。抑制组转染24、48 h后的细胞活力较未转染组和空白对照组减弱(P<0.05);与未转染组和空白对照组相比,抑制组的Vimentin和α-SMA水平降低,而PTEN和E-cadherin水平升高(P<0.05)。未转染组和空白对照组上述指标的差异无统计学意义(P>0.05)。双荧光素酶活性检测结果显示,miR-130b模拟物与PTEN野生型质粒共转染细胞后,荧光素酶活性降低(P<0.05),而与PTEN突变型质粒共转染后的荧光素酶活性无明显变化(P>0.05)。结论OS组织中miR-130b水平升高且下调miR-130b水平可抑制细胞增殖和EMT过程并诱导凋亡,可能通过靶向PTEN来发挥促癌作用,在OS靶向治疗中有一定价值。

Objective To investigate the effect of microRNA-130b(miR-130b)on expression of phosphatase and tensin homology deleted on chromosome 10(PTEN)and epithelial mesenchymal transition of osteosarcoma(OS)cells.Methods The series matrix data of GSE39058 and GSE79181 were downloaded from the GEO database of National Biotechnology Information Center of the United States to analyze the miR-130b level in OS tissues and its relationship with prognosis.The miR-130b inhibitor was transfected into MG-63 cells(Inhibition group)by liposome method,and the Untransfected group and Blank control group were set up.Cell count kit(CCK-8)and flow cytometry with Annexin V-FITC/PI double staining were used to detect the proliferation activity and apoptotic rate.Real-time quantitative PCR and Western blotting were used to detect the levels of PTEN,epithelial phenotype marker(E-cadherin)and interstitial phenotypic markers(Vimentin)andα-smooth muscle actin(α-SMA).The targeted binding sequence of miR-130b and PTEN was predicted online.The double luciferase reporter gene assay was used to verify the targeting relationship between miR-130b and PTEN.Results GSE39058 data set showed that the miR-130b level in 65 patients with OS was 11.040±1.008,higher than 9.614±1.154 of 26 patients with resected tissues after chemotherapy(P<0.05).GSE79181 data set showed that the median overall survival of miR-130b low-level group was 2823 days,better than 1463 days of miR-130b high-level group(P<0.05).The miR-130b level was 0.038±0.006 in the Inhibition group,lower than 1.160±0.137 in the Untransfection group and 1.049±0.089 in the Blank control group.The apoptotic rate was(17.672±1.664)%in the Inhibition group,higher than(7.975±0.364)%in the Untransfection group and(7.438±0.560)%in the Blank control group(P<0.05).The cell viability in the Inhibition group was weaker than those in Untransfection group and Blank control group at 24 and 48 h after transfection(P<0.05).Compared with Untransfection group and Blank control group,levels of Vimentin andα-SMA in Inhibition group were decreased,while levels of E-cadherin and PTEN were increased(P<0.05).There was no significant difference between Untransfection group and Blank control group(P>0.05).Results of double luciferase activity assay showed that the luciferase activity decreased after co-transfection of miR-130b mimics with PTEN wild-type plasmid(P<0.05),but there was no significant change in luciferase activity after co-transfection with PTEN mutanted plasmid(P>0.05).Conclusion The increased level of miR-130b is observed in OS tissues.Down-regulation of miR-130b level can inhibit cell proliferation and EMT process and induce apoptosis.It may play a role in promoting cancer by targeting PTEN,which has certain value in targeted therapy of OS.