The Methylation State Of PTPROt And Its Significance In Lymphoma Cells

Objective :Lymphoma is one of the most common malignant tumor in blood system. The study shows that the genome of low methylation and local CpG island abnormally high methylation exist in lymphoma cells. Many genes with high methylation in promoter are associated with lymphoma,as calcitonin, the adjustment factors that controlled the growth of cells as p15,p16, and the well known suppressor factors as ARF and BLU. These genes are closely related with the cell proliferation, differentiation, growth, embryonic development and biological immune function .PTPRO is one of the newly discovered members of protein tyrosine phosphastase family (PTPs) which can be a negative control factor to JAK-TAT signaling pathways.It plays an important role in regulating cell growth, proliferation, differentiation, embryonic development and immunity. It is a potential tumor-suppressor factor. The truncation type PTPROt of PTPRO only expresses in hematopoietic cells, espacially in lymphocytes. The chip and tissue microarray technology prove that the gene expresses obviously decreased in malignant lymphoma cells and some leukemia cells. The silencing of this gene can lead to the activity of relevant signal transduction molecules so as to promote the malignant tumor cell proliferation.This experiment regards the lymphoma patients' nodes and the lymphoma lines as the research object. The study is to investigate the expression of PTPROt and the methylation state in lymphoma patients. Meanwhile, give methylation drug treatment to lymphoma cells to compare the expression and the changes of methylation state of PTPROt, proliferation and apoptosis in lymphoma cells.Methods:1 Collected lymph nodes of the lymphoma patients who pathology diagnosis clear but untreated.Regarded inflammatory and reactive hyperplasia patients as a control group.Using the methods of fluorescence real-time quantitatie polymerase chain reaction (FQ-PCR) and semi-quantitative polymerase chain reaction to detect the expression in lymphoma patients, inflammatory patients and reactive hyperplasia patients.2 Lymphoma lines Raji and Jurkat were cultured in RPMI1640 medium, supplemented with 10% (v:v) heat-inactivated fetal bovine serum (Hyclone), 100U/ml penicillin G and 100μg/ml streptomycin sulfate, at 37℃in an atmosphere of 5% CO2. The cells were passaged every two or three days. Logarithmically growing cells with viability≥95% were used in the experiment. Lymphoma lines were cultivated and treated with different concentrations of methylation durg 5-aza-CdR when they reached a certain density. Collected the cells in different periods to detect the expressions of PTPROt and changes of the methylation state.3 Extracted DNA from lymph node and treated lymphoma lines to detect the methylation state in lymphoma patients,inflammatory and reactive hyperplasia patients,lymphoma lines by methylation specific polymerase chain reaction (MSP).4 Raji and Jurkat cells in exponential growth state were planted in 96-well plates at density of 5.0×105 cells/ml in RPMI1640 medium, with 200μl cell suspension in each well. The concentrations of 5-aza-CdR in Raji were 1.0μmol/L,5.0μmol/L,10.0μmol/L.The concentrations of 5-aza-CdR in Jurkat were 1.0μmol/L,3.0μmol/L,5.0μmol/L(all three holes);no durg in one hole and only RPMI1640 as a zero hole.Cultured with different concentrations of 5-aza-CdR for 24 hours,48 hours and72 hours.Then cells were harvested, detected the proliferation in lymphoma lines condition by MTT after the drug interention.5 Raji and Jurkat cells in exponential growth state were planted in 6-well plates at density of 5.0×105 cells/ml in RPMI1640 medium. Cultured with different concentrations of 5-aza-CdR for 24 hours,48 hours and72 hours. Flow cytometric detected apoptosis.by the way of AnnexinV/PI. Results: 1 In the controls that inflammatory and reactive hyperplasia patients it showed obvious 213bp strips in semi-quantitative polymerase chain reaction, but in lymphoma patients the strips were decreased or disappeard, then used fluorescence real-time quantitative polymerase chain reaction (FQ-PCR) for further testing the expression level of PTPROt mRNA. The expression level of PTPROt in inflammatory and reactive hyperplasia of patients was 1.174±0.586 but in lymphoma patients is 0.557±0.34 0(P=0.01<0.05),it had statistical significance.2 When the lymphoma lines Raji were treated with different concentrations 1.0μmol/L,5.0μmol/L,10.0μmol/L of 5-aza-CdR for 24 hours,48 hours and72 hours the expressions of PTPROt mRNA were 1.03±0.02,1.103±0.051,1.263±0.064,(P=0.009<0.05)1.027±0.022,1.203±0.064,1.41±0.113(P=0.014<0.05), 1.07±0.02, 1.36±0.107, 1.54±0.04(P=0.02<0.05) When Jurkat were treated with different concentrations 1.0μmol/L ,3.0μmol/L,5.0μmol/L of 5-aza-CdR for 24 hours,48 hours and 72 hours the expressions of PTPROt mRNA were 1.08±0.152,1.246±0.057,1.263±0.064(P=0.03<0.05)1.306±0.03,1.633±0.182,1.773±0.064(P=0.042<0.05),1.478±0.112,1.923±0.68,2.066±0.208 (P=0.01<0.05),with the elevation of the drugs concentration and the extension of time the expression of PTPROt mRNA was increased.3 It exsited methylation sites at PTPROt gene promoter for 16 in lymphoma patients in 23, but none of the 14 inflammatory and reactive hyperplasia patients. For the lymphoma lines Raji and Jurkat, the methylation states existed, after the treatment of 5-aza-CdR ,the methylation states were decreased or disappeard.4 After being treated with different concentration of 5-aza-CdR the proliferation of lymphoma lines was restrained,with the elevation of the drugs concentration and the extension of time the inhibition rate increased.5 The drug 5-aza-CdR could promote the apoptosis of lymphoma lines, with the elevation of the drugs concentration and the extension of time apoptosis rate increased. Conclusions: The expression of PTPROt mRNA in Lymphoma patients is decreased or disappeard. It exsited high methylation site at PTPROt gene promoter. When the lymphoma lines were treated with 5-aza-CdR ,the high methylation site at PTPROt gene promoter disappeared ,the expression of PTPROtmRNA was increased or expressed again.And when the lymphoma lines were treated with different concentrations of 5-aza-CdR for differences times it showed significant differences With the elevation of the drugs concentration and the extension of time the expression of PTPROt mRNA was increased.And at the same time the methylation state was decreased or disappeard.The inhibition rate and apoptosis rate of tumor cells were also increasesed. It possibly can be an important inhibitory factor to JAK/STAT channel ,to inhibit lymphoma cells by inhibit JAK/STAT pathways.