Methylation Status Of ZNF382 And Its Biology Function In Multiple Myeloma

BackgroundMultiple myeloma(MM)is a malignant plasma cell disease,ranking second in hematological malignancies,and its molecular pathogenesis mechanism is not fully understood.Compared with traditional chemotherapy,the marketing of new drugs and the application of hematopoietic stem cell transplantation(HSCT),CAR-T and other therapeutic technologies have significantly improved the long-term survival rate of MM patients.However,MM still remains most often incurable due to the development of drug resistance and relapse.Its treatment is still controversial and challenging.The KRAB zinc finger proteins(KRAB-ZFPs)family is one of the largest and most widely distributed families of transcription factors.Zinc-finger protein 382(ZNF382)is a member of KRAB-ZFPs protein family,located at 19p13.12,and has a KRAB domain.Previous studies have identified that ZNF382 could play a tumor suppressor role in various malignant tumors,and due to the high frequency methylation of its promoter CpG island,its expression was down-regulated in these tumors.The exact role of ZNF382 in carcinogenesis,however,remains elusive.Moreover,its epigenetic alterations and biological functions/mechanism in MM are still unknown.Therefore,the research is to detect the methylation status of ZNF382 promoter region in MM,and preliminarily discussed its biological function.ObjectiveIn this study,the mRNA expression and promoter methylation status of ZNF382 in MM bone marrow mononuclear cells(MM-BMMNC)and its cell line U266 cells were detected,the correlation between the promoter methylation status of ZNF382 and the clinicopathological characteristics of MM patients was analyzed.The demethylation drug Decitabine(DAC)was applied to treat the U266 cells,then the mRNA expression and the promoter methylation status of ZNF382 were detected.Furthermore,the effect of ZNF382 on the biological functions of MM cells such as proliferation and apoptosis were investigated by overexpression ZNF382.Methods1.The BMMNC and clinicopathological characteristics of MM patients were collected,RT-PCR and WB were used to detect the mRNA and protein expression levels of ZNF382 in MM-BMMNC and normal control bone marrow mononuclear cells(N-BMMNC).RT-PCR and WB were also used to identify the mRNA and protein expression levels of ZNF382 in MM cell line U266 cells.2.Methylation specific polymerase chain reaction(MSP)was used to detect the promoter methylation status of ZNF382 in MM-BMMNC,N-BMMNC and U266 cells,and to explore the relationship between the promoter methylation status of ZNF382 and the clinicopathological characteristics of MM patients.The MM cell line U266 was treated with DAC,then RT-PCR and MSP were used to investigate the changes of ZNF382 mRNA expression and promoter methylation status after DAC treatment.3.The ZNF382 overexpression lentivirus vectors were transfected into human embryonic kidney epithelial cells(293T)and then packaged into lentivirus particles.The lentivirus particles expressing ZNF382 were infected into U266 cells and screened by puromycin.RT-PCR and WB were used to identify the ZNF382 expression.The biological function of ZNF382 in MM was investigated using CCK-8 and flow cytometry.Results1.Compared with N-BMMNC,the mRNA and protein expression levels of ZNF382 in MM-BMMNC were significantly down-regulated(P<0.05),the mRNA and protein expression levels of ZNF382 in MM cell line U266 cells were also decreased significantly(P<0.05).2.The MSP experiment results indicated that the incidence of abnormal hypermethylation of the ZNF382 promoter was 43.48% in MM-BMMNC,whereas the incidence of N-BMMNC was only 11.11%,and the promoter methylation status of ZNF382 was significantly correlated with the disease stage.Moreover,the promoter methylation level of ZNF382 was significantly decreased in U266 cells after DAC treatment,and the mRNA expression of ZNF382 was partially recovered.3.The ZNF382 overexpression lentivirus vector was constructed and the U266 cells that stably expressed ZNF382 were successfully established.The results indicated that overexpression of ZNF382 could significantly inhibit the cell proliferation and increased the apoptosis of MM cells.ConclusionsThe results indicated that compared with N-BMMNC,the expression level of ZNF382 in MM-BMMNC and U266 cells were decreased,the abnormal hypermethylation of the ZNF382 promoter was increased,and the promoter methylation status of ZNF382 was negatively correlated with the disease stage of MM patients.The demethylation drug DAC can reduce the promoter methylation level of ZNF382 and partially restore the expression of ZNF382.ZNF382 can play a biological function of inhibiting cell proliferation and promoting apoptosis in MM cells.In conclusion,ZNF382 may function as a tumor suppressor gene in MM and thus affect the biological function of MM,and further study will be needed to provide certain theoretical basis for targeted therapy of MM in the future.