MiR-196a Promoted Cell Metastasis Of Human Colorectal Cancer By Modulating Epithelial-Mesenchymal Transition

Increasing evidence indicated that microRNAs(miRNAs)played critical roles in cancer metastasis by regulating epithelial-mesenchymal transition(EMT)process.Here,we found miR-196 a was upregulated in human colorectal cancer(CRC)cell lines and clinical tumor tissues by qRT-PCR.Result of wound healing and cell migration assays revealed that miR-196 a promoted cell metastasis,whereas miR-196a-in showed the opposite effect.EMT-associated makers(E-cadherin,?-catenin,and vimentin)were evaluated using qRT-PCR and Western Blots.miR-196 a overexpression upregulated vimentin expression,while downregulated E-cadherin and ?-carenin expression.In conclusion,miR-196 a promoted CRC cell metastasis by modulating EMT,and miR-196 a might be used as therapeutic target for CRC.Objective1.QRT-PCR was used to test the expression of miR-196 a in colorectal cancer cell lines and colorectal cancer tissues and paracancerous tissues,select the appropriate colorectal cancer cells to be transfected and establish the control group,use wound healing and cell migration assays to revealed that miR-196 a promoted cell metastasis.2.EMT-associated makers(E-cadherin,?-catenin,and vimentin)were evaluated by qRT-PCR and Western Blots.Analysis of whether miR-196 a regulates the EMT process.Methods1.QRT-PCR was used to detect the miR-196 a expression of eight colorectal cancer and adjacent tissues and HT-29,SW620 and SW480 in the colorectal cancer cell lines,the corresponding cell lines were selected for cell culture according to the results ofqRT-PCR.2 Colorectal cancer cell lines were selected for plasmid transfection(miR-196a-in miR-196a)and the corresponding negative control(NC)and(NC-in),a total of 4 groups and continue to culture.3.Wound healing cell scratch test,the migration distance of the 4 groups of cells were observed under microscope,and analyzed by using the image Pro Plus 6 software.4.Migration assays migration analysis,the use of Transwell membrane migration experiment,observe the staining of cells under the microscope and count.5.Western-blots experiments,using-actin as reference to analysis expression of E-cadherin,?-catenin and vimentin.Result MiR-196 a expression was upregulated in colorectal cancer clinical tissues and cell linesTo determine the expression of miR-196 a in human colorectal cancer,we detected miR-196 a expression in 8 patients,with paired CRC tissues(T)and corresponding adjacent non-tumor tissues(ANT)using qRT-PCR.It showed that the expression of miR-196 a was significantly up-regulated in the CRC tissues compared to the corresponding adjacent non-tumor tissues(Figure 1A).Further experiment to detect the miR-196 a in a panel of CRC cell lines(HT-29,SW620 and SW480)by qRT-PCR analysis,the results indicated that the expression of miR-196 a in SW620 was lower than that in HT-29,was higher than that in SW480(Figure 1B).Given the above results,it was decided to use the SW620 cells for the below experiments.MiR-196 a promoted cell metastasis of colorectal cancerTo further explore the functional role of miR-196 a in colorectal cancer,the colorectal cancer SW620 cells were transfected with miR-196 a or miR-196a-in or the relative controls.The migration and invasion capacities of SW620 cells were evaluated by using wound healing and transwell assays.Results of wounding healing assays demonstrated that SW620 cells after transfected with miR-196 a had stronger wound healing ability than those in the relative control group(Figure 2A).In contrast,SW620 cells after transfected with miR-196a-in into the wound was much slower than that of the cell after transfected withcontrols(Figure 2A).Furthermore,result of migration assay revealed that cell migration of SW620 cells was dramatically enhanced by miR-196 a overexpression,while miR-196a-in decreased cell migration(Figure 2B).Collectively,these results demonstrated that miR-196 a overexpression promoted CRC metastasis in vitro.MiR-196 a upregulated vimentin expression,while downregulated E-cadherin and?-carenin expressionFinally,we determined the mechanism that underlies the metastasis responsesregulated by miR-196 a via regulating the levels of EMT markers(E-cadherin,vimentin and?-catenin).As shown in Figure 3,result of RT-PCR and western blots revealed that miR-196 a caused a decrease in the mRNA and protein expression of E-cadherin and?-catenin and meanwhile greatly increase in the expression of the mesenchymal marker vimentin,whereas the miR-196a-in had the opposite effect.Conclusions1.MiR-196 a was highly expressed in colorectal cancer tissues and cells.2.MiR-196 a can promote the metastasis of colorectal cancer cells.3.MiR-196 a can promote the metastasis of colorectal cancer cells may be related to the up regulation of vimentin expression and down-regulation of E-cadherin and ?-catenin expression.