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目的通过综合分析肿瘤标本中基因和蛋白表达,及体外生物学作用的检测,来评价胶质瘤中Nix蛋白的作用以及Nix蛋白与miR-520h的关系。方法选取46例胶质瘤标本,RT-PCR检测miR-520h基因的表达,Western blotting检测Nix蛋白的表达;培养U251胶质瘤细胞,Western blotting检测Nix蛋白的表达;构建靶向Nix基因的shRNA敲除U251细胞中Nix基因(Nix-kn),Western blotting检测Nix,IKKα,i-κBα和p-NF-κB/p65等蛋白的表达水平;has-miR-520h抑制剂转染U251细胞,Western blotting检测Nix,IKKα,i-κBα和p-NF-κB/p65等蛋白的表达水平。结果胶质瘤标本的基因和蛋白检测表明,miR-520h的高表达伴随着Nix蛋白的高表达;体外实验结果表明,与正常培养的U251相比,缺氧培养的U251细胞Nix表达较高;与Nix-kn组相比,Nix-wt组胶质瘤细胞Nix和p-NF-κB/p65表达均明显增高,而NF-κB活性抑制剂蛋白i-κBα表达降低;200 nmol/L的has-miR-520h抑制剂处理细胞后,与正常对照组比较,Nix蛋白表达显著减少,而p-NF-kappaB/p65蛋白表达显著增加。结论在神经胶质瘤的发生发展中miR-520h与Nix蛋白的表达相关;miR-520h可能通过促进Nix合成,从而成为一个强大的肿瘤激活剂。
Objective To evaluate the role of Nix protein in gliomas and the relationship between Nix protein and miR-520h by comprehensively analyzing the expression of genes and proteins in tumor samples and detecting the biological effects in vitro. Methods Forty-six glioma specimens were selected and the expression of miR-520h was detected by RT-PCR. The expression of Nix protein was detected by Western blotting. The U251 glioma cells were cultured and the expression of Nix protein was detected by Western blotting. The expression of Nix, IKKα, i-κBα and p-NF-κB / p65 protein in U251 cells was knocked down by Nix-kn and the expression of Nix, IKKα, i-κBα and p-NF- The expressions of Nix, IKKα, i-κBα and p-NF-κB / p65 were detected by Western blot. Results The gene and protein of glioma specimens showed that the high level of miR-520h was accompanied by the high expression of Nix protein. The results of in vitro experiments showed that the expression of Nix in U251 cells was higher than that in U251 cells. Compared with the Nix-kn group, Nix and p-NF-κB / p65 expression of Nix-wt group glioma cells were significantly increased, while NF-κB activity inhibitor protein i-κBα expression decreased; 200 nmol / L has After treated with miR-520h inhibitor, the expression of Nix protein was significantly decreased and the expression of p-NF-kappaB / p65 protein was significantly increased compared with the normal control group. Conclusion miR-520h is associated with the expression of Nix protein in the development of glioma. MiR-520h may be a powerful tumor activator by promoting the synthesis of Nix.