Mechanisms Of Epithelial-mesenchymal Transition Regulation By MicroRNA And Its Effect On The Invasion And Migration Of Nasopharyngeal Carcinoma Cells

Aims:1. To investigate the correlation biological characteristics of drug resistancenasopharyngeal carcinoma cells and the phenotype of epithelial-mesenchymaltransition (EMT), and to explore the relationship among EMT, drug resistance andmetastasis in nasopharyngeal carcinoma.2. To investigate the expressive changes of miRNAs and to further determine andverify differentially expressed miRNA in drug resistance nasopharyngeal carcinomacells.3. To explore the mechanisms of EMT regulation by miRNA and its effect on theinvasion and migration of nasopharyngeal carcinoma cells.Methods:1. HNE1, HNE1/DDP cells were treated with different concentrations of cisplatin(DDP) for24,48,72h, respectively, the viability rate was detected in order to makeclear the dose-effect relationship and resistance index (RI) by MTT assay. Thenumber of cells was counted at24,48,72,96,120,144,168h in HNE1andHNE1/DDP cells by cell growth assay, respectively. The protein expression levels ofMRP, Mcl-1, Bcl-2, Bax, Puma in HNE1and HNE1/DDP cells were detected byWestern blot. The mRNA levels of MDR1, MRP, Bcl-2, Bax in HNE1andHNE1/DDP cells were detected by quantitative real-time RT-PCR (qRT-PCR).2. Cell migration was assessed in HNE1and HNE1/DDP cells using a wound healingassay. To detect the ability of migration and invasion in vitro, transwell chambers forcell migration experiments and the reconstituted basement membrane for invasionassay was adopted. The morphological characteristics of both cell lines were observed under a light microscopy. The expression levels of EMT-related proteins E-cadherin,β-catenin, Vimentin, Fibronectin, MMP-9and EMT-related transcription factors Snail, Slug,Twist and ZEB1were determined in HNE1and HNE1/DDP cells by Western blot. ThemRNA expression levels of EMT-related makers E-cadherin, β-catenin, Vimentin, Fibronectin,MMP-9and EMT-related transcription factors Snail, Slug, Twist and ZEB1were determinedin HNE1and HNE1/DDP cells by qRT-PCR.3. To detect the expressive changes of miRNAs and to further select differentiallyexpressed miRNA in in HNE1and HNE1/DDP cells by a miRNA microarray.QRT-PCR was applied to verify the selected miRNA. The expression levels of HIF-1αinHNE1and HNE1/DDP cells were determined by Western blot and qRT-PCR.4. Cell was transfected with miRNA mimics/inhibitors, the level of miRNA byqRT-PCR, cell migration using a wound healing array, the capacity of migration andinvasion by Transwell Boyden chamber assays, the expression levels of E-cadherin,Vimentin, MMP-9by Western blot and qRT-PCR, and the morphological characteristicsunder a light microscopy.5. To predict potential target genes of miRNA and effect on target genes regulation ofmiRNA by western blot and qRT-PCR. After transfection with small interfering RNA(siRNA), the levels of protein and mRNA by Western blot and qRT-PCR, cellmigration using a wound healing array, the capacity of migration and invasion byTranswell Boyden chamber assays, the morphological characteristics under a lightmicroscopy, and the expression levels of E-cadherin, Vimentin, MMP-9by Western blot andqRT-PCR.Results:1. HNE1/DDP cells exhibit chemoresistance to DDP(1) The proliferation of NPC cells could be inhibited by different concentrations of DDP (2,4,8,16,32μmol·L-1) for24,48,72h by MTT assay, and the inhibitory effect was morepronounced when the concertrations of DDP was increased. The IC50values of HNE1/DDPand HNE1cells were29.04±2.82,19.44±1.77,11.39±1.89μmol·L-1and6.84±1.59,4.25±0.36,2.35±0.96μmol·L-1for24,48, and72h, and RI were4.25,4.57,4.85, respectively.In addition, HNE1/DDP cells were less sensitive to treatment with DDP than HNE1cells, and HNE1/DDP cells were less efficiently proliferative under normal culture conditions,compared with HNE1cells.(2) The expression levels of MRP, Mcl-1and Bcl-2were higher in HNE1/DDP cellscompared with HNE1cells by western blot. But the expression levels of Bax and Puma werelower in HNE1/DDP cells. In addition, the mRNA expression levels of MDR1, MRP, Bcl-2were up-regulated and Bax were down-regulated in HNE1/DDP cells by qRT-PCR.2. HNE1/DDP cells have an increased invasion and migration potential and theacquisition of EMT(1) The capacity of cell migration was obviously enhanced in HNE1/DDP cellscompared with HNE1cells by wound healing assay. The ability of migration andinvasion was increased in HNE1/DDP cells compared with HNE1cells byTranswellBoyden chamber assays in vitro.(2) Cell morphology was observed by microscopy. The HNE1cells showed a cell-cellconnection, colony growth, rounded and contained little formations of pseudopodia, similar toepithelial-like. In contrast, the phenotypic changes observed in HNE1/DDP cells includedcell-matrix connection, narrow shape, dispersed growth, and increased formation ofpseudopodia, similar to mesenchymal-like.