Epigenetic Changes Of ZNF382 And Its Biology Function In Diffuse Large B Cell Lymphoma

BackgroundAs one of the most common types of non-Hodgkin’s lymphoma,diffuse large B cell lymphoma(DLBCL)has the characteristics of high heterogeneity,strong invasiveness and poor prognosis.In-depth exploration of the pathogenesis of DLBCL and finding new potential therapeutic targets have important clinical and practical significance.KRAB zinc finger proteins(Krüppel-associated box domain-zinc finger protein,KRAB-ZFPs)is the most widely distributed transcription factor family,one of its members is ZNF382,which located at 19p13.12.Many previous studies have literature that ZNF382 is inactivated in many malignant tumors due to high-frequency methylation of the Cp G island in the promoter region.However,its expression in DLBCL and its correlation with patient prognosis have not been reported previously.Therefore,this study started from detect the expression of ZNF382 and clarified the methylation level of the gene promoter region in DLBCL,and deeply explored its epigenetic changes and biological functions in DLBCL.ObjectiveThe study mainly detects the expression level and methylation status of ZNF382 in DLBCL tissues and cell lines,and analyzes the correlation between ZNF382 expression and the clinicopathological characteristics,treatment response rate and prognosis of DLBCL patients.Then use the demethylation drug decitabine(DAC)to treat DLBCL cell lines,after 72 h,the expression level and the promoter methylation status of ZNF382 were detect.And further construct the ZNF382 overexpression cell lines to explore the effects of ZNF382 on the biological function of DLBCL cells.Methods1.RT-PCR to detect the m RNA expression level of ZNF382 in DLBCL cell lines OCILY10 and U2932.Collect the pathological lymph-node tissues of patients diagnosed with DLBCL and the lymph-node tissues as control,IHC to detect the difference of ZNF382 protein expression,and explore the correlation between ZNF382 expression and clinical characteristics treatment effect and prognosis of DLBCL patients.2.Methylation-specific PCR(MSP)was used to detect the methylation status of ZNF382 in the DLBCL cell lines.Then the above cell lines were treated with DAC for 72 hours and use RT-PCR and MSP to investigate the methylation status of the ZNF382 promoter region and m RNA expression levels.3.Lentiviral packaging system was used to transfect human embryonic kidney cells293 T to prepare lentiviral particles overexpressing ZNF382 and infected into DLBCL cell lines.After 2 weeks of puromycin screening,the purity were detectd by flow cytometry.Through CCK-8,transwell migration and clone formation experiment we analyze the effect of overexpression ZNF382 on the biological function of DLBCL cells.Results1.Compared with the control group,the protein expression level of ZNF382 in the pathological tissues of DLBCL patients were significantly down-regulated(P<0.05),and the expression level of ZNF382 was positively correlated with 2-year event-free survival(EFS)rate of DLBCL patients(P<0.001).The m RNA expression levels of ZNF382 were also decreased significantly in DLBCL cell lines OCI-LY10 and U2932(P<0.05).2.The methylation status of ZNF382 promoter region in DLBCL cell lines were significantly higher than that of normal lymph-node tissues.After 72 hours of treatment with the demethylating drug DAC,the methylation level of the ZNF382 promoter was lower than before,and the m RNA expression level was partially recovered(P<0.05).3.The lentiviral particles overexpressing ZNF382 were obtained and then infected OCI-LY10 and U2932 cells.The results showed that ZNF382 overexpression can significantly inhibits the proliferation,migration and colony formation ability of DLBCL cell lines(P<0.05).ConclusionsThe results of this study indicate that the expression level of ZNF382 in DLBCL cell lines were decreased,the abnormal hypermethylation of the ZNF382 promoter was increased,and the expression level of ZNF382 was positively correlated with the prognosis of DLBCL 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,migration and colony formation ability.In conclusion,ZNF382 may function as a tumor suppressor gene in DLBCL.The research is expected to provide laboratory and theoretical basis for the targeted and precise treatment of DLBCL through further research in the future.