miR-382-5p过表达介导PTEN的表达下调对人脑胶质瘤U251细胞恶性生物学行为的影响

Overexpression of miR-382-5p Overexpression Mediated Downregulation of PTEN on Malignant Biological Behavior of Human Glioma U251 Cells

目的探究miR-382-5p过表达对人脑胶质瘤U251细胞恶性生物学行为的影响。方法将miR-382-5p mimic转染入U251细胞,逆转录-聚合酶链反应检测miR-382-5p、磷酸酶-张力蛋白同源物(PTEN) mRNA水平;生物信息学预测miR-382-5p与PTEN存在碱基结合位点,构建PTEN pcDNA载体过表达,荧光素酶报告实验检测miR-382-5p与PTEN靶向关系,将细胞随机分为4组:Control组、mimics组、pc-PTEN组和mimics+pc-PTEN组进行后续实验,逆转录-聚合酶链反应检测各组细胞PTEN水平;克隆形成法检测细胞增殖;逆转录-聚合酶链反应检测Ki67、 Survivin、 c-Myc mRNA水平;Transwell检测细胞侵袭能力;蛋白免疫印迹检测E-cadherin、N-cadherin、Vimentin蛋白表达水平。结果 miR-382-5p和PTEN存在直接靶向作用关系,与Control组相比较,mimics组miR-382-5p mRNA水平升高(P<0.05),PTEN mRNA水平降低(P<0.05),细胞克隆形成率降低(P<0.05),Ki67、Survivin mRNA水平降低(P<0.05),c-Myc mRNA水平升高(P<0.05),细胞侵袭数降低(P<0.05),E-cadherin蛋白水平升高(P<0.05),N-cadherin、Vimentin蛋白水平降低(P<0.05)。pc-PTEN组miR-382-5p mRNA水平降低(P<0.05),PTEN mRNA水平升高(P<0.05),细胞克隆形成率升高(P<0.05),Ki67、Survivin mRNA水平升高(P<0.05),c-Myc mRNA水平降低(P<0.05),细胞侵袭数升高(P<0.05),E-cadherin蛋白水平降低(P<0.05),N-cadherin、Vimentin蛋白水平升高(P<0.05);与pc-PTEN组相比较,mimics+pc-PTEN组miR-382-5p mRNA水平升高(P<0.05),PTEN mRNA水平降低(P<0.05),细胞克隆形成率降低(P<0.05),Ki67、Survivin mRNA水平降低(P<0.05),c-Myc mRNA水平升高(P<0.05),细胞侵袭数降低(P<0.05),E-cadherin蛋白水平升高(P<0.05),N-cadherin、Vimentin蛋白水平降低(P<0.05)。结论 miR-382-5p过表达介导PTEN的表达下调可抑制胶质瘤U251细胞增殖、侵袭、生长和上皮细胞-间充质转化。

Objective To investigate the effect of overexpression of miR-382-5p overexpression on malignant biological behavior of human glioma U251 cells. Methods U251 cells were transfected with miR-382-5 pmimic. Then miR-382-5p and PTEN mRNA levels were detected by reverse transcription-polymerase chain reaction(RT-PCR) after transfection. Used bioinformatics to predicted the presence of base binding sites between miR-382-5p and PTEN, and constructed PTEN pcDNA vector overexpression plasmid was constructed. Luciluciase reporting experiment was used to detect the targeting relationship between miR-382-5p and PTEN. Cells were randomly divided into four groups: control group, mimics group, pc-PTEN group and mimics+pc-PTEN group for follow-up experiments. RT-PCR was carried out to detect the level of PTEN mRNA in each group. Cell proliferation was detected by clone formation method. The mRNA levels of Ki67, Survivin and c-Myc were detected by RT-PCR. Transwell experiment was used to assayed cell invasion ability. The expression levels of E-cadherin, N-cadherin and Vimentin were determined by Western blot. Results Results showed that miR-382-5p directly targeted PTEN. Compared with the control group, miR-382-5 p and c-Myc mRNA levels and E-cadherin protein level were increased(P<0.05), PTEN, Ki67 and Survivin mRNA levels were decreased(P<0.05),cell clonal formation rate and cell invasion number were decreased(P<0.05), N-cadherin and Vimentin protein levels were decreased(P<0.05) in the mimics group;In pc-PTEN group, miR-382-5p mRNA and c-Myc mRNA levels and Ecadherin protein level were decreased(P<0.05), PTEN, Ki67 and Survivin mRNA levels were increased(P<0.05), cell clonal formation rate and cell invasion number were increased(P<0.05), N-cadherin and Vimentin protein levels were increased(P<0.05). Compared with pc-PTEN group, PTEN, Ki67 and Survivin mRNA levels, the cell clone formation rate, the number of invasion cells and the N-cadherin and Vimentin levels of mimics+PC-PTEN group decreased significantly(P<0.05), while the c-Myc mRNA level and E-cadherin protein level increased significantly(P<0.05).Conclusion Overexpression of miR-382-5p mediates the downregulation of PTEN expression, causing the inhibition of the proliferation, invasion, growth and EMT of U251 glioma cells.