| Pancreatic ductal adenocarcinoma(PDAC)is one of the most commonly diagnosed cancer.Due to its hidden physical location,early diagnosis is tough.Most patients are not diagnosed until PDAC has metastasized to other organs,therefore the 5-year relative survival is only 5-8%.Gemcitabine(2′,2′-difluorodeoxycytidine),a deoxycytidine analogue,represents the first line intervention for the treatment of advanced PDAC,however,the total efficiency is less than 20%,about 80% of the patients died of tumor metastasis within 1 year.Emerging evidence suggests the association between PDAC chemoresistance and the acquisition of epithelial-mesenchymal transition(EMT)phenotype and/or the existence of a sub-population of cancer stem-like cells(CSC)within the tumor mass.These chemoresistant cancer cells are refractory to gemcitabine and highly prone to metastasize and relapse.Therefore,to reveal the mechanisam of the CSCs key molecules would provide theoretical and experimental basis for reverting gemcitabine resistance and accurate target strategy for PDAC.To investigate the function and mechanism of long noncoding RNA(lnc RNA)in PDAC gemcitabine resistance,EMT and CSCs,a gemcitabine resistant cell line(Bx PC-3-Gem)was established through exposing parental Bx PC-3 cells to increased concentrations of gemcitabine for 16 months.As only a small population with EMT and/or CSC phenotypes remained after each selection,The EMT/CSC properties were compared between Bx PC-3-Gem cells and Bx PC-3.ZEB1,which initiates the EMT program through downregulation of E-cadherin and upregulation of vimentin,was increased in Bx PC-3-Gem cells,along with decrease of E-cadherin and increase of vimentin.All above phenomena proved that the pancreatic cancer cells underwent EMT and CSCs in the evolution process from gemcitabine sensitive to resistant.Through analysis of lnc RNA microarray,the longe intergenic noncoding RNA DYNC2H1-4(linc-DYNC2H1-4)was found that significantly high-regulated in pancreatic gemcitabine resistant cells than that in sensitive cells.The closest gene to linc-DYNC2H1-4 in the sense strand is DYNC2H1,which it was named after.No significant difference of DYNC2H1 expression was found between Bx PC-3-Gem and Bx PC-3.In contrast,the expressions of nearby genes in the antisense strand,MMP1,MMP3 and MMP27,were significantly different,among which MMP3 showed the most significant difference.To address the role of linc-DYNC2H1-4 in the formation of EMT and CSC phenotypes in gemcitabine resistant cells,siRNAs which targeting linc-DYNC2H1-4 were transfected into Bx PC-3-Gem cells.After that,the levels of MMP3,ZEB1 and vimentin,as well as Oct4,Lin28,Nanog and Sox2 were significantly decreased,while the level of E-cadherin was increased.Relative to these molecular alterations,knockdown of linc-DYNC2H1-4 inhibited the EMT and stem cell properties of Bx PC-3-Gem cells,and the opposite results were obtained after the overexpression of linc-DYNC2H1-4.These results demonstrated that linc-DYNC2H1-4 promoted EMT and CSCs properties in pancreatic cancer cells.Furthermore the molecular mechanism of linc-DYNC2H1-4 to promote the ability of EMT and CSCs in pancreatic cancer cells was discussed.First,the cytosolic localization of linc-DYNC2H1-4 supported the possibility that linc-DYNC2H1-4 acts as a sponge of miRNAs.The dual luciferase report system showed that linc-DYNC2H1-4 interacted with miR-145 through complementary binding sites.The expression level of linc-DYNC2H1-4 and miR-145 were tested after overexpression or knockdown,as well as the endogenous expression levels in the pancreatic cancer cells.The results showed that linc-DYNC2H1-4 functioned as sponge of miR-145 which inhibited EMT and CSCs in pancreatic cancer cells.It is also reported that miR-145 targeted CSCs markers Lin28,Oct4,Sox2,Nanog and EMT marker ZEB1.As a transcription factor,ZEB1 upregulates vimentin and inhibits the expression of E-cadherin.Luciferase reporter gene assay showed that MMP3 3’UTR reporter activity dropped with miR-145 co-transfection.The expression level of MMP3 after miR-145 was down-regulated as well.These results demonstrated that miR-145 targets MMP3 though MMP3 3’UTR.Rescure experiemnt confirmed that linc-DYNC2H1-4 could revert the functions of miR-145 which inhibited EMT and CSCs in pancreatic cancer cells.In summary,a pancreatic caner cell line Bx PC-3-Gem with acquired gemcitabine resistance has been established.Linc-DYNC2H1-4 expression is significantly different in the pancreatic resistant and sensitive cells.Linc-DYNC2H1-4 acts as a sponge of miR-145 which inhibits EMT and CSCs progression of pancreatic caner cells in both molecular and cellular level s. |
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