Mir-27a Oncogene Function In Pancreatic Cancer Cell Lines And Targeted Ubiquitination Degradation Of K-ras Oncoprotein To Inhibit The Growth Of Pancreatic Cancer Cell Line Panc-1

Partâ… Mir-27a regulates the growth, colony formation and migration of pancreatic cancer cells by targeting Spry2Aims MicroRNAs are short regulatory RNAs that participate in the development of cancers. Among these, miR-27a was abnormally up-regulated in several types of cancers and identified as an oncogene. Although overexpressed in pancreatic adenocarcinoma, the role of miR-27a has not yet been reported. In this study, we aimed to detect the function of miR-27a in pancreatic cancer cell lines and explore the possible mechanisms.Methods 1. Observe the effect of miR-27a inhibition on the biological behavior of pancreatic cancer cell lines PANC-1 and MIA PaCa-2; 2. Screen and identify the target genes of miR-27a by using the reporter assays; 3. Explore the possible mechanisms of miR-27a in pancreatic cancer cells by performing immunohistochemistry and western blotting.Results 1. Inhibition of miR-27a suppressed the growth, colony formation and migration of pancreatic cancer cells; 2. The 3'UTR of Spry2 carried a putative miR-27a binding site; furthermore, the Spry2 protein, which had low expression in pancreatic adenocarcinoma tissues, was up-regulated by transfection with the miR-27a inhibitor; 3. Inhibition of miR-27a in pancreatic cancer cells down-regulated the phosphorylation level of Erk1/2.Conclusions 1. MiR-27a plays an oncogenic role by modulating the malignant biological behavior of pancreatic cancer cells; 2. MiR-27a abnormally regulates the Ras/MAPK signal pathway by targeting Spry2 gene in pancreatic cancer cells.Partâ…¡Suppression of the proliferation of PANC-1 cells by targeted ubiquination and degradation of K-ras oncoproteinAims Ubiquitin-proteasome pathway (UPP) can degrade specific proteins in eukaryotic cells. A technique named "Targeting Specific Proteins for Ubiquitination and Degradation" (also called "protein knockdown") harnesses the theory of UPP to engineer certain chimeric E3s, and can specially "knockdown" the targeted proteins effectively, which supplies an alternative therapy strategy for diseases. In this study, we tried to "knockdown" K-ras oncoprotein by using this strategy and evaluated the effect of it on the proliferation of pancreatic cancer cell PANC-1 in vitro.Methods 1. Construct the chimeric E3 ligase expressing vectors by using molecular clone technique; 2. Select the potent chimeric E3 expressing vector by performing western blotting; 3. Evaluate the activity of E3 ligase which targets K-ras oncoprotein by employing the immunoprecipitation and ubiquitination assay in vivo; 4. Test the effect of chimeric E3 ligase on the proliferation of PANC-1 cells in vitro.Results 1. Chimeric E3 ligase degraded K-ras oncoprotein via a ubiquitin-dependent, proteasome-mediated degradation pathway; 2. Transient transfection of chimeric E3 ligase vector suppressed the proliferation of PANC-1 cells in vitro.Conclusions Our study offered experimental foundation for gene therapy of pancreatic adenocarcinoma by "knockdown" of K-ras oncoprotein using an alternative method named "Targeting Specific Proteins for Ubiquitination and Degradation".