Methylation Status Of PCDH10 Gene Promoter In Diffuse Large B-cell Lymphoma And Its Clinical Significance

BackgroundDiffuse large B-cell lymphoma is the most common non-Hodgkin’s lymphoma,and its incidence is gradually increasing in China in recent years.The standard first-line treatment of DLBCL is R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine and prednisone).60%-70% of the patients can be clinically cured after treatment with this regimen,but about 1/3 of the patients are still poorly treated,so there is an urgent need to further study the molecular mechanism of DLBCL and explore new therapeutic drugs and targets.In recent years,researchers have found that the down-regulation of some tumor suppressor genes driven by promoter hypermethylation plays an important role in the generation and progression of tumor.DNA promoter methylation is reversible,so epigenetic drug therapy based on DNA promoter demethylation has become a research hotspot.Decitabine is an epigenetic drug that can reverse the methylation of DNA promoter.Studies have shown that decitabine has a good therapeutic effect on DLBCL.Changes in the methylation level of some genes promoter may be involved in the anti-tumor process of DLBCL,but it is not entirely clear which target molecules play an important role in this process.It has been found that the expression of tumor suppressor gene procadherin 10 is down-regulated due to promoter hypermethylation in many lymphoma cell lines,and the down-regulation of PCDH10 expression mediated by promoter hypermethylation can promote the chemoresistance of lymphoma cells.However,the expression and promoter methylation status of PCDH10 in DLBCL and its correlation with clinicopathological features,and whether it participates in the anti-tumor effect of decitabine on DLBCL is not completely clear.ObjectivesThe purpose of this study is to explore the expression and promoter methylation status of PCDH10 in DLBCL tissues,and to explore the effects of DAC on the proliferation and apoptosis of LY8 cells,and the changes of DAC on the methylation status of PCDH10 promoters,in order to provide new theoretical basis for the study of molecular mechanism and clinical diagnosis and treatment of DLBCL.Methods1.The expression of PCDH10 protein in DLBCL paraffin specimens was detected by immunohistochemical method,and the methylation status of PCDH10 gene promoter in DLBCL paraffin specimens was detected by methylation specific PCR method.The correlation between promoter methylation status and clinicopathological features of patients was analyzed.2.The proliferation inhibition rate of LY8 cells treated with different concentrations of decitabine(0.625 μmol/L,1.25 μmol/L,2.5 μmol/L,5 μmol/L)was detected by CCK-8method,and the apoptosis rate of LY8 cells was detected by flow cytometry.3.MSP method was used to detect the changes of methylation status of PCDH10 gene promoter in LY8 cells treated with decitabine,and fluorescence quantitative PCR was used to detect the changes of PCDH10 gene m RNA expression in LY8 cells treated with decitabine;Western Blotting was used to detect the changes of PCDH10 protein and Caspase-3 and other apoptosis-related proteins in LY8 cells treated with decitabine.Results1.The positive rate of PCDH10 expression in DLBCL paraffin samples was 29.0%(16/55),which was significantly lower than that in hyperplastic lymph nodes 65.2%(15/23)(P(27)0.05).2.The methylation positive rate of PCDH10 gene in DLBCL paraffin samples was72.7%(40/55),which was significantly higher than that in hyperplastic lymph nodes26.1%(6/23)(P(27)0.05).The methylation of PCDH10 gene in DLBCL was correlated with LDH level,Ki-67 expression level,Ann Arbor stage and international prognostic score index(P(27)0.05).It was not related to sex,age and bone marrow invasion(P(29)0.05).3.The positive rate of PCDH10 expression in DLBCL tissues with methylation of PCDH10 gene promoter was 7.5%(3/40),which was significantly lower than that in unmethylated DLBCL tissues of PCDH10 gene promoter 86.7%(13/15).The difference was statistically significant(P(27)0.05).4.CCK-8 assay: the proliferation inhibition rates of LY8 cells treated with0.625μmol/L,1.25μmol/L,2.5μmol/L and 5μmol/L decitabine for 24 h were(12.5±0.29)%,(21.97±0.12)%,(34.53±0.33)%,(39.93±0.25)%,(29.83±0.83)%,(43.27±0.25)%,(55.8±0.36)%,(62.9±0.29)%,respectively.(35.97±0.29)%,(57.47±0.31)%,(69.03±0.74)%,(79.17±0.82)% at 72 h.suggesting that the proliferation inhibition rate of LY8 cells increased in a time and concentration-dependent manner after the intervention of decitabine(P(27)0.05).5.Apoptosis was detected by flow cytometry: the apoptosis rate of LY8 cells treated with 2.5 μmol/L decitabine for 48 hours was(38.41 ? 0.08)%,which was significantly higher than that of the untreated group(8.36?0.13)%(P(27)0.05).6.After LY8 cells were treated with decitabine,the unmethylation status of PCDH10 gene promoter and the expression of PCDH10 gene m RNA and protein were significantly increased.The expression of apoptosis-related proteins Caspase-3 and Bax increased,while the expression of Bcl-2 decreased.Conclusions1.Hypermethylation of PCDH10 gene promoter with low expression exists in DLBCL tissue,and hypermethylation of PCDH10 gene promoter is associated with poor prognosis of DLBCL patients.2.Methylation of PCDH10 gene promoter can down-regulate the expression of PCDH10,and decitabine can inhibit proliferation and promote apoptosis by reversing the methylation of PCDH10 gene promoter in LY8 cells.