反义miR-30a-5p抑制人脑胶质瘤细胞U87生长的体外实验研究

Inhibitory effect of knocking - down miR - 30a - 5p on growth of U87 glioma cell

目的前期发现miR-30a-5p在胶质瘤中表达明显上调，本实验探究敲低miR-30a-5p对人脑胶质瘤细胞U87细胞生物学特征的影响。方法real—timePCR检测转染miR-30a-5pI（miR-30a-5p抑制物）后U87细胞的miR-30a-5p表达水平，流式细胞术、MTT、Transwell、AnnexinV法检测细胞周期、生长、侵袭及凋亡的改变；Western blot检测相关蛋白。结果real—timePCR结果显示miR-30a-5pI可有效降低U87细胞中miR-30a-5p表达，抑制细胞增殖活性，使细胞周期阻滞在G0／G1期，侵袭能力明显受抑，凋亡增加，促增殖蛋白PCNA、促细胞周期进展蛋白Cyclin D1及促侵袭蛋白MMP-9的表达明显下调，而抑制侵袭蛋白TIMP-1及凋亡相关蛋白p53表达明显上调。结论敲低U87细胞的miR-30a-5p表达可抑制胶质瘤的增殖及侵袭，诱导凋亡；miR-30a-5p可成为人脑胶质瘤基因治疗的潜在候选靶点。

Objective Our previous study had shown that there was overexpression of miR -30a -5p in malignant glionka cell lines. In the present study, we aim to investigate the effect of knocking- down miR-30a -5p on the biological characteristics of U87 glioblastoma cells. Method The U87 cells were divided into three groups： control cells,cells transfected with scramble oligonucleotides and cells transfected with miR-30a-5p inhibitors（miR -30a- 5p I）. Oligonucleotides mediated by lipofectmnine were transfected to U87 cells. Real -time PCR was conducted to detect the expression of miR- 30a- 5p in transfected cells. The cell proliferation was determined by 3 - （4,5 - Dimethylthiazol - 2 - yl） - 2,5 - diphenyltetrazolium bromide （ MtT ） assay and flow cytometry. The cell invasion was evaluated by Transwell assay and cell apoptosis was detected with Annexin V staining. Moreover, the relevant molecules regulating proliferation, invasion, cell cycle progression and apoptosis were examined by Western blot analysis. Results The expression of miR - 30a - 5p in the cells transfected with miR - 30a - 5p I was significantly reduced. The cell proliferation activity of U87 cell was inhibited. The cell cycle was arrested in G0/G1 phase, cell invasive ability was attenuated and apoptotic cells were increased in cells transfected with miR - 30a - 5pI as compared to those of the cells transfected with scrambled siRNA and control cells. The expression of proliferating cell nuclear antigen （PCNA）, matrix metallopeptidase 9 （ MMP - 9） and Cyclin D1 were downregulated while the tissue inhibitor of metalloproteinasel （TIMP - 1） and p53 were upregulated. Condusions Transfection of miR -30a -Sp I into glioma cells could inhibit the proliferation activity and invasive ability of U87 cell and induce cell apoptosis, miR- 30a- 5p is a potential target of gene therapy for glioma.