Long Noncoding RNA MIR31HG Functions As An Oncogene In Pancreatic Ductal Adenocarcinoma And Is Negatively Regulated By MiR-193b

Pancreatic cancer is the fourth-lead ing cause of cancer-related deaths in the United States and remains one of the most lethal malignancies worldwide. Although notable improvements in survival have been made for most cancers over the past three decades, the prognosis for pancreatic cancer patients has shown the least improvement. Accumulating data from whole-genome and transcriptome studies have revealed that only a very small percentage (1%-2%) of the genome encodes proteins, but the majority of the mammalian genome is transcribed. It has become increasingly apparent that much of the genome, far more than expected, encodes vast numbers of noncoding RNAs. Long noncoding RNAs (lncRNAs) are a new class of regulatory RNAs that are>200nucleotides in length and do not have the potential to encode proteins. Recent studies have demonstrated that LncRNAs play central roles in a wide range of biological processes, and appear to be involved in the progression of a variety of human diseases. Furthermore, it has been shown that several lncRNAs are deregulated in many tumors and may be involved in carcinogenesis or cancer progression.An analysis of pancreatic cancer patient gene profiling results (GSE28735) from pubmed showed that, among45matched pairs of pancreatic cancer tumor and adjacent non-tumor tissues, MIR31HG was more highly expressed in pancreatic tumors. To further validate this result, we examined the expression level of MIR31HG in pancreatic cancer cell lines (AsPC-1, PANC-1, CFPAC-1, Hs766T, SW1990, BxPC-3, MIA PaCa-2) and hTERT-HPNE cells, an immortalized, normal human pancreatic ductal cell line, using real-time qPCR. Compared with hTERT-HPNE cells, pancreatic cancer cells exhibited significantly higher levels of MIR31HG expression. We then evaluated MIR31HG expression level in13paired pancreatic cancer and adjacent pancreatic tissue samples using real-time qPCR. The results showed that MIR31HG levels were significantly higher in pancreatic cancer tissues. Collectively, these results suggest that MIR31HG is upregulated in pancreatic cancer. CPAT software was used to calculate the coding potential of MIR31HG, and the results showed that the possible ORF of MIR31HG is very short, and the coding probability of MIR31HG is very low(<0.05). Then the possible ORF of MIR31HG was cloned into the eukaryotic expression vector pcDNA3.1with C-terminal Flag tag, and Western blot confirmed that MIR31HG does not have the coding capacity. The full sequence of miR-31is located within the intron of MIR31HG, and the transcriptional activity of miR-31is under the control of MIR31HG. Nonetheless, siRNA-mediated knockdown of MIR31HG did not alter the levels of miR-31, and inhibition or overexpression of miR-31had no effect on MIR31HG expression. Despite the interrelated location and co-transcription of MIR31HG and miR-31, they did not regulate each other’s expression. Thus, M1R31HG plays a role in biological processes independent of miR-31.Applying loss-of-function and gain-of-function approaches, we investigated the role of MIR31HG in pancreatic cancer cell proliferation and invasion. Downregulation of MIR31HG significantly decreased AsPC-1and PANC-1cell growth, induced apoptosis and G1/S arrest and inhibited migration and invasion in vitro, whereas overexpression of this lncRNA had the opposite effects. These findings indicate that MIR31HG may function as a tumor oncogene, and its overexpression could contribute to pancreatic cancer development.Recently study have found that all types of RNA transcripts, including lncRNAs, could be targeted by miRNAs though "microRNA response elements". Using Online software, we found that miR-193b could target MIR31HG. miR-193b, a putative tumor suppressor, is downregulated in a variety of tumors, including pancreatic cancer. Our study showed that, whereas MIR31HG was upregulated in pancreatic cancer tissues, miR-193b was downregulated in the same tumor specimens, resulting in a significant inverse correlation between MIR31HG and miR-193b. Moreover, overexpression of miR-193b reduced MIR31HG expression whereas inhibition of miR-193b expression significantly upregulated MIR31HG, suggesting that MIR31HG is negatively regulated by miR-193b. Luciferase reporter assays showed that miR-193b mimic significantly reduced luciferase reporter activity and inhibition of miR-193b induced a remarkable increase in luciferase activity, while both effects were abrogated by directed mutagenesis of the miR-193b-binding seed region in MIR31HG. These results indicate that miR-193b exerts inhibitory effects on MIR31HG expression by directly targeting MIR31HG. RIP assays found that whereas MTR31HG was detected in AG02immunoprecipitates from control group, its levels were drastically reduced in AG02complexes purified from cells treated with miR-193b inhibitor, indicating that miR-193b regulates MIR31HG in an AGO2-dependent manner. Finally, using CCK8and Transwell assays, we confirmed that miR-193b could inhibit pancreatic cancer cell growth and invasion, also it suppressed the proliferation and invasion promoted by MIR31HG. In summary, miR-193b negatively regulated MIR31HG expression and function.Collectively, lncRNA MIR31HG is upregulated in pancreatic cancer. The effects of this lncRNA on cell proliferation, cell-cycle regulation, migration and invasion suggest that it functions as an oncogene in pancreatic cancer tumorigenesis. In addition to promoter methylation, MIR31HG could also be subjected to post-transcriptional regulation by miR-193b in a manner similar to miR-mediated silencing of protein-coding genes. The high expression of MIR31HG in pancreatic cancer may be partly attributable to a reduction in the tumor suppressor miR-193b. The discovery of a direct regulatory link between miRNAs and lncRNAs provides a new avenue for the investigation of mechanisms underlying tumorigenesis.