MicroRNA-30e-5p通过下调泛素特异性蛋白酶22抑制非小细胞肺癌的发生和发展

目的 探讨microRNA-30e-5p(miR-30e-5p)是否可通过下调泛素特异性蛋白酶22(USP22)抑制非小细胞肺癌(NSCLC)的发生和发展.方法 采用qPCR、蛋白质印迹法和免疫组织化学染色法检测miR-30e-5p、USP22在NSCLC组织和癌旁组织中的表达.NSCLC细胞株H460转染miR-30e-5p模拟物或miR-30e-5p抑制物后,利用qPCR和蛋白质印迹法检测细胞中miR-30e-5p、USP22的表达.构建USP22突变载体,采用荧光素酶报告基因检测miR-30e-5p在USP22基因中的结合位点.采用MTT法检测转染后H460细胞的增殖情况,并采用异种移植法检测裸鼠体内肿瘤生长情况.流式细胞术检测转染后H460细胞的周期阻滞和凋亡情况.结果 MiR-30e-5p、USP22在肿瘤组织中的表达均高于癌旁组织(P均＜0.01).在H460细胞中过表达miR-30e-5p后USP22 mRNA和蛋白的表达均下调(P均＜0.01),而抑制miR-30e-5p表达后USP22 mRNA和蛋白的表达均上调(P均＜0.01).NSCLC组织中miR-30e-5p与USP22的表达呈负相关(P＜0.01).miR-30e-5p可通过结合在3'UTR的特异序列负调控USP22的表达.过表达miR-30e-5p可抑制H460细胞的增殖、诱导细胞周期阻滞和凋亡,并抑制裸鼠体内肿瘤的生长(P＜0.05,P＜0.01);而抑制miR-30e-5p的表达可促进H460细胞的增殖、抑制细胞周期阻滞和凋亡(P＜0.05,P＜0.01).结论 MiR-30e-5p可以下调USP22的表达,从而抑制NSCLC的发生和发展,提示其可作为NSCLC患者的潜在治疗靶点.

MicroRNA-30e-5p inhibits development and progression of non-small cell lung cancer by down-regulating ubiquitin-specific protease 22 expression

Objective To investigate whether microRNA-30e-5p (miR-30e-5p) can inhibit tumor growth by down-regulating the expression of ubiquitin-specific protease 22 (USP22) in non-small cell lung cancer (NSCLC). Methods We detected the expressions of miR-30e-5p and USP22 in NSCLC tissues and adjacent non-tumor tissues by qPCR, Western blotting, and immunohistochemistry. After transfecting with miR-30e-5p mimic or miR-30e-5p inhibitor, the expressions of miR-30e-5p and USP22 in H460 cells was detected by qPCR and Western blotting. USP22 mutant vector was constructed and used to identify miR-30e-5p binding sites in the USP22 gene by luciferase reporter assay. The proliferation of H460 cells was measured in vitro by MTT assay and the tumor growth in nude mice was measured in vivo by xenograft assay. The cell cycle arrest and apoptosis of H460 cells were detected by flow cytometry. Results The expressions of MiR-30e-5p and USP22 in NSCLC tissues were significantly higher than those in adjacent non-tumor tissues (both P<0.01). Overexpression of miR-30e-5p significantly down-regulated the expressions of USP22 mRNA and protein in NSCLC tissues (both P<0.01), while inhibition of miR-30e-5p expression significantly up-regulated the expressions of USP22 mRNA and protein (both P<0.01). MiR-30e-5p was negatively related to USP22 in NSCLC tissues (P<0.01). MiR-30e-5p could negatively regulate the expression of USP22 by binding to the specific sequence of 3''UTR. Overexpression of miR-30e-5p significantly inhibited proliferation of H460 cells, induced cell cycle arrest and apoptosis, and inhibited tumor growth in nude mice (P<0.05, P<0.01); while inhibition of miR-30e-5p significantly promoted the cell proliferation and inhibited cell cycle arrest and apoptosis (P<0.05, P<0.01). Conclusion MiR-30e-5p can down-regulate the expression of USP22, thereby inhibiting the development and progression of NSCLC, suggesting that it can be used as a potential therapeutic target for NSCLC.