Study Of MiR-223 Regulating Drug Resistance Of Gemcitabine In Human Pancreatic Cancer Cells

ObjectiveWe established gemcitabine-resistant(GR)pancreatic cancer cell lines and Subsequently observed the difference of the cell phenotype and malignant biological behavior between GR cell and parental cell.We also explored the potential role of miR-223 in governing EMT in GR cells.Moreover,we explored whether there was the synergistic effect between miR-223 inhibitor and genistein on the increasing sensitivity of the GR cells to gemcitabine.Methods1.The pancreatic cancer cell lines were treated with increasing concentrations of gemcitabine for more than 6 months to established GR cells.MTT assay was used to detect the gemcitabine sensitivity for confirmation of successful establishment of GR cells.Cell morphology was observed by microscopy in parental and GR cells.Cell migration and invasion capacity was measured by Wound healing assays,transwell migration and invasion assay between parental and GR cells.Cell attachment and detachment assays were assessed in parental and GR cells to analyze the cell attachment and detachment capacity.qRT-PCR and western blotting assays were performed to detect mRNA and protein expression of EMT markers in parental and GR cells,including E-cadherin,Vimentin,Snail,Slug,ZEB1,ZEB2.2.To define whether miR-223 is involved in GR-induced EMT in PC cells,we detected the expression of miR-223 in GR cells and their parental cells.Cell morphology was taken by microscopy in GR cells transfected with miR-223 inhibitor in AsPC-1 GR and PANC-1 GR cells transfected with miR-223 inhibitor.qRT-PCR and western blotting assays were performed to detect the mRNA and protein levels of EMT markers.Moreover,wound healing assay and transwell assay were conducted to detect the migration capacity and invasion capacity in AsPC-1 GR and PANC-1 GR cells transfected with miR-223 inhibitor.Cell attachment and detachment assays were measured in AsPC-1 GR and PANC-1 GR cells transfected with miR-223 inhibitor.Furthermore,FBW7 and Notch1 expressions were measured in GR cells.More importantly,to determine whether down-regulation of miR-223 enhances GR cells to gemcitabine sensitivity,we performed MTT assay in GR cells treated with miR-223 inhibitor.3.MTT assay was implemented to detect the cells proliferation after genistein treatment in AsPC-1 GR and BxPC-3 GR cells.Moreover,we performed RT-PCR analysis to measure the expression of miR-223 in GR cells after genistein treatment.To investigate whether there was the synergistic effect between miR-223 inhibitor and genistein cells,we set up the experimental groups as follows,including control group,miR-223 inhibitor transfection group,genistein treatment group,both miR-223 inhibitor transfection and genistein treatment group.The cell morphology and EMT markers expression were detected in GR cells with different treatment as mentioned above.Moreover,FBW7 and Notch1 expressions were measured in GR cells with different treatments as mentioned above.Furthermore,we also observed the influence of combination of miR-223 inhibitor and genistein on the malignant biological behavior in AsPC-1 GR and Bx PC-3 GR cells.Similarly,we also detected the sensitivity to gemcitabine by MTT assay in AsPC-1 GR and Bx PC-3 GR cells treated with miR-223 inhibitor or genistein or the combination of miR-223 inhibitor and genistein.Results1.After incubated with increasing concentrations of gemcitabine for more than 6 months,the cells reached to steady growth with the final concentration of gemcitabine.The final gemcitabine concentrations were 100 μM for AsPC-1 cells,5 μM for PANC-1 cells and 6 μM for Bx PC-3 cells,respectively.MTT assay has showed the final gemcitabine concentration treatment caused significantly growth inhibition in parental pancreatic cancer cells.However,the final concentration of gemcitabine did not cause the cell growth inhibition in AsPC-1 GR,PANC-1 GR and Bx PC-3 GR,respectively.Cell morphology has showed that the parental cells displayed a rounded shape,whereas GR cells exhibited EMT phenotype carrying with elongated,fibroblastoid morphology.Wound healing assay and transwell migration assay have demonstrated that that GR cells have higher numbers of cells migrating across the wound than the parental cells.We also measured the cell invasion capacity using transwell invasion assay,which exhibited that GR cells have significantly increased numbers of invaded cells through a Matrigel-coated membrane compared with parental cells,suggesting that GR cells obtained enhanced invasion.In line with these findings,we identified that GR cells have increased attached and detached activities compared with parental cells.To identify whether GR cells have EMT molecular marker changes,we measured the mRNA and protein levels of EMT markers using RT-PCR and westein blotting in paired parental cells and GR cells.We found that epithelial molecule E-cadherin mRNA and protein levels were down-regulated,while the mRNA