激活素A通过甲基转移酶1诱导骨髓瘤细胞系NS-1细胞凋亡

发布时间：2018-07-05 02:20

本文选题：DNA甲基化转移酶1 + 激活素A　；参考：《吉林大学》2017年博士论文

【摘要】：研究背景激活素A属于TGF-β超家族成员,其受体属于丝氨酸/苏氨酸激酶(Ser/Thr)型受体,分为I型受体及II型受体,激活素与II型结合,再招募I型受体,进而通过细胞内Smads信号蛋白,或非Smads依赖的MAPK通路、Rho GTPase通路和PI3K/Akt通路传导信号至细胞核,激活靶基因转录。有关研究显示,激活素A的表达与肿瘤的发生发展密切相关,参与多种肿瘤细胞增殖及凋亡调控。如大肠癌及骨肉瘤等患者外周血激活素A水平升高,大肠癌及卵巢癌等组织高表达激活素A,激活素A可以诱导肺腺癌细胞凋亡、促进前列腺癌细胞转移,激活素A表达异常还与肿瘤恶液质形成有关等。DNA甲基化是由DNA甲基化转移酶DNMTs(DNA methyltransferases,Dnmts)催化并维持的,DNMTs家族包括DNMT1、DNMT2、DNMT3a、DNMT3b及DNMT3L。DNMTs是调控表观遗传学DNA甲基化的重要分子,DNMT1主要起维持性甲基化酶作用。以往的研究显示,DNMTs在肿瘤细胞中高表达,DNMTs的表达还能影响胚胎发育过程,推测细胞核潜能越高与DNMTs的调控关系越密切。激活素也具有调控中胚层细胞发育及肿瘤细胞增殖的作用,但是,激活素A能否通过调控DNMTs表达,影响肿瘤细胞增殖及凋亡,仍不清楚。因此,本研究通过预实验,选择对激活素A敏感的小鼠骨髓瘤细胞系NS-1细胞作为靶细胞,探讨激活素A调控NS-1细胞增殖及凋亡与DNMT1表达的关系。课题分为四方面进行论述,第一部分分析了激活素A对多种肿瘤细胞的生物学作用,探讨激活素A作用与DNMT1关系。第二部分阐述了激活素A调控小鼠骨髓瘤细胞NS-1增殖及凋亡作用及其信号机制,分析激活素信号蛋白Smad3 Knock-down及过表达对DNMT1的影响。第三部分通过DNMT1Knock-down,进一步探讨改变DNMT1表达对激活素A抑制NS-1细胞增殖及诱导凋亡的影响及其机制。第四部分建立NS-1荷瘤鼠模型,评价激活素A及DNMT1 Knock-down对小鼠体内NS-1细胞成瘤的影响。研究方法RT-PCR及Western blotting检测DNMT1在NS-1细胞的表达。MTT法、CFSE染色、Hochest33342染色及Annexin V-7AAD评价NS-1肿瘤细胞活力及凋亡。构建pLVX-U6/Puro-DNMT1 sh RNA表达质粒,转染NS-1细胞评价敲低DNMT1基因对细胞增殖及凋亡的影响。建立NS-1细胞荷瘤鼠模型,HE染色、免疫组织化学染色、Tunel法评价DNMT1基因Knock-down对NS-1细胞成瘤的影响。研究结果第一部分激活素A对多种肿瘤细胞活性及DNMT1表达的影响DNMT1在肿瘤细胞高表达,而在正常组织来源细胞低表达。Giemsa染色法显示激活素A作用于SW480、Hela、A549、NS-1细胞后发生明显的细胞形态学改变,提示激活素A可以影响NS-1细胞形态变化。且在低浓度5ng/ml时激活素A即可影响NS-1细胞形态变化。同时可见激活素A可以显著抑制骨髓瘤细胞系NS-1细胞DNMT1表达。因此,实验采用NS-1细胞作为激活素A敏感细胞,进行后续研究。第二部分激活素A诱导小鼠骨髓瘤细胞系NS-1凋亡为了确定激活素A对NS-1细胞的作用,实验首先采用MTT法检测NS-1细胞活力,结果显示激活素A可以抑制NS-1细胞活力。CFSE染色进一步显示,激活素A抑制了NS-1细胞增殖。Hocheast染色和Annexin V-7AAD结果显示激活素A可以促进NS-1细胞凋亡。Western blotting结果显示激活素A上调NS-1细胞Bax、Cyc C及Caspase3表达,而抑制Bcl2及DNMT1表达,提示激活素A通过线粒体凋亡途径诱导NS-1细胞凋亡,其作用可能与下调DNMT1表达有关。