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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USP22mRNA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。
Objective To investigate whether microRNA-30e-5p (miR-30e-5p) can inhibit the occurrence and development of non-small cell lung cancer (NSCLC) by down-regulating ubiquitin-specific protease 22 (USP22). Methods The expression of miR-30e-5p and USP22 was detected by qPCR, Western blotting and immunohistochemistry in NSCLC tissues and adjacent tissues. After miR-30e-5p mimics or miR-30e-5p inhibitors were transfected into NSCLC cell line H460, the expression of miR-30e-5p and USP22 was detected by qPCR and Western blot. The USP22 mutant vector was constructed and the luciferase reporter gene was used to detect the binding site of miR-30e-5p in the USP22 gene. The proliferation of H460 cells was detected by MTT assay and the tumor growth in nude mice was detected by xenograft. Flow cytometry was used to detect the cycle arrest and apoptosis of H460 cells after transfection. Results The expression of MiR-30e-5p and USP22 in tumor tissue were higher than that in paracancerous tissues (all P <0.01). The expression of USP22 mRNA and protein were down-regulated in H460 cells after overexpression of miR-30e-5p (all P <0.01), while the expression of USP22 mRNA and protein was up-regulated after miR-30e-5p expression was inhibited (all P <0.01). The expression of miR-30e-5p and USP22 in NSCLC tissues was negatively correlated (P <0.01). miR-30e-5p negatively regulates the expression of USP22 by binding to specific sequences at the 3’UTR. Overexpression of miR-30e-5p can inhibit the proliferation of H460 cells, induce cell cycle arrest and apoptosis, and inhibit the growth of tumor in nude mice (P <0.05, P <0.01), while inhibit the expression of miR-30e-5p It can promote the proliferation of H460 cells and inhibit cell cycle arrest and apoptosis (P <0.05, P <0.01). Conclusions MiR-30e-5p down-regulates the expression of USP22 and thus inhibits the occurrence and development of NSCLC, suggesting that MiR-30e-5p may serve as a potential therapeutic target for NSCLC patients.