Researches Of Mir-675-5p On Regulating Epithelial-mesenchymal Transition Of Pancreatic Cancer Cells Via UBQLN1-ZEB1-mir-200

Objective:The research was aimed at detecting the expression of mir-675-5p in pancreatic cancer cells,analyzing the relationship between the expression of mir-675-5p in tumor tissues and overall survival of pancreatic cancer,investigating the effects of mir-675-5p on cell proliferation,apoptosis,migration and invasion of pancreatic cancer and exploring the underlying molecular mechanisms of mir-675-5p on EMT.Methods:1.Analyzing the clinical significance of mir-675 by interrogating the TCGA datasets.Making statistical analysis on the relationship between the expression of mir-675 and overall survival of pancreatic cancer,TNM stage and maximum tumor dimension using SPSS Statistics 20 software and Graphpad Prism5.2.Using qRT-PCR to examine the expression of mir-675-5p on different pancreatic cancer cells and verify the efficiency of transfection with mir-675-5p mimics into Patu8988 cells or inhibitor into SW1990 cells by small RNA interfering technology.3.We used CCK-8 assay to examine the cell proliferation activity,plate colony formation assay to detect the colony formation ability and the transwell assay to detect the migration or invasion ability of pancreatic cancer cells.The apoptotic ability and the change on cell cycle were examined by flow cytometry assay.All the experiments were conducted to explore the effects of mir-675-5p on cell function.4.Detecting the mRNA level of RB1,ZEB1 and mir-200 family by qRT-PCR.Examining the expression of proliferation and apoptotic related protein PCNA,Bcl-2,Bax,cleaved-caspase3,cell invasion related protein MMP2,MMP9,EMT related protein E-cadherin,N-cadherin,ZEB1,Snail,Slug and Vimentin by Western blotting.5.Using UBQLN1 si RNAs to decrease the expression of UBQLN1 in Patu8988 cells and SW1990 cells.The Patu8988 cells were tranfected with UBQLN1 siRNAs and mir-675-5p mimics while the SW1990 cells were tranfected with UBQLN1 siRNAs and mir-675-5p inhibitor.The transfection efficiency of UBQLN1 and the mRNA or protein expression of ZEB1 were detected by qRT-PCR after cotransfection.Results:1.The higher expression of mir-675 in tumor tissues can provide longer survival time and smaller maximum tumor size for pancreatic cancer patients.Moreover,there was no relationship between the expression of mir-675 and TNM stage on PDAC patients.2.Mir-675-5p expressed in four pancreatic cancer cells(Patu8988< Panc-1<Bxpc-3< SW1990)differentially.SW1990 cells had the highest miR-675-5p expression while Patu8988 cells had the lowest miR-675-5p expression.3.The increased expression of mir-675-5p in Patu8988 cells can impair the cell proliferation,inhibit the ability of cell colony formation,promote cell apoptosis and cause cell cycle arrest at the G1/S phase.In Patu8988 cells transfected with miR-675-5p mimics,PCNA and the ratio of Bcl-2 to Bax were decreased.On the other hand,the cleaved caspase-3 was activated.The cell invasion related protein MMP2 and MMP9 were downregulated and the protein levels of E-cadherin were increased whereas the expression levels of N-cadherin,ZEB1,Vimentin,Snail and Slug were significantly decreased by miR-675-5p mimics in Patu8988 cells.The decreased expression of mir-675-5p in SW1990 cells can promote the cell proliferation and cell colony formation.In SW1990 cells transfected with miR-675-5p inhibitor,PCNA and the ratio of Bcl-2 to Bax were increased.On the other hand,the cleaved caspase-3 was suppressed.The cell invasion related protein MMP2 and MMP9 were upregulated and the protein levels of E-cadherin were decreased whereas the expression levels of N-cadherin,ZEB1,Vimentin,Snail and Slug were significantly increased by miR-675-5p inhibitors in SW1990 cells.4.MiR-675-5p mimics increased the relative expression of ZEB1 mRNA and RB1 mRNA,but decreased miR-200 a,miR-200 b and miR-200 c in Patu8988 cells.In contrast,miR-675-5p inhibitors decreased ZEB1 mRNA and RB1 mRNA,but increased miR-200 a,miR-200 b and miR-200 c in SW1990 cells.5.Both the relative mRNA and protein levels of UBQLN1 were increased by miR-675-5p mimics in Patu8988 cells;in contrast,miR-675-5p inhibitors suppressed UBQLN1 in SW1990 cells6.Interestingly in Patu8988 cells,miR-675-5p mimics increased the ZEB1 mRNA but this upregulation can be reversed by suppressing the expression of UBQLN1.In SW1990 cells,miR-675-5p inhibitors decreased the mRNA level of ZEB1 and UBQLN1 siRNAs maintained the results.Western blot showed that the protein levels of ZEB1 and Snail were also decreased by mi R-675-5p mimics in Patu8988 cells and the trend can be reversed by UBQLN1 siRNAs.In contrast,the protein levels of ZEB1 and Snail were increased by miR-675-5p inhibitors in SW1990 cells and the trend can be maintained by UBQLN1 siRNAs Conclusion:1.The higher expression of mir-675 can lead to better overall survival.2.Mir-675-5p can impair the proliferation,migration,invasion and promote apoptosis of pancreatic cancer.3.Mir-675-5p may affect ZEB1 in a post-transcriptional level which was verified to be regulated by UBQLN1 protein.4.Mir-675-5p can promote the expression of UBQLN1 which can decrease the protein level of ZEB1.Mir-200 can be regulated by mir-675-5p through the intermediate gene ZEB1.MiR-675-5p regulates the progression and development of pancreatic cancer via the UBQLN1-ZEB1-mir200 axis.