进一步研究显示,激活素A可以促进NS-1细胞表达激活素信号蛋白Smad3,但过表达和Knock-down Smad3不能影响NS-1细胞DNMT1表达,提示激活素A可能通过Smad3信号非依赖途径调控DNMT1表达。第三部分DNMT1 Knock-down对NS-1细胞的活力及凋亡的影响Western blotting结果显示构建的pLVX-DNMT1干涉质粒转染NS-1细胞成功敲低了DNMT1表达,同时上调NS-1细胞Cyc C及Caspase3表达及Bax/Bcl2比值。MTT结果显示NS-1细胞转染0.5μg pLVX-DNMT1质粒24h后,与pLVX对照组质粒转染NS-1细胞组比较细胞活力明显下降;添加5ng/ml激活素A组,较激活素A处理的pLVX对照组质粒转染NS-1细胞组细胞活力进一步下降。Annexin V-7AAD结果显示转染pLVX-DNMT1质粒可以诱导NS-1细胞发生凋亡。第四部分DNMT1 Knock-down对小鼠NS-1细胞成瘤的影响为了进一步确定DNMT1 Knock-down对激活素A诱导NS-1细胞凋亡的影响,实验首先采用不同浓度的NS-1细胞接种Balb/c小鼠背部,观察成瘤情况。结果显示2.0x106 NS-1细胞/0.1ml接种小鼠背部皮下,成功构建了NS-1细胞荷瘤鼠模型。进一步分析DNMT1 Knock-down对小鼠NS-1细胞体内成瘤影响。实验采用预转染pLVX-NC空质粒、转染pLVX-DNMT1 sh RNA表达质粒及未转染质粒的野生型NS-1接种Balb/c小鼠背部,体内测量肿瘤体积结果显示,与pLVX-NC组相比,pLVX-DNMT1组的NS-1细胞成瘤能力明显减弱,且体积增长减慢。提示DNMT1 Knock-down可以抑制NS-1细胞体内成瘤能力。待肿瘤生长到第18天处死Balb/c小鼠取肿瘤,结果显示与pLVX-NC组相比,pLVX-DNMT1组的NS-1细胞肿瘤体积明显降低,具有统计学差异。提取肿瘤组织总蛋白Westernblotting结果显示pLVX-DNMT1组成功敲底了DNMT1的表达,且HE染色结果和免疫组织化学染色结果显示与pLVX-NC组相比,pLVX-DNMT1组细胞核浆比例明显增加、排列紊乱、局部出现脓肿和坏死,提示DNMT1 Knock-down可以诱导NS-1细胞瘤发生凋亡,可能具有骨髓瘤治疗价值。Tunnel染色显示与激活素A处理的pLVX-NC组相比,激活素A处理的DNMT1 Knock-down的NS-1细胞肿瘤组织存在更多的染色阳性细胞,提示DNMT1 Knock-down促进了激活素A诱导NS-1细胞凋亡作用。综上所述,DNMT1是DNA甲基化表观遗传学调控的重要分子蛋白,降低NS-1细胞DNMT1的表达水平,可以通过线粒体凋亡途径抑制NS-1细胞增殖、促进NS-1细胞凋亡。激活素A可以调控肿瘤细胞DNMT1表达,对治疗DNA甲基化修饰异常所引发的肿瘤疾病具有重要的意义。
[Abstract]:Background activin A belongs to the member of the TGF- beta superfamily, whose receptor belongs to the serine / threonine kinase (Ser/Thr) receptor, which is divided into I type and II receptor. Activin is combined with II type, and then recruitment of I type receptor, and then through intracellular Smads signal protein, or MAPK pathway of non Smads dependent Lai, Rho GTPase pathway and