RMP Promotes DNA Damage Repair Through ATM/NFκB Pathway In Hepatocellular Carcinoma

Objective:In this study, we explore the mechanism of RMP in regulating the ability of DNA damage repair.Methods:1. After the treatment of irradiation, the m RNA expression level of RMP in both hepatocellular carcinoma(HCC) cells and normal liver cells was detected by Real-time PCR.2. After the treatment of irradiation, the protein expression level of RMP in both hepatocellular carcinoma(HCC) cells and normal liver cells was detected by Western blot.3. The m RNA expression level of Rad50, Mre11 and Ku70 was detected by Real-time PCR.4. The protein expression level of Rad50, Mre11, Ku70, ATM, p-ATM, p65, p-p65, AKT, p-AKT and IκB was detected by Western blot.5. The protein expression level of p65, p-p65 and IκB was detected by Western blot followed by transfection with different dose of p CDNA3.1-RMPo plasmid.6. After the treatment of Ku55933, the protein expression level of ATM, p-ATM and p65 was detected by Western blot.7. After the treatment of Bay 11-7082, the protein expression level of p65 and p-p65 was detected by Western blot.8. After the treatment of Bay 11-7082, the change of DNA damage was measured by comet assay.Results:1. After DNA damage in HCC cells, the m RNA and proterin expression of RMP increased, which is detected by Real-time PCR and Western blot. However, in normal liver cells, there is no significant change on the expression of RMP.2. Whether the overexpression or the konwdown of RMP did not change the m RNA expression level of Rad50, Mre11, Ku70.3. Whether the overexpression or the knowdown of RMP did not change the protein expression level of Rad50, Mre11, Ku70, ATM, p65, AKT, p-AKT.4. The overexpression groups increased the protein expression level of p-ATM and p-p65 but the knowdown groups decreased the expression level.5. Western blot analysis demonstrated that the expression of RMP promoted the protein level of p-p65 in dose-dependent manner.6. Immunoprecipitation analysis demonstrated that RMP overexpression promoted the interaction between RMP and DNA damage repair factor Mre11.7. After the treatment of Bay 11-7082, the ability of DNA damage repair was decreased.8. The protein expression level of p65 in the nuclear was reduced after the treatment of Ku55933.9. The protein expression level of p-p65 was reduced after the treatment of Bay 11-7082.Conclusion:RMP can promote DNA damage repair partly via ATM/NFκB signal pathway.