| 【Objective】: The study focused on the effect of cytokine Transforming Growth Factor-β(TGF-β)on cell migration and Epithelial to Mesenchymal Transition(EMT)in Colorectal Cancer(CRC).And we further revealed the function of RUNX1 in promoting CRC cells migration among TGF-β signaling pathway.【Methods】: Immunoblotting and immunochemistry were applied to detect the expression of RUNX1 in 10 paired tumor tissues and normal epithelial tissues from CRC patients.Transwell cell migration assay was used to show changes in migration after TGF-βtreatment in CRC cell lines.The m RNA or protein levels of RUNX1,EMT markers,EMT transcriptional factors and matrix metalloproteins(MMPs)after TGF-β treatment were tested by immunoblotting and Real Time Quantitative PCR(RTq-PCR).RUNX1 specific siRNA was transfected to knockdown RUNX1 expression in CRC cell line.Later,the migration ability of transfected cells was tested under the stimulation of TGF-β.Immunoblotting revealed the changes in protein levels of EMT markers and EMT transcriptional factors with the knock-down effect of RUNX1.【Results】:(1)The results of Immunoblotting and immunochemistry showed a trend that tumor tissues from CRC patients express higher RUNX1 protein levels than normal epithelial tissues.And cancer cell nucleus expressed most RUNX1 protein,wile a few stromal cells could also express RUNX1 in their nucleus.(2)HT-29,SW116 and LoVo CRC cell lines showed an increased migration under 10ng/mL TGF-βtreatment(p<0.05).(3)The mRNA levels of N-Cadherin show a significant increase(p<0.05)in HT-29 and SW1116 after treated with 5ng/mL TGF-βfor 24 hours and 48 hours.The protein levels of N-Cadherin and Vimentin also enhanced within the Immunoblotting experiment.These results indicated that TGF-β do trigger EMT in CRC cell lines.Meanwhile,the MMP-9was found to have an increase in protein levels after TGF-β treatment.(4)Several CRC cells lines presented an enhanced expression of RUNX1 in both mRNA(p<0.05)and protein levels after the stimulation of TGF-β.(5)SW1116 cell line was transfected with RUNX1 specific siRNA,and the migration ability of the transfected cells were detected by Transwell cell migration assay.The RUNX1 knock-down effect showed to attenuate the inducement of TGF-β in cell migration(p<0.05).And the transfected cells showed a decrease in the expression of SNAI1 and N-Cadherin.The addition of TGF-β could not reverse the knock-down effect.【Conclusion】: RUNX1 regulated SNAI1 expression in TGF-β signaling pathway,and induced CRC cells migration through EMT process. |
