Zhang Rui, Li Min, Zang Wenqiao, Chen Xudong, Wang Yuanyuan, Li Ping, Du Yuwen, Zhao Guoqiang, Li Li
Department of Emergency, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe Road, Zhengzhou, Henan, 450052, China.
Tumour Biol. 2014 Jan;35(1):837-44. doi: 10.1007/s13277-013-1115-2. Epub 2013 Aug 23.
Our previous studies have revealed that miR-148a is downregulated in pancreatic cancer. Bioinformatics analysis has shown cholecystokinin-B receptor (CCKBR) and B cell lymphoma (Bcl-2) to be potential targets of miR-148a. But the pathophysiologic role of miR-148a and its relevance to the growth and development of pancreatic cancer are yet to be investigated. The purpose of this study is to elucidate the molecular mechanisms where miR-148a acts as a tumor suppressor in pancreatic cancer. Our results showed significant downregulation of miR-148a in 28 pancreatic cancer tissue samples and five pancreatic cancer cell lines, compared with their non-tumor counterparts by qRT-PCR. MiR-148a was found to not only inhibit the proliferation of pancreatic cancer cells (PANC-1 and AsPC-1) in vitro by MTT assay and colony formation assay, but also to promote cells apoptosis in vitro by Annexin V-FITC apoptosis detection and caspase activity assay. Using western blot and luciferase activity assay, CCKBR and Bcl-2 were identified as targets of miR-148a. Moreover, we also found that the expression of Bcl-2 lacking in 3'UTR could abrogate the pro-apoptosis function of miR-148a. These findings suggest the importance of miR-148a's targeting of CCKBR and Bcl-2 in the regulation of pancreatic cancer growth and apoptosis.
我们之前的研究表明，miR-148a在胰腺癌中表达下调。生物信息学分析显示，胆囊收缩素B受体（CCKBR）和B细胞淋巴瘤（Bcl-2）是miR-148a的潜在靶点。但miR-148a的病理生理作用及其与胰腺癌生长发育的相关性尚待研究。本研究的目的是阐明miR-148a在胰腺癌中作为肿瘤抑制因子发挥作用的分子机制。我们的结果显示，通过qRT-PCR检测，与非肿瘤对照相比，28例胰腺癌组织样本和5种胰腺癌细胞系中miR-148a均显著下调。通过MTT法和集落形成试验发现，miR-148a不仅在体外抑制胰腺癌细胞（PANC-1和AsPC-1）的增殖，还通过Annexin V-FITC凋亡检测和caspase活性试验促进体外细胞凋亡。利用蛋白质免疫印迹法和荧光素酶活性测定法，确定CCKBR和Bcl-2为miR-148a的靶点。此外，我们还发现，缺少3'UTR的Bcl-2的表达可消除miR-148a的促凋亡功能。这些发现表明，miR-148a靶向CCKBR和Bcl-2在调节胰腺癌生长和凋亡中具有重要作用。