and protein levels of mesenchymal markers including Vimentin,Snail,Slug,ZEB1 and ZEB2 were up-regulated in GR cells.2.To defne whether miR-223 is involved in GR-induced EMT in PC cells,we detected the expression of miR-223 in GR cells and their parental cells.We observed that miR-223 was highly expressed in GR cells,suggesting that GR-mediated EMT could be partly due to over-expression of miR-223.Furthermore,We found that miR-223 inhibitor treatment caused round cell-like morphology in AsPC-1 GR and PANC-1 GR cells.Additionally,we found that the expression of E-cadherin was signifcantly increased in GR cells after inhibition of miR-223,whereas the expression of mesenchymal markers including Vimentin,Snail,Slug,ZEB1 and ZEB2 was markedly deceased in AsPC-1 GR and PANC-1 GR cells with miR-223 inhibitor treatment.These fndings identifed that down-regulation of miR-223 could reverse EMT to MET in GR cells.Wound healing assay and transwell assay demonstrated that inhibition of miR-223 signifcantly retarded the migration and invasion in AsPC-1 GR and PANC-1 GR cells.Furthermore,mi R-223 inhibitor treatment reduced the capacity of attachment and detachment in GR cells.To further define whether Fbw7 plays a key role in GR-mediated EMT,we measured the expression of Fbw7 at mRNA and protein levels in GR cells and their parental cells using real-time RT-PCR and Western blotting,respectively.We observed that the expression of Fbw7 at mRNA and protein was markedly decreased in AsPC-1 GR and PANC-1 GR cells compared with parental cells.Consistently,the expression of Fbw7 substrate Notch-1 was signifcantly increased.Moreover,we found that miR-223 inhibitor treatment led to upregulation of Fbw7 in AsPC-1 GR and PANC-1 GR cells.Furthermore,Notch-1 expression was downregulated in GR cells treated with miR-223 inhibitor.These fndings indicated that the acquisition of EMT could be in part due to down-regulation of Fbw7 and subsequent overexpression of Notch-1 in As PC-1 GR and PANC-1 GR cells.Additionally,we observed that inhibition of miR-223 signifcantly increased sensitive to gemcitabine-induced cell growth inhibition.3.We found that genistein inhibited cell growth in a dose-dependent manner in both AsPC-1 GR cells and BxPC-3 GR cells.Moreover,we performed RT-PCR analysis to measure the expression of miR-223 in GR cells after genistein treatment for 72 h.The results showed that miR-223 level was remarkably down-regulated in AsPC-1 GR cells and Bx PC-3 GR cells treated with genistein.Furthermore,we found that both genistein treatment and mi R-223 inhibitor caused round cell morphology change from elongated,fbroblastoid morphology in GR cells.More importantly,combination of genistein with mi R-223 inhibitor led to the remarkable morphology change in GR cells.In addition,we observed that both mRNA and protein expressions of E-cadherin were obviously up-regulated by genistein and miR-223 inhibitor treatments compared with the single treatment.However,the expressions of mesenchymal markers,such as Slug,Vimentin,Snail,ZEB1 and ZEB2,were down-regulated to a greater degree in the combination treatment.Furthermore,we observed that genistein plus miR-223 treatment caused up-regulation of Fbw7 and down-regulation of Notch-1 in a deeper degree compared with the single treatment.Our wound healing assay and transwell migration and invasion assays showed that genistein and mi R-223 inhibitor inhibited cell migration and invasion capacity.Consistently,genistein and miR-223 inhibitor treatment inhibited the cells detachment and attachment capacity to a greater degree compared to single treatment alone.Our MTT results showed that both genistein and miR-223 inhibitor caused the more signifcant growth inhibition than genistein alone and miR-223 inhibitor alone in GR cells.ConclusionsGR cells displayed mesenchymal features and acquired increased motility and invasiveness in our results,suggesting that GR cells acquire EMT characteristics.These results indicated that the drug resistance to gemcitabine was associated with EMT in pancreatic cancer.Notably,miR-223 played a key role in regulation EMT associated drug resistance.inhibition of miR-223 led to the reversal of EMT phenotype and inhibition of migration and invasion.Furthermore,inhibition of miR-223 led to the enhanced the cell sensitivity to gemcitamine.More importantly,mi R-223 governs GR-induced EMT in part due to down-regulation of its target Fbw7 and subsequent upregulation of Notch-1 in pancreatic cancer.Our study implied that down-regulation of miR-223 could be a novel therapy for pancreatic cancer.Moreover,combination of miR-223 inhibitor and genistein synergistically reduced cell motility and invasion and enhanced gemcitabine sensitivity in GR cells.In addition,we further observed that miR-223 inhibitor and genistein reversed EMT features in GR cells.This study suggests that the combination of miR-223 inhibitor and genistein may be a potential therapeutic strategy for the treatment of pancreatic cancer.