Study On P73 MRNA Gene Expression And Methylation Of Non-Hodgkin's Lymphoma With Bone Marrow Involvement

Background and Objective:p73 gene, a member of p53 gene family, has been implicated playing role of tumor suppressor gene (anti-oncogene) in lymphoma/leukemia. It was also shown loss and low of p73 gene expression in leukemia/ lymphoma due to hypermethylation in exon. However the study conclusion rooted in data of cell lines and acute lymphocyte leukemia. In malignant lymphoma (ML) the study involved p73 gene was very seldom. Non-Hodgkin's lymphoma (NHL) were most of ML, bone marrow involvement (BMI) were very common in clinic. It suggested that NHL with bone marrow infiltration were critical condition,rapid advance,poor treated effect and severe prognosis.The aim of this study was to investigate the relationships between expression of p73 mRNA and oncogenesis in non-Hodgkin's lymphoma with bone marrow involvement, the relationships between p73 Gene inactivation and hypermethylation. To explore the influencing factors of p73 gene inactivation,relationships between expression of p73 mRNA and therapeutic efficacy, suggest possible molecular biologic mechanism of NHL of BMI. .Methods: 1 Subjects: The subjects were 28 NHL patients with BMI, who include sixteen males and twelve females, and who had been diagnosed by clinic,cell morphologic,pathology and immunohistochemistry, including seven Lymphoma cell leuke- mia(LMCL). The average age is forty-one. The patients included thirteen B-NHL, fifteen T-NHL. Eight patients whose bone marrow were diagnosed no tumor were done as normal control. .2 Methods:We routinely collect the bone marrow from all NHL patients and normal control people, isolated mononuclear cells (MNCs) and extracted total RNA by TRIZOL reagent. Then we used Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus,Point Mutant (M-MLV RNaseH-) to synthesize first-strand cDNA from RNA molecules with random hexamer. The primers were designed based on the p73 gene cDNA sequence provided by NCBI. We used a pair of sequence-specific primers and reverse transcription Polymerase Chain Reaction (RT-PCR) method to amplify a part of p73 gene. At the same time the primers of house-keeping geneβ-actin were designed and synthesized as endogenous control. PCR products were run on a gel and visualized by staining with EB. After extracted DNA by RelaxGene Blood DNA System, we digested it with either methylcytosine sensitive enzyme ,HpaII, or its methylation resistant isoschizomers, MspI. Then amplify the p73 DNA fragments covering exon 1 to assess the methyl- lation status of the p73 gene locus. We analyzed relationship between expression of p73 mRNA and results of treatment for NHL of BMI in clinical work. .3 Statistical methods: Difference of p73 mRNA expression rate between NHL and control sample were tested by Fisher's exact probability test in 2×2 table. The same statistical method was used to find the relationship between p73 mRNA expression and remission rate by initial therapy in NHL. We also used Mann-Whitney U test, one of nonparametric test methods, to analyze the difference of therapeutic efficacy of p73-negative NHL and p73-positive NHL. The data were analyzed using Statistical software Package of the SPSS 11.5.The p-values are two-tailed and a p-value of less than 0.05 being considered to be statistically significant. .Results:1 The expression of p73 mRNA gene was negative in 20 of 28 (71.4%) non-Hodgkin′s lymphoma (NHL) with bone marrow involvement (BMI). The expression of p73 mRNA gene was found in 8of 28 cases of NHL patients and 8 people whose bone marrow were diagnosticated no tumor. The difference of the expression of p73 mRNA gene between NHL and control sample was significant(P<0.001).2 All study subjects were not found hypermethylation in exon 1. The data suggest that hypermethylation cannot regulate the expression of p73mRNA.3 The total remising rate (RR) of the p73-negative NHL patients of BMI is 40% (8/20) and the RR of the p73-positive NHL patients is 87.5% (7/8) when the patients got initially treatment. The difference of two groups was significant(P= 0.038).4 p73 mRNA-negative NHL patients with BMI had worse therapeutic efficacy than p73 mRNA-positive NHL patients. The difference of two groups analyzed with Mann-Whitney U test was significan(tU=2.373,P=0.018). .Conclusion:1 The p73 gene inactivation may contribute to development and/or progression of BMI NHL.The total remising rate of the p73-negative NHL patients of BMI is lower than the p73- positive NHL patients after initially treatment. There were correlations between expression of p73 mRNA and the thera- peutic efficacy and prognosis of BMI NHL.2 Hypermethylation in exon 1 was not main influencing factor of p73 gene inactivation in BMI NHL. The mechanisms of p73 gene inactivation should be taken great light on and certificated.