The Role Of Histone Methylase G9a In The Development Of Mantle Cell Lymphoma

[Background]Mantle cell lymphoma(MCL)is a group of highly heterogeneous non-Hodgkin lymphoma characterized by the t(11;14)(q13;q32)transposition and the over expression of CyclinDl and accounts for 5%-10%of non-Hodgkin lymphoma.MCL,which is easy to relapse,is the lowest long-term survival of lymphoma subtype and the median survival is 3-5 years.G9a(Euchromatic histone-lysine N-methyltransferase 2,also known as G9a)is a histone lysine methylation transferase,which is abnormally expressed in many malignant tumors and is closely related to the development of tumors.But it has not been studied in mantle cell lymphoma.The purpose of this study is to investigate the expression of G9a in mantle cell lymphoma and its role in the development of mantle cell lymphoma.Part I The Expression of G9a in Mantle Cell Lymphoma and Related Methylation Chips Analysis[Methods]1.Specimens including 39 cases with mantle cell lymphoma and 20 cases with reactive hyperplasia of lymph node during 2006 to 2017 were collected at Qi Lu Hospital of Shandong University.The expression of G9a protein in experimental group and control group was detected by immunohistochemical method.2.We respectively selected 4 cases of high expression of G9a from the experimental group and 4 cases from control group and analyzed the difference of gene methylation in whole genome was analyzed by Illumina methylation 850 Bead Chips.And then we screen for genes with significant methylation differences.3.Through more rigorous screening,we selected three tumor suppressor genes that were significantly hypermethylated,namely EDNRB,GATA3,and EPHA7.And through the BSP,we verify the chip results.[Results]1.The expression of G9a was mainly localized nucleus in mantle cell lymphoma and lymph node reactive hyperplasia.The expression rate of G9a in mantle cell lymphoma was 69.2%(27/39),and that of G9a in lymph node hyperplasia was 0%(0/20).Compared with lymph node reactive hyperplasia,G9a was significantly up-regulated in mantle cell lymphoma with statistical significance.2.Methylation gene chip shows that abnormal DNA methylation is a very common event in the global genome of MCL tumor tissue.Abnormal methylation outside the gene body is mostly happened in the promoter region.Abnormal methylation is more common in CpG Island region.3.GO analysis shows that abnormal methylation is mainly associated with biological functions such as anabolism,growth and development,and transmembrane transport.By KEGG analysis,abnormal methylation enriched in MAPK,P13K-Akt,cAMP,Rapl and other signaling pathways.4.The methylation sites suggested by the chip in the EDNRBS'UTR region were detected by BSP,but the group of experimental and control did not show significant differences.Other sites still need further testing.[Conclusions]1.G9a expression is up-regulated in mantle cell lymphoma compared to reactive lymph node hyperplasia.2.In mantle cell lymphoma,aberrant methylation of genes is ubiquitous.Aberrant methylation is most commonly found in the promoter region,the 3'UTR region,the CpG island and the CpG Shore region.Aberrant gene methylation is mainly associated with functions such as anabolism and transmembrane transport.And it is mainly enriched in MAPK,PI3K-Akt and other signaling pathways.Part ? The affect of BIX01294 on the Histone Methylation in MCL Cell Lines and tumor Cell-related Functions[Methods]1.Mino cells were treated with 0,1,2,4 and 6mol/1 of drug concentration gradient.Protein was extracted after 48h.Western blot was used to detect the expression of G9a and the methylation of H3K9me2,H3K9me3,H3K4me2 and H3K27me2.2.CCK8 method was used to detect the effect on the proliferation of Mino cells after treatment and the cell growth curve was drawn.3.After using PI staining,the flow cytometry was used to test the effect on the cell cycle after treatment.4.WB was used to detect the expression of cyclinD1,CDK4 and P21 protein with different concentrations.5.After using Annexin V-FITC/PI double staining cells,we use flow cytometry to detect changes in apoptosis after treatment.6.Western blot was used to detect the expression of apoptosis proteins BAX and caspase-3 under different drug concentration gradients.[Results]1.After drug effect,the expression of G9a protein did not change significantly with the change of drug concentration.It explains that drugs only affect G9a activity.The methylation levels of H3K9me2,and H3K27me2 decreased with the increase of drug concentration.And H3K4me2 methylation levels gradually increased,while the methylation level of H3K9me3 did not change significantly.2.The results of cell growth curve showed that the cell proliferation ability decreased significantly after 24 hours of treatment,and with the increase of drug concentration,the trend of inhibiting proliferation is more obvious.3.After 48h treatment,the number of cells in G1 phase was induced and the number of S phase cells was reduced.The cells were arrested in G1 phase.With the drug concentration increased,the number of cells which arrested in G1 phase was increased.Western results showed that with the increase of drug concentration,the expression of cyclin D1,CDK4 and P21 protein was gradually decreased.4.After 48h of drug treatment,the apoptotic rate was 8.9%,11.2%,14.3%,26.1%and 76.6%at 0,1,2,4 and 6?mol/1 drug concentration respectively.The difference was statistically significant.Western blot results show that with the drug concentration increased,the expression of apoptotic proteins BAX and capase-3 gradually was increased.[Conclusions]1.BIX01294 can inhibit G9a activity but has no effect on its expression.After drug treatment,the methylation levels of H3K9me2 and H3K27me2 were decreased.And H3K4me2 methylation levels gradually increased,but it had no effect on the methylation level of H3K9me3.2.BIX01294 inhibits cell proliferation.It is positively correlated with drug concentration and duration of action.BIX01294 can affect G1 and S phase transformation by inhibiting cyclinD1 and CDK4 protein,and arrest cells in G1 phase.In addition,it can promote apoptosis by up-regulating BAX and caspase-3 proteins expression.Part ? Searching for G9a interacting proteins and researching related downstream target genes[Methods]1.Through CO-IP experiments,we find the proteins that interact with G9a.2.A total of four ShRNAs targeting the G9a gene were designed and synthesized.The liposome was used to transfect cervical cancer cell line Hela cells to interfere with G9a gene expression.3.After transfection,We collected the cells and extracted the RNA and protein.RT-qPCR and western blot were used to detect the mRNA level and protein expression level.The interference efficiency of G9a was detected,and the best shRNA was selected.4.Viral(LV-G9a-RNAi),which transfected into the mantle cell lymphoma cell line Jeko-1,was synthesized based on the selected ShRNA sequence.The mRNA level was detected by RT-PCR and the transfection efficiency was verified.5.When the transfection efficiency reached more than 50%,gene expression microarrays were used to screen for genes that were significantly different in the interference and control groups.[Results]1.The results showed that in MCL,G9a could interact with HDAC1?HDAC2 and UHRF1 proteins to form a complex and act on downstream target genes.But there are no interaction with HDAC3.2.Expression microarray results showed that after G9a knockdown,compared with the NC group,a total of 611 genes were altered of which 266 genes were up-regulated and 345 genes were suppressed.After the interference,several cancer-related signaling pathways were inhibited,of which the inhibition of Tec kinase signaling pathway was the strongest.The specific mechanism of action needs further study.[Conclusions]1.In MCL,G9A interacts with UHRF1 and various histone deacetylases.2.Interfering with G9a may affect multiple gene expressions and affect related signaling pathways.