EIF4A3 Binds And Stabilizes MMP11 To Promote Invasion,Migration And EMT Of Colorectal Cancer

Objective: To investigate the effect of EIF4A3/MMP11 on invasion,migration and EMT of colorectal cancer.Methods: The expression level of MMP11 in colorectal cancer and the relationship between MMP11 and EIF4A3 were predicted by bioinformatics.A total of 20 patient samples,including 20 colorectal cancer tissues and 20para-cancer tissues,were obtained from the Second Affiliated Hospital of Chengdu Medical College,Nuclear Industry 416 Hospital.The expression of MMP11 in those tissues was detected by q RT-PCR.To further study the expression profile of MMP11 in normal human colonic epithelial cells and colorectal cancer cells.QRT-PCR and western blot were used to analyze the expression levels of MMP11 m RNA and protein,respctively.In colorectal cancer cells HCT15 and DLD1 which have the highest expression level of MMP11,the interaction between EIF4A3 and MMP11 was detected by RNA binding protein immunoprecipitation(RIP)experiment.To investigate the function of EIF4A3 on MMP11 RNA expression,si RNA specific to EIF4A3(si-EIF4A3)and its negative control were transfected into HCT15 and DLD1 cells,and after 24 hours,cells well treated with actinomycin D to inhibit RNA synthesis and harvested at 0h,3h,6h,9h and 12 h,and then the total RNA were extracted to detect the half-life of MMP11 m RNA in each group.Furthermore,the function of EIF4A3 was confirmed by overexpression this protein in HCT15 cells.HCT15 cells were divided into EIF4A3 inhibition group(cells transfected with si-EIF4A3),and control group(cells transfected with si RNA),EIF4A3 overexpression group(cells transfected with p EGFP1EIF4A3)and control group(cells transfected with p EGFP1-NC).The expression levels of MMP11 protein in each group were detected using Western blot.In order to detect the effects of MMP11 and EIF4A3 on the invasion and migration of CRC cells,HCT15 cells were divided into EIF4A3 inhibition group(cells transfected with si-EIF4A3),and control group(cells transfected with si-NC),EIF4A3 inhibitor group + MMP11 overexpression group(cells transfected with si-EIF4A3 + p EGFP1 MMP11),and control group(cells transfected with si-EIF4A3 + p EGFP1-NC).Then the expression of MMP11 protein in each group was detected by Western blot;Transwell and scratch test were used to detect the changes of cell invasion and migration ability in each group.Finally,the expression of EMT related proteins such as E-cadherin,vimentin and n-adherin were detected by Western blot.Results: MMP11 was one of the top25 highly expressed genes in patients with colorectal cancer.Compared with normal human adjacent tissues and epithelial cells,the expression of MMP11 in colorectal cancer tissues and cells was higher(all P < 0.001).The expression level of MMP11 was the highest in HCT15,and secondly in DLD1 cells.RIP experiment suggested that EIF4A3 could interact with MMP11 RNA;RT-q PCR results demonstrated that the half-life of MMP11 RNA was shortened after reducing the expression of EIF4A3(P < 0.05).Western blot results showed that the expression level of MMP11 protein was decreased after inhibiting EIF4A3 expression,while overexpression of EIF4A3 increased the expression level of MMP11 protein;Transwell and scratch experiments showed that interfering with the expression of EIF4A3 inhibited the invasion and migration of colorectal cancer cell HCT15(all P < 0.05).Western blot results implied that after interfering with the expression of EIF4A3,the expression level of E-cadherin protein was increased and the expression levels of vimentin and N-adherin protein were decreased.The decreased invasion and migration ability of HCT15 cells caused by EIF4A3 was recovered by overexpression of MMP11,and MMP11 overexpression also decreased the expression of E-cadherin protein and slightly increased the expression of vimentin and N-cadherin protein.Conclusion:1.The expression of MMP11 is significantly increased in colorectal cancer tissues and cells,especially in colon cancer HCT15 cells.2.EIF4A3 can interact with MMP11 RNA to enhance its stability.The expression level of EIF4A3 was reduced by inhibiting the expression of EIF4A3,and overexpression of EIF4A3 can increase the protein expression level of MMP11.3.EIF4A3/MMP11 axis can promote the invasion,migration and EMT process of colorectal cancer cells.Interfering with the expression of EIF4A3 can inhibit the invasion,metastasis and EMT of colorectal cancer cells,while overexpression of MMP11 can reverse the effects,In conclusion,targeting EIF4A3 or MMP11 may have an important impact on colorectal cancer treatment.