Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Urol Oncol. 2013 Jan;31(1):115-23. doi: 10.1016/j.urolonc.2010.09.017. Epub 2011 Mar 10.
We previously demonstrated that miR-133a is a tumor-suppressive microRNA (miRNA) and is commonly down-regulated in human bladder cancer (BC). The aim of this study is to determine a novel oncogenic gene targeted by miR-133a in BC.
To identify genes targeted by miR-133a, an oligo-microarray analysis was performed using the miR-133a-transfected BC cell lines. For gain/loss-of-function studies, miR-133a/si-glutathione S-transferase π1 (GSTP1)-transfectants were subjected to XTT assay and flow cytometry to evaluate their cell viability and apoptosis status. The luciferase reporter assay was used to confirm the actual binding sites between miR-133a and GSTP1 mRNA. The mRNA and protein expression of GSTP1 in BC cell lines and clinical samples were evaluated by real-time RT-PCR and Western blot, respectively.
MiR-133a transfection induced cell viability inhibition and apoptosis in BC cell lines. We focused on the GSTP1 gene that was the top 7 down-regulated one in the gene profile from the miR-133a-transfectants. MiR-133a transfection repressed expression levels of mRNA and protein levels of GSTP1. A luciferase reporter assay suggested that the actual binding may occur between miR-133a and GSTP1 mRNA. Cell viability inhibition and apoptosis were induced in the si-GSTP1 transfectants compared with the controls (P < 0.005). GSTP1 mRNA expression levels in 43 clinical BCs were significantly higher than those in eight normal bladder epitheliums (P = 0.0277).
Our data suggest that tumor suppressive miR-133a directly regulated oncogenic GSTP1 gene in BC, and that an anti-apoptotic effect mediated by GSTP1 is maintained by miR-133a down-regulation in human BC.
我们之前证明 miR-133a 是一种肿瘤抑制性 microRNA（miRNA），在人类膀胱癌（BC）中普遍下调。本研究旨在确定 miR-133a 在 BC 中靶向的新型致癌基因。
为了鉴定 miR-133a 靶向的基因，我们使用 miR-133a 转染的 BC 细胞系进行了寡聚微阵列分析。为了进行增益/失能研究，将 miR-133a/si-谷胱甘肽 S-转移酶 π1（GSTP1）-转染细胞进行 XTT 测定和流式细胞术，以评估其细胞活力和凋亡状态。使用荧光素酶报告基因检测证实 miR-133a 与 GSTP1 mRNA 之间的实际结合位点。通过实时 RT-PCR 和 Western blot 分别评估 GSTP1 在 BC 细胞系和临床样本中的 mRNA 和蛋白表达。
miR-133a 转染诱导 BC 细胞系的细胞活力抑制和凋亡。我们专注于 GSTP1 基因，它是 miR-133a 转染物的基因谱中下调最明显的基因之一。miR-133a 转染抑制了 GSTP1 的 mRNA 和蛋白水平表达。荧光素酶报告基因检测表明，miR-133a 与 GSTP1 mRNA 之间可能发生实际结合。与对照组相比，si-GSTP1 转染细胞的细胞活力抑制和凋亡诱导作用更为明显（P<0.005）。43 例临床 BC 中的 GSTP1 mRNA 表达水平明显高于 8 例正常膀胱上皮（P=0.0277）。
我们的数据表明，肿瘤抑制性 miR-133a 直接调节 BC 中的致癌 GSTP1 基因，并且 GSTP1 的抗凋亡作用通过 miR-133a 在人 BC 中的下调得以维持。
