Department of Urology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
Central Laboratory, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
Asian J Surg. 2017 Sep;40(5):396-406. doi: 10.1016/j.asjsur.2016.07.001. Epub 2016 Aug 9.
Prostate cancer (PCa) is a leading cause of cancer-related death in men, which emphasizes the need for novel therapeutic approaches. Targeting microRNA (miRNA) has been considered as a therapeutic strategy against cancers. Human miR-204-5p potentially targeting BCL2 has been reported to be downregulated in various cancers. We hypothesized that miR-204-5p overexpression induces cancer cell apoptosis by repressing BCL2 expression.
A vector harboring mature miR-204-5p was constructed and delivered into human PCa cells. The expression level of miR-204-5p was determined by miRNA quantitative polymerase chain reaction (QPCR). Luciferase reporter assays were performed to verify the function of mature miR-204-5p and its direct binding to BCL2 transcripts. The expression levels of BCL-2 messenger RNA (mRNA) and protein samples were measured by QPCR and Western blot, respectively. Cell viability was detected by WST-1 assays. Induction of apoptosis was determined by increased levels of cleavage caspase 3 and caspase 3/7 activity.
The expression levels of miR-204-5p were downregulated in PCa cells compared with normal prostate epithelial cells. Transfection of pSM-204 resulted in up to 6.2-fold higher expression of miR-204-5p when compared with pSM control. The mRNA levels of several potential target genes of miR-204-5p were decreased in pSM-204-transfected PC3 and Rv1 cells. BCL2 mRNA and protein expression decreased in miR-204-5p-transfected cells, which led to cytochrome C release from mitochondria. It subsequently increased cleaved caspase 3 and caspase 3/7 activities and reduced cell viability. Cotransfection of a reporter vector harboring the BCL2 3'-untranslated region to compete with endogenous transcripts partially rescued miR-204-5p-induced apoptosis.
Human miR-204-5p targets BCL2 in PCa cells. Restoration of miR-204-5p in PCa could therefore be considered as a novel strategy by targeting antiapoptotic BCL2.
前列腺癌（PCa）是男性癌症相关死亡的主要原因，这强调了需要新的治疗方法。靶向 microRNA（miRNA）已被认为是针对癌症的治疗策略。已经报道，人类 miR-204-5p 可能靶向 BCL2 在各种癌症中下调。我们假设 miR-204-5p 的过表达通过抑制 BCL2 表达诱导癌细胞凋亡。
构建了含有成熟 miR-204-5p 的载体，并将其递送至人 PCa 细胞中。通过 miRNA 定量聚合酶链反应（QPCR）测定 miR-204-5p 的表达水平。进行荧光素酶报告基因测定以验证成熟 miR-204-5p 的功能及其与 BCL2 转录本的直接结合。通过 QPCR 和 Western blot 分别测量 BCL-2 信使 RNA（mRNA）和蛋白质样品的表达水平。通过 WST-1 测定检测细胞活力。通过增加切割半胱天冬酶 3 和 caspase 3/7 活性来确定细胞凋亡的诱导。
与正常前列腺上皮细胞相比，PCa 细胞中的 miR-204-5p 表达水平降低。与 pSM 对照相比，pSM-204 的转染导致 miR-204-5p 的表达水平高达 6.2 倍。在 pSM-204 转染的 PC3 和 Rv1 细胞中，几种潜在的 miR-204-5p 靶基因的 mRNA 水平降低。miR-204-5p 转染的细胞中 BCL2 mRNA 和蛋白表达降低，导致线粒体细胞色素 C 释放。这随后增加了切割的半胱天冬酶 3 和 caspase 3/7 活性，并降低了细胞活力。转染含有 BCL2 3'-非翻译区的报告载体以与内源性转录物竞争可部分挽救 miR-204-5p 诱导的细胞凋亡。
人类 miR-204-5p 在 PCa 细胞中靶向 BCL2。因此，通过靶向抗凋亡 BCL2 来恢复 PCa 中的 miR-204-5p 可以被认为是一种新的策略。