(3) The results from western blot and qRT-PCR showed that the expression of epithelialmarkers, such as E-cadherin and β-catenin, was lower in HNE1/DDP cells compared withHNE1cells. The expression of mesenchymal markers, such as Vimentin, Fibronectin, andMMP-9was higher in HNE1/DDP cells. Moreover, the mRNA and protein levels of theEMT-related transcription factors Snail, Slug, Twist and ZEB1were higher in HNE1/DDPcells compared with HNE1cells.3. The expression profile of miRNAs in NPC cellsMicroarray analysis showed a significant upregulation of18miRNAs and downregulation of9miRNAs in HNE1/DDP compared with HNE1cells. Among all miRNAs examined,miR-10b was the most significantly dysregulated miRNA, which was up-regulated25-fold inHNE1/DDP compared with HNE1cells by qRT-PCR. Meanwhile, the expression of HIF-1αwas upregulation in HNE1/DDP cells by Western blot and qRT-PCR.4. MiR-10b regulates EMT in NPC cells and effect on invasion and migration (1) The HNE1cell line, which expressed lower level of miR-10b, was selected fortransfection with miR-10b mimics. HNE1/DDP cell line, which showed higher level ofmiR-10b, was chosen for transfection with miR-10b inhibitors. The levels of miR-10b weresignificantly elevated by the miR-10b mimics and reduced by the miR-10b inhibitors byqRT-PCR.(2) The capacity of cell migration was significantly enhanced in HNE1cells by miR-10bmimics, and reduced in HNE1/DDP cells by miR-10b inhibitors by wound healing assay. Inaddition, the relative migrated and invaded cell numbers were significantly increased inHNE1cells by miR-10b mimics, and decreased in HNE1/DDP cells by miR-10b inhibitors byTranswell Boyden chamber assays.(3) The expression of E-cadherin was reduced in the HNE1cells by transfected with miR-10bmimics by qRT-PCR and western blot, but the expression of vimentin and MMP-9werehigher, respectively. Moreover, the mRNA and protein levels of E-cadherin were increased inHNE1/DDP cells by transfected with miR-10b inhibitors, but the expression of vimentin andMMP-9were lower, respectively. Morphology study showed that the HNE1cells bytransfected with miR-10b mimics had more mesenchymal cells. However, the HNE1/DDPcells by transfected with miR-10b inhibitors seemed to change the morphology frommesenchymal-like spindle-cell shape to epithelial-like.5. MiR-10b regulates EMT through modulating KLF4and Notch1in NPC cells(1) By miRTarBase to predict targets of miR-10b in human, and KLF4and Notch1wereselected among them for further analysis. The mRNA and protein levels of KLF4werereduced in HNE1/DDP cells compared with HNE1cells, but the mRNA and protein levels ofNotch1were higher.(2) The expression of KLF4was significantly reduced in HNE1cells transfected withmiR-10b mimics and increased in HNE1/DDP cells transfected with miR-10b inhibitors byWestern blot. But the mRNA level of KLF4showed that over-expression or inhibition ofmiR-10b had no effect on KLF4level by qRT-PCR. Interestingly, the results from Westernblot and qRT-PCR showed that the levels of activated Notch1were significantly increased inHNE1cells transfected with miR-10b mimics, and decreased in HNE1/DDP cells transfectedwith miR-10b inhibitors. (3) Notch1siRNA and control siRNA were transfected into HNE1/DDP cells. After48hincubation, the mRNA and protein levels of Notch1were decreased by Western blot andqRT-PCR. The capacity of cell migration was significantly reduced in HNE1/DDP cellstransfected with Notch1siRNA by wound healing assay. In addition, the relative migratedand invaded cell numbers were significantly decreased in HNE1/DDP cells transfected withNotch1siRNA by Transwell Boyden chamber assays.(4) Morphology study showed that the HNE1/DDP cells transfected with Notch1siRNAseemed to change the morphology from mesenchymal-like spindle-cell shape toepithelial-like. Moreover, the mRNA and protein levels of E-cadherin were increased, but theexpression of vimentin and MMP-9were decreased, respectively.Conclusion:1. The development of DDP resistance in NPC cells was accompanied by inducible EMT-likechanges with increased potential to invasion and migration in vitro.2. The miRNAs were differentally expressed in NPC cells, which maybe plays an importantrole in the underlying mechanisms of DDP resistance and induction of EMT-like properties.3. Over-expression of miR-10b could promote invasion and migration and the acquisition ofEMT in HNE1cells. In contrast, inhibition of miR-10b expression reversed EMT. Moreover，the post-transcriptional regulation of miR-10b on KLF4level, and miR-10b may regulateEMT through KLF4in human NPC cells.4. MiR-10b over-expression or inhibition was consistent with the level of Notch1.Knockdown of Notch1decreased cell migration and invasion, and reversed EMT inHNE1/DDP cells. Decreased expression of Notch1could reverse EMT in NPC cells byregulation of miR-10b.