Xie Hanbing, Nie Ling, Zhang Mengni, Su Zhengzheng, Chen Xueqin, Xu Miao, Gong Jing, Chen Ni, Zhou Qiao
Pathology Department and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan 610041, P.R. China.
Exp Ther Med. 2020 Mar;19(3):1806-1816. doi: 10.3892/etm.2019.8406. Epub 2019 Dec 31.
Overexpression of α-methylacyl-coenzyme A racemase (AMACR/P504S) is a major abnormality that has been observed in prostate cancer, whereas microRNA (miRNA/miR) 200c, is downregulated. The aim of the present study was to explore whether miR200c was able to exert any regulatory effects on AMACR. To meet this aim, bioinformatics analysis was performed to identify potential binding sites for miR200c in the 3'-untranslated region (3'-UTR) of AMACR. Recombinant adenoviral and dual reporter gene assays were designed to examine the binding of miR200c to the potential seed sequences in the AMACR 3'-UTR. Conventional reverse transcription (RT)-PCR, RT-quantitative (q)PCR and western blotting were also used to examine the regulatory effects of miR200c on AMACR at the mRNA and protein levels. Furthermore, Cell Counting Kit-8, wound healing and Transwell assays were performed to investigate the biological effects of miR200c-AMACR deregulation on prostate cancer cell proliferation, migration and invasion. It was revealed that miR200c post-transcriptionally suppressed AMACR expression by interacting with the 90-97 nucleotide sequence of the AMACR mRNA 3'-UTR. Artificial overexpression of miR200c significantly downregulated the mRNA and protein levels of AMACR in DU145 and PC-3 prostate cancer cells. Knockdown of AMACR by RNA interference, or overexpression of miR200c by recombinant adenoviral Ad-miR200c, inhibited prostate cancer cell proliferation, migration and invasiveness. Taken together, the results of the present study revealed that miR200c may suppress the AMACR expression level post-transcriptionally. The results also indicate that perturbation of the miR200c-AMACR regulatory mechanism may be involved in prostate carcinogenesis and that this may be exploited in future therapeutic approaches to prostate cancer.
α-甲基酰基辅酶A消旋酶（AMACR/P504S）的过表达是在前列腺癌中观察到的主要异常情况，而微小RNA（miRNA/miR）200c则下调。本研究的目的是探讨miR200c是否能够对AMACR发挥任何调节作用。为实现这一目的，进行了生物信息学分析，以确定AMACR的3'-非翻译区（3'-UTR）中miR200c的潜在结合位点。设计了重组腺病毒和双报告基因检测，以检查miR200c与AMACR 3'-UTR中潜在种子序列的结合。还使用常规逆转录（RT）-PCR、RT-定量（q）PCR和蛋白质印迹法，在mRNA和蛋白质水平上检查miR200c对AMACR的调节作用。此外，进行了细胞计数试剂盒-8、伤口愈合和Transwell检测，以研究miR200c-AMACR失调对前列腺癌细胞增殖、迁移和侵袭的生物学影响。结果显示，miR200c通过与AMACR mRNA 3'-UTR的90-97核苷酸序列相互作用，在转录后抑制AMACR表达。miR200c的人工过表达显著下调了DU145和PC-3前列腺癌细胞中AMACR的mRNA和蛋白质水平。通过RNA干扰敲低AMACR，或通过重组腺病毒Ad-miR200c过表达miR200c，可抑制前列腺癌细胞的增殖、迁移和侵袭性。综上所述，本研究结果表明，miR200c可能在转录后抑制AMACR表达水平。结果还表明，miR200c-AMACR调节机制的紊乱可能参与前列腺癌的发生，这可能在未来前列腺癌的治疗方法中得到应用。
