Key Laboratory of Tumour Molecular Biology in Binzhou Medical University, Department of Biochemistry and Molecular Biology, Binzhou Medical University, YanTai, ShanDong, PR China.
PLoS One. 2012;7(10):e46090. doi: 10.1371/journal.pone.0046090. Epub 2012 Oct 5.
microRNAs (miRNAs) are small noncoding RNAs that regulate genes and contribute to many kinds of human diseases, including cancer. Two miRNAs, miR-511 and miR-1297, were investigated for a possible role in adenocarcinoma based on predicted binding sites for the TRIB2 oncogene by microRNA analysis software, and the pcDNA-GFP-TRIB2-3'UTR vector was constructed to investigate the interaction between TRIB2 and miR-511/1297 in the adenocarcinoma cell line A549. Green fluorescent protein (GFP) expression was estimated by fluorescence microscopy and flow cytometry after A549 cells were co-transfected with miR-511 (or miR-1297) and pcDNA-GFP-TRIB2-3'UTR vector. The expression of GFP in the miR-511- and miR-1297-treated cells was significantly downregulated in contrast with the negative-control (NC) miRNA-treated cells. The decreased expression of TRIB2 was further detected after miR-511 (or miR-1297) treatment by western blotting. The MTT test showed inhibition of A549 cell proliferation and Annexin V-FITC/PI dual staining showed increased apoptosis in the miR-511- and miR-1297-treated cells compared to the NC cultures. A transcription factor downstream of TRIB2, the CCAAT/enhancer-binding protein alpha (C/EBPα), was expression at higher levels after miR-511 (or miR-1297) decreasing TRIB2 expression. Our results illustrate that miR-511 and miR-1297 act as tumor suppressor genes, which could suppress A549 cell proliferation in vitro and in vivo by suppressing TRIB2 and further increasing C/EBPα expression.
微小 RNA(miRNA)是一种调节基因的小非编码 RNA,参与多种人类疾病,包括癌症。基于 miRNA 分析软件对 TRIB2 癌基因预测的结合位点,研究了两种 miRNA(miR-511 和 miR-1297)在腺癌中的可能作用,并构建了 pcDNA-GFP-TRIB2-3'UTR 载体,以研究 TRIB2 与 miR-511/1297 在腺癌 A549 细胞系中的相互作用。通过荧光显微镜和流式细胞术估计 A549 细胞共转染 miR-511(或 miR-1297)和 pcDNA-GFP-TRIB2-3'UTR 载体后 GFP 的表达。与阴性对照(NC)miRNA 处理的细胞相比,miR-511 和 miR-1297 处理的细胞中 GFP 的表达明显下调。western blot 进一步检测到 miR-511(或 miR-1297)处理后 TRIB2 的表达降低。MTT 试验显示 miR-511 和 miR-1297 处理的 A549 细胞增殖受到抑制,Annexin V-FITC/PI 双重染色显示与 NC 培养物相比,miR-511 和 miR-1297 处理的细胞凋亡增加。TRIB2 下游的转录因子 CCAAT/增强子结合蛋白 α(C/EBPα)在 miR-511(或 miR-1297)降低 TRIB2 表达后表达水平更高。我们的结果表明,miR-511 和 miR-1297 作为肿瘤抑制基因,通过抑制 TRIB2 进一步增加 C/EBPα 的表达,在体外和体内均可抑制 A549 细胞的增殖。