| Objective: We used qRT-PCR to detect the expression of Hsa-mi R-590-3p(hereinafter referred to as miR-590-3p)in pancreatic cancer(hereinafter referred to as PC).To investigate the effect of mi R-590-3p on the malignant biological behavior of pancreatic cancer,and to explore the target genes and pathways directly affected by miR-590-3p,so as to provide new therapeutic ideas and targets for the study of the diagnosis and treatment of pancreatic cancer.Methods: 1.MiR-590-3p expression quantities relatively expressed higher in PC tissues than paired normal pancreas measured by qRT-PCR in 42 paired PC and normal pancreas tissues(p<0.05).The transfection efficiency of mi R-590-3p mimics and inhibitors was detected by qRT-PCR.After mimics and inhibitors of mi R-590-3p transfected into pancreatic cancer cells,changes in proliferation capacity of Capan-2and PANC-1 cells were detected by MTT and clonal formation assay.Cell cycle changes of Capan-2 and PANC-1 cells were detected by flow cytometry.Transwell assay was used to detect the migration and invasion of Capan-2 and PANC-1 cells.2.We used the bioinformatics tool TargetScan(http://www.targetscan.org)to identify the possible target genes of miR-590-3p.Immunohistochemistry revealed the clinicopathological significance of PPP2R2 A,p27 and miR-590-3p in the expression of pancreatic cancer.Western blot was used to detect the expression changes of PPP2R2 A,p27 and G1/S cell cycle pathway-related proteins CDK2,cyclinE2 and p21 after transfection of mimics and inhibitors of miR-590-3p.The binding region of miR-590-3p to the 3 ’UTR region of PPP2R2 A and p27 mRNA was predicted by bioinformatics website.Wild-type and mutant dual-fluorescent reporter plasmids of the 3 ’UTR region of PPP2R2 A and p27 m RNA were constructed,and the direct binding of miR-590-3p to PPP2R2 A and p27 was detected by double-luciferase reporter gene assay.PPP2R2 A and p27 overexpression plasmids were constructed and co-transfected with miR-590-3p mimics.We used MTT,colony formation and transwell assay to test and verify changes in pancreatic cancer cell proliferation,migration and invasion ability.Results: 1.The expression level of miR-590-3p in pancreatic cancer tissues was higher than that in the paired normal pancreatic tissues.The high expression of miR-590-3p was significantly correlated with tumor size(p = 0.042)and preoperative ca19-9 level(p = 0.046).MiR-590-3p was relatively high in Capan-2 cells and relatively low in PANC-1 cells in 6 pancreatic cancer cell lines.Capan-2 cells and PANC-1 cells were transiently transfected with miR-590-3p mimics and inhibitors for cell functional experiments.Over expression of miR-590-3p can promote cell proliferation,improve cell migration and invasion ability,shorten cell G0/G1 phase,and prolong S and G2/M phase.Interference with the expression of miR-590-3p will weaken cell proliferation ability,reduce cell migration and invasion ability,prolong cell G0/G1 phase,and shorten S and G2/M phase.2.Western blot analysis revealed that the overexpression of mi R-590-3p down-regulated the expression of PPP2R2 A,p27 and G1/S cell cycle pathway-related proteins p21 in pancreatic cancer cells,and up-regulated the expression of CDK2 and cyclinE2.Conversely,inhibition of miR-590-3p up-regulated the expression of PPP2R2 A,p27 and G1/S cell cycle pathway-related proteins p21 in pancreatic cancer cells,and down-regulated the expression of CDK2 and cyclinE2.Mi R-590-3p was also negatively correlated with the expression of p27 and PPP2R2 A in clinical tumor samples.Dual-luciferase reporter gene assay revealed that mi R-590-3p can bind directly to the 3 ’UTR region of p27 and PPP2R2 A.The overexpression of P27 and PPP2R2 A reversed the promoting effect of miR-590-3p on cell proliferation,migration and invasion.Conclusion: 1.MiR-590-3p promotes the proliferation,migration and invasion of pancreatic cancer cells.2.MiR-590-3p directly downregulated p27 and PPP2R2 A and via the G1/S cell cycle pathway to promote the development of pancreatic cancer. |
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