On was increased in RMG-II cells upon inhibition of miR-21. (B
On was increased in RMG-II cells upon inhibition of miR-21. (B) MiR-21 directly targets the 3′-UTR of PTEN mRNA. The activity of luciferase in the pGL3 wild-type PTEN 3-UTR was downregulated compared to pGL3 mutant-type PTEN 3′-UTR and the pGL3 control in RMG-II cells. P <0.05 according to the t-test.miR-21, and its expression is regulated by miR-21 in CCC (Figure 3B). Several potential miR21 targets that could have implications in CCC were identified using web-based computational approaches to predict gene targets (miRBase Targets BETA Version 1.0, PicTar predictions, and TargetScan). Three putative target genes, PDCD4, SMARCA4, and SPRY2, were predicted by 3 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27527552 different programs. This result indicates that tumor suppressor genes are potentially regulated by miR21. Therefore, we performed real-time RT-PCR for PDCD4, SMARCA4, SPRY2 in the miR21 knockdown experiments in RMG-II cells. We found that miR-21 knockdown increased the expression of these mRNAs (Additional file 5: Figure S5). To investigate the regulation of PTEN expression by miR-21 in JHOC9 cells, we overexpressed miR21 using miR21 mimics in JHOC9 cell. Quantitative real-time PCR analysis confirmed the level of miR21 was significantly overexpressed. As expected, the level of PTEN mRNA was downregulated in JHOC9 cells. Expression of PDCD4, SMARCA4, and SPRY2 mRNA was also decreased by the overexpression of miR-21 in response to miR-21 mimics in JHOC9 cells (Additional file 6: Figure S6).Discussion DNA copy number aberrations are a frequent event in many malignant tumors, leading to altered expression and function of genes residing within the affected genome region. Such genomic abnormalities can harbor either oncogenes or tumor suppressor genes depending on the original gene function and whether the copy number is amplified or deleted. Previous studies have identified a high frequency of copy number amplifications in CCC, including 17q23-25 (18-40 ), 20q13 (22-25 ), and 8q21q- 24q. Additionally, deletions at chromosome 9q and 19p have been also reported in CCC [9,18-20]. Of the chromosomal alterations associated with CCC, 17q23-25 is one of the most frequently amplified regions and is reported to be associated with patient outcome [9]. So far, PPM1D and APPBP2 have been identified as potential targets of 17q23-25 amplification in CCC. However, a recent report suggests there might be new driver genes other than PPM1D and APPBP2 in this region [11]. More than half of miRNAs have been aligned to genomic fragile sites or frequently deleted or amplified regions in several malignancies [21,22]. MiRNAs are a class of small, noncoding RNA molecules that AZD-8055 web regulate gene expressionHirata et al. BMC Cancer 2014, 14:799 http://www.biomedcentral.com/1471-2407/14/Page 8 ofthrough translational repression or cleavage of target mRNA. Among them, miR-21, located on 17q23.2, is unique in that it is overexpressed in many cancers as an oncogene. Previous studies have revealed several significant miR-21 targets that might be related to carcinogenesis. Based on this evidence, miR-21 is a potential candidate for 17q23-25 amplification in CCC oncogenesis. We analyzed DNA copy number alterations at chromosome 17 in a panel of 28 primary CCCs using CGH array. In our data set, 17q23-25 amplification was observed at a frequency similar to that of previous reports. In addition, we confirmed that 17q23-25 amplification correlated negatively with patient prognosis, suggesting that the chromosomal alteration migh.