【作者】 王珺；

【导师】 张翠娟；

【作者基本信息】 山东大学 ， 病理学与病理生理学， 2018， 硕士

【摘要】 [研究目的]套细胞淋巴瘤(Mantle cell lymphoma,MCL)为非霍奇金B细胞淋巴瘤,经检测发现,在大量病人中特异性存在t(11;14)(q13;q32),并因此导致cyclinDl过度表达。套细胞淋巴瘤病发率偏低,仅为5%-10%,但其预后极差,容易复发,是中位生存期最短的淋巴瘤亚型,大约为3-5年。G9a(Euchromatic histone-lysine N-methyltransferase 2,也称作EHMT2)是研究比较广泛的组蛋白赖氨酸甲基化转移酶,其在多种肿瘤中出现异常表达增高,并与肿瘤增殖、凋亡、侵袭等生物学功能有关,但其在MCL中尚未有研究。本实验旨在研究G9a在MCL中表达情况及其在套细胞淋巴瘤发生发展中的作用机制。第一部分G9a在套细胞淋巴瘤中表达情况及甲基化芯片分析[方法]1.收集山东大学齐鲁医院2006年至2017年之间套细胞淋巴瘤病例及淋巴结反应性增生病例,分别收集到39例及20例病例,利用免疫组化方法检测实验组与对照组中G9a蛋白的表达。2.在实验组中选取4例G9a高表达病例,4例淋巴结反应性增生病例,使用Illumina甲基化芯片分析全基因组中基因甲基化差异,筛选具有显著甲基化差异的基因。3.通过更严格的筛选,我们选取了 3个发生显著高甲基化的抑癌基因,分别是EDNRB、GATA3及EPHA7。并通过BSP,验证芯片结果。[结果]1.在套细胞淋巴瘤中G9a表达率为69.2%(27/39),淋巴结反应性增生中G9a表达率为0%(0/20)。与淋巴结反应性增生相比,在套细胞淋巴瘤中,G9a表达明显上调,差异具有统计学意义。p<0.0012.分析甲基化芯片可知,基因甲基化异常是MCL全基因组中常见事件。除基因Body区外,异常甲基化最常发生于Promoter区和3’UTR区。根据CpG注释分类结果显示,异常甲基化多见于CpG Island和CpG shore区域。3.通过GO分析可知,异常甲基化主要与合成代谢、生长发育、跨膜转运等生物学功能相关。通过KEGG分析可知,异常甲基化富集于MAPK、PI3K-Akt、cAMP、Rapl等信号通路。4.目前通过BSP检测了 EDNRB5’UTR区芯片提示的甲基化位点,但实验组与对照组未显示出有明显的差异,其它高甲基化位点正在进一步检测中。[结论]1.与淋巴结反应性增生相比,在套细胞淋巴瘤中,G9a存在异常表达。2.在套细胞淋巴瘤中,基因异常甲基化普遍存在。异常甲基化最常见于启动子区、3’13UTR区、CpGIsland内和CpG Shore区。异常基因甲基化主要与合成代谢、跨膜转运等功能相关,并且主要富集于MAPK、PI3K-Akt等信号通路。第二部分G9a特异性抑制剂BIX01294对MCL细胞系组蛋白甲基化情况及对肿瘤细胞相关功能影响。[方法]1.按药物浓度梯度0、1、2、4、6μmol/l作用于Mino细胞,48h后提取蛋白,利用 western blot 检测 G9a 蛋白表达及 H3K9me2、H3K9me3、H3K4me2 及H3K27me2甲基化情况。2.利用CCK8方法检测用药后对Mino细胞增殖的影响并绘制细胞生长曲线。3.经周期检测试剂盒处理后,使用流式细胞技术,检测细胞周期情况。4.利用Western blot检测不同浓度梯度下CyclinD1、CDK4、P21蛋白表达情况。5.AnnexinV-FITC/PI双染细胞后,利用流式细胞技术,检测用药后对细胞凋亡的改变。6.利用Western blot检测不同药物浓度梯度下细胞凋亡蛋白BAX、caspase-3蛋白表达情况。[结果]1.药物作用后,随着药物浓度的改变,G9a蛋白表达无明显改变,说明药物只影响G9a活性。而随着药物浓度升高,H3K9me2、H3K27me2甲基化水平随着药物浓度增高而降低,H3K4me2甲基化水平逐渐升高,而H3K9me3甲基化水平没有明显改变。2.细胞生长曲线结果显示,在作用24h后细胞增殖能力出现明显下降,并且随着药物浓度的升高,抑制增殖的趋势越明显。3.药物作用48h后,流式结果显示G1期细胞增多,S期细胞减少,并且随着药物浓度的增加,被阻滞于G1期的细胞增多。Western blot结果显示,随着药物浓度增加,cyclinD1、CDK4和P21蛋白表达逐渐降低。4.药物作用48后,在0、1、2、4、6μmol/l药物浓度下,细胞凋亡率分别为8.9%、11.2%、14.3%、26.1%和 76.6%,差异具有统计学意义。(P<0.05)Western blot结果表明,随着药物浓度增加,凋亡蛋白BAX和capase-3表达逐渐增多。[结论]1.BIX01294可抑制G9a活性但对其表达没有影响。加药后可降低H3K9me2、H3K27me2甲基化水平,H3K4me2甲基化水平升高,对H3K9me3甲基化水平无影响。2.BIX01294可抑制细胞增殖,并与药物浓度和作用时间呈正相关。BIX01294可通过抑制cyclinD1、CDK4表达而影响细胞G1/S期的转化,将细胞阻滞于G1期,并且可通过上调BAX及caspase-3蛋白发挥促进细胞凋亡的作用。第三部分寻找G9a相互作用蛋白及相关下游靶基因研究[方法]1.通过CO-IP实验,寻找与G9a相互作用的蛋白。2.设计并合成针对G9a基因的ShRNA,以脂质体为介导转染子宫颈癌细胞株Hela细胞。3.转染48及72h后收集细胞,提取RNA及蛋白质,利用RT-qPCR及western blot检测mRNA水平及蛋白质表达水平,检测G9a干扰效率,选取干扰效率最好的一条ShRNA。4.根据干扰情况,挑选ShRNA序列,合成病毒LV-G9a-RNAi,转染套细胞淋巴瘤细胞系Jeko-1细胞,通过RT-PCR检测mRNA水平,验证转染效率。5.当转染效率达到50%以上时,通过基因表达谱芯片,筛选干扰后,表达有显著差异的基因。[结果]1.CO-IP结果显示在MCL中G9a可与HDAC1、HDAC2、UHRF1蛋白相互结合形成复合体,作用于下游靶基因,但与HDAC3未有结合。2.表达谱芯片结果显示,敲低G9a后,与NC组相比,共有611个基因发生改变,其中有266个基因表达上调,345个基因被抑制。干扰后,有多条与癌症相关的信号通路被抑制,其中对Tec激酶信号通路抑制作用最强,具体作用机制尚待进一步研究。[结论]1.在MCL中,G9a会与UHRF1及多种组蛋白去乙酰化酶结合共同发挥作用。2.干扰掉G9a后会影响多种基因表达,并影响相关信号通路。更多还原

【Abstract】 [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 Ⅱ 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 6μmol/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 Ⅲ 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.更多还原