Effect Of ART1 On UPA Promoter Methylation And Mouse CT26 Cells Invasion And Metastasis Potential

Objective:To search for the effect of ART1 on DNA methylation status and probe the effect and possible mechanism of ART1 on metastasis and metastasis of mouse colon CT26 cells.Methods:In this experiment, we set up GFP-ART1 group and GFP-shARTl group as experimental group, GFP-Vector group and un-transfection group as control group.Expression of DNA methyltransferase1(DNMT1) was detected by western blotting in vitro.The methylation status of uPA gene was detected by bisulfite sequencing PCR.The combination in GFP-ART1,GFP-Vector and un-transfection group between ART1 and DNMT1 was assessed by co-immunoprecipitation.BALB/C mouse model of liver metastasis was successfully established through injecting the mentioned group CT26 cells.Expression of DNA methyltransferasel(DNMT1) was detected by western blotting in vivo.GFP-shART1 CT26 cells were treated with 5-aza-dc,abilities of metastasis and invasion were determined by Wound healing assay and Transwell invasion assay respectively between treated and untreated group.5-aza-dc treated and untreated GFP-shART1 CT26 cells were injected into BALB/C mouse respectively,and we have successfully built BALB/C mouse model of liver metastasis.Liver metastasis was observed.Expression of uPA in GFP-shART1,ART1 GFP-ARTl,Un-transfection,GFP-Vector group was detected by western blot in vitro and in vivo.Meanwhile,the uPA expression between 5-aza-2-dc treated and untreated group in vitro and in vivo.Results:(1) Effect and possible mechanism of ART1 on uPA promoter methylaytion status:It was demonstrated by western blot that the expression of DNMT1 significantly increased in GFP-shARTl group,while decreased in GFP-ART1 group compared with the control group(p<0.01) in vivo and in vitro.BSP showed that a significant increase of uPA promoter methylastion status appeared in GFP-shARTl group,and a relative decrease in GFP-ART1 group.CO-IP revealed that there was no combination between ART1 and DNMT1 in GFP-ART1,GFP-Vector and un-transfection group.meanwhile,(2) Effect of ART 1 on methylation status of uPA promoter thus its expression and on CT26 cells metastatic and invasion potential:We treated GFP-shARTl group with 5-aza-2-dc,the treated group promoted CT26 cells abilities of invasion and metastasis in vitro(p<0.05) compared with the untreated group.We successfully built the BALB/C mouse model of liver metastasis through under the splenic capsule with GFP-shARTl group CT26 cells with or without 5-aza-2-dc injection.The weight of liver was much heavier in treated group compared with untreated group(p<0.05),and the numer of metastasis nodules showed an increase in treated group compared with untreated group in vivo(p<0.05).Western blot was taken to test the expression of uPA in GFP-shART1, ART 1 GFP-ART1,Un-transfection GFP-Vector group,it significantly increased in GFP-ART1 group in vivo and in vitro,while decreased in GFP-shART1 group(p<0.01).Meanwhile,we treated GFP-shARTl group with 5-aza-2-dc,it was illustrated that expression of uPA increased in treated group in vivo and in vitro compared with the untreated group(p<0.01).Conclusion:This study reveals that ART1 silencing induces hypermethylation of uPA promoter thus causing the downregulation of its expression and ART1 over-expression causes hypomethylation of uPA promoter thus causing its overexpression.It probably relates to the feedback mechanism of NF-κB to PARP1 thus mediating the expression and activity of DNMT1.To some extent,the uPA gene could be a certain factor of invasion and metastasis in colorectal carcinoma.Meanwhile,inhibition of DNMT1 results in upregulation of uPA expression in order to promote the metastatic and invasive potential.It reveals that ART1 could inhibit uPA promoter methylation status thus increasing its expression,so as to play a role in CT26 cell metastasis and invasion.In a word,the relationship between ART1 and DNA methlation might offer a new perspective of the therapeutic target for the enhanced treatment of this illustrious world-wide cancer.However,the exact mechanism need to be taken into research in the near future.