The Expressive Relevance And Regulatory Mechanism Of HIF-1and MiR-185、miR-191、miR-212in The Pancreatic Cancer

HIF-1up-regulates a lot of genes in low-oxygen conditions, involving certain microRNAs(miRs). In the preliminary experiment, we found that the miR-185, miR-191and miR-212as a target gene of HIF-1via searching the promoter regions by UCSC Genome Browser. Furthermore, we had confirmed the over-expression of miR-185, miR-191and miR-212in hypoxic conditions. The aim of study was to investigate the association and regulatory mechanism of HIF-1expression and miR-185, miR-191and miR-212in pancreatic tumor tissues. Then, the relationships between the expression of HIF-1, miR-185, miR-191and miR-212and clinicopathologic characteristics, prognosis were analyzed, which may provide the new direction of diagnosis, predicting prognosis of pancreatic cancer patients.Methods1. Using Western blotting and RT-PCR, it was observed that the expression of HIF-1, miR-185, miR-191and miR-212of MiaPaCa2and AsPcl cells increased significantly after hypoxic cultured6h and12h in comparing with control group.2. In the normoxia, we used HIF-1over-expressing plasmid or siRNA to transfected the MiaPaCa2and AsPcl cells to change the expression of HIF-1. The cells which had expected effect were selected by Western blotting, and then, the expression of HIF-1, miR-185, miR-191and miR-212were analyzed by RT-PCR.3. ChIP sequencing identifies the binding sites of HIF-1and miR-185, miR-191, miR-21. Whether HIF-1participate in the up-regulation of transcription of miR-185, miR-191, miR-212will be analyzed by plasmids co-transfected and luciferase assay.4. Tumor and normal tissues samples were obtained from pancreatic cancer patients, then extract total RNA form tissues. The expression of HIF-1, miR-185, miR-191, miR-212were investigated by qRT-PCR. Then, the relationships between the expression of HIF-1, miR-185, miR-191and miR-212and clinicopathologic characteristics, prognosis were analyzed.Results1. The expression of HIF-1protein of two cell lines increased significantly after hypoxic cultured for6h and12h, the most overpression after12h. The expression of HIF-1of two cell lines increased significantly, the most obvious increasing was after12h. The expression of miR-185and miR-191of cell lines cells were up-regulated after these cells were hypoxic cultured. Especially, after hypoxic cultured for12h, significant up-regulation were detected in comparing with control group(P<0.05).2. After transfected Aspc-1and MiaPaCa2by HIF-1over-expressing plasmid, the over-expression of HIF-1, miR-185, miR-191, miR-212can be observed (P<0.05). After transfected the MiaPaCa2by HIF-1siRNA, the significant decreasing of HIF-1and miR-185, miR-191, miR-212expression were investigated(.P<0.05). HIF-1were significantly associated with miR-185、miR-191、miR-212in two cell lines(P<0.05).3. In MiaPaCa2cells, HIF-1binds the miR-185, miR-191, miR-212in the2nd,5th,1st hypoxia response elements sequence of the promoter in5’flanking region of them. So that, HIF-1can up-regulate transcription of miR-185, miR-191, miR-212.4. Expression of HIF-1mRNA and miR185, miR191, miR212were significant higher in tumor tissues in comparing with normal tissues. The expression of HIF-1mRNA was positively correlated with miRs in tumor tissues, the expression of HIF-1, miR-185and miR-191closely associated with the tumor size, pTNM stage, lymph node metastasis and perneural invasion (P<0.05), and HIF-1was related with histological grade, too. In survival analysis, patients with higher expression of HIF-1, miR185and miR-191inclined to have shorter survival time (P<0.05)Conclusion1. Expression of HIF-1, miR-185, miR-191, miR-212in pancreatic cancer cells increased under chronic hypoxia. The expression of HIF-1closely related with miR-185, miR-191and miR-212. The mechanism on this correlation may caused by binding of HIF-1and Promoters of miRs and initiate a transcription of these miRs.2. The expression of HIF-1was positively correlated with miR-185, miR-191and miR-212in tumor tissues. The expression of HIF-1, miR-185and miR-191closely associated with the tumor size, pTNM stage, lymph node metastasis and perneural invasion. Patients with over-expression of HIF-1, miR-185and miR-191in pancreatic tissues tend to have shorter survival time, which imply the important values of HIF-1, miR185and miR-191in predicting the prognosis of cancer.