Study On The Effect Of MiCroRNA-23a On EMT In Pancreatic Cancer And Its Mechanism

MiCroRNA-23 a regulates epithelial-mesenchymal transition of pancreatic cancer cells via ESRP1Objective1 To investigate the relationship between the expression of MiCroRNA-23 a in pancreatic cancer tissues and its clinicopathological data.2 To study the expression of MiCroRNA-23 a in different pancreatic cancer cell lines(Aspc-1,Bxpc-3,Cfpac-1,Panc-1).3 To investigate the promotion effect of MiCroRNA-23 a on invasion and metastasis of pancreatic cancer cells in vitro and in vivo.4 To study whether mi Cro RNA-23 a can change the expression of CD44 subtypes,promote epithelial mesenchymal transition(EMT)of pancreas cancer cells,and promote invasion and metastasis of pancreatic cancer cells by target inhibition of epithelial splicing regulator protein 1(ESRP1).Methods1 Pancreatic cancer specimens and clinicopathological data of 47 patients were collected,who received hepatobiliary surgeries in the Department of Hepatobiliary Surgery of Southwest Hospital from January 2013 to December 2013.FOS and OS of all the patients were recorded.The expression of MiCroRNA-23 a in primary pancreatic cancer tissues,metastatic lymph nodes and adjacent normal pancreatic tissues was detected by RT-PCR,and the expression of MiCroRNA-23 a in these three types of tissues was compared.The relative expression of MiCro RNA-23 a in cancer tissues was calculated.All samples were classified into MiCroRNA-23 a high expression group and low expression group according to the value.The relationship between MiCroRNA-23 a expression and clinicopathological data was analyzed.Kaplan-Meier software was applied for survival analysis,and the effect of different expression levels of MiCro RNA-23 a on prognosis of patients with pancreatic cancer was analyzed.2 qRT-PCR and Western blot were used to detect the expressions of E-cadherin(E-cad),N-cadherin(V-cad)and vimentin(VIM),the morphology of cells was observed,TGF-?1 was used to induce EMT treatment,and the epithelial and mesenchymal phenotype of pancreatic cancer cell lines Aspc-1,Bxpc-3,Cfpac-1 and Panc-1 were differentiated.The expression of mi RNA-23 a in normal pancreatic ductal epithelial cells(PDC)and pancreatic cancer cells(Aspc-1,Aspc-1 + TGF-?1,Bxpc-3,Bxpc-3 + TGF-?1,Cfpac-1,Panc-1)was detected by qRT-PCR,and the relationship between its expression and EMT phenotype of pancreatic cancer cells was observed.The effect of interference of miR-23 a on TGF-?1 in inducting EMT was observed.3 In order to observe the changes of invasion and migration abilities,MiR-23 a mimic and mi R-23 a inhibitor were transfected into Aspc-1 and Panc-1,respectively;the mi R-23 a sponges lentiviral expression vector was constructed,Panc-1 cells were transfected,stable cell lines were established,and the effect of MiR-23 a in promoting metastasis of pancreatic cancer was observed in in-vivo experiments.4.The results of microarray analysis and bioinformatics website(TargetScan.com)were cross-compared and dual-luciferase reporter plasmid experiments were used to verify that ESRP1 is a direct target gene of mi R-23 a.5 The effect of interference of miR-23 a on the expression of ESRP1 was investigated,qRT-PCR was used to detect the expression of ESRP1 in pancreatic cancer tissues,and its relationship with the mi R-23 a expression was analyzed;the effect of interference or overexpression of ESRP1 on the invasion and metastasis of pancreatic cancer cells was observed;and ESR1 response assay was applied to observe the reversal effect of mi R-23 a on EMT of pancreatic cancer cells.6 Western blot and qRT-PCR were used to detect the effect of mi R-23 a on the expression of ESRP1 and its downstream CD44 s / CD44 v,FGFRIIIb / IIIc spliciced isoforms.ESR1 response assay was applied to observe the effect of interfering miR-23 a expression on the expression of downstream splicing molecules.Result1 The expression of mi R-23 a in metastatic pancreatic cancer lymph nodes tissues was significantly higher than that in primary pancreatic cancer tissues and adjacent normal tissues.The expression of MiCroRNA-23 a in pancreatic cancer tissues was significantly correlated with tumor differentiation,tumor infiltration depth(T stage)and pancreatic cancer lymph node metastasis(N stage),but it is not associated with age,gender,tumor sites and distant metastasis(M stage).Survival analysis revealed that mi CroRNA-23 a expression was correlated with the prognosis of patients with pancreatic cancer.Patients with high expression mi CroRNA-23 a had poor prognosis and the survival time was lower than that of the low expression group,with significant statistical difference.2.Aspc-1 and Bxpc-3 cells were epithelial phenotype,Cfpac-1 and Panc-1 were interstitial phenotypes.EMT occurred after TGF-?1 induced Aspc-1 and Bxpc-3.The expression of miR-23 a was significantly increased in pancreatic cancer cells with mesenchymal phenotype.MiR-23 a expression was increased in pancreatic cancer metastatic lymph node tissues,suggesting that mi R-23 a may promote EMT of pancreatic cancer cells and their invasion and metastasis abilities.Interference of mi R-23 a inhibited the occurrence of EMT of TGF-?1-induced pancreatic cancer cells with epithelial phenotype.3 In vitro experiments showed that miR-23 a mimic transfected with Aspc-1 enhanced its invasion and migration abilities,and that mi R-23 a inhibitor transfected with Panc-1 decreased its invasion and migration abilities.In vivo experiments showed that mi R-23 a sponges lentiviral expression vector transfected with Panc-1 cells could inhibit the function of mi R-23 a,thus inhibiting the growth and metastasis of pancreatic cancer cells.4 Gene chip analysis and bioinformatics retrieval confirmed that ESRP1 was a direct downstream target gene of miR-23 a,and luciferase reporter plasmid experiments demonstrated that ESRP1 was a direct target gene of mi R-23 a.5 ESRP1 maintained the epithelial phenotype of pancreatic cancer cells,an d interfering mi R-23 a induced the expression changes of ESRP1.The expression of ESRP1 was negatively correlated with the expression of mi R-23 a in pancreatic cancer tissues.ESRP1 si RNA transfected with Aspc-1 enhanced its invasion and migration abilities.The ESRP1 overexpression plasmid transfected with Panc-1 weakened its invasion and migration abilities.ESRP1 recovery assay could partially reverse the effect of interfering mi R-23 a expression on the EMT of pancreatic cancer cells.6 qRT-PCR and WB assays confirmed that miR-23 a inhibited the expression of downstream splicing molecules CD44 s / CD44 v and FGFRIIIb / IIIc through target inhibition of the expression of ESRP1,and ESRP1 recovery assay could partially reverse the effect of interfering mi R-23 a expression on the expression of downstream splicing molecules.Conclusion1 The expression of MiCroRNA-23 a in metastatic pancreatic cancer lymph nodes tissues was significantly higher than that in primary pancreatic cancer tissues and adjacent normal pancreatic tissues.The expression of MiCroRNA-23 a was significantly positively correlated with tumor differentiation,depth of tumor invasion and lymph node metastasis of pancreatic cancer.MiCroRNA-23 a expression was significantly correlated with the prognosis of pancreatic cancer.2 MiCroRNA-23 a was highly expressed in pancreatic cancer cells with epithelial phenotype,and MiCroRNA-23 a was highly expressed in pancreatic cancer cells with mesenchymal phenotype.The expression of MiCroRNA-23 a was significantly increased when the pancreatic cancer cells with epithelial phenotype occurred EMT,hence,MiCroRNA-23 a may promote occurrence of EMT of pancreatic cancer cells.3 Overexpression of MiCroRNA-23 a can promote the transition of pancreatic cancer cells from epithelial phenotype to mesenchymal phenotype.Inhibition of MiCroRNA-23 a expression can promote the transition of pancreatic cancer cells from mesenchymal phenotype to epithelial phenotype.4 MiCroRNA-23 a can enhance the invasion and metastasis abilities of pancreatic cancer cells in vitro and in nude mice.5 MiCroRNA-23 a can regulate the EMT of pancreatic cancer cells through target inhibition of the expression of ESRP1,thus promoting the invasion and metastasis abilities of pancreatic cancer cells.6 ESRP1 affects the epithelial mesenchymal transition of pancreatic cancer cells by regulating the alternative splicing of CD44 s / CD44 v and the expression of FGFR2 IIIb / IIIc molecules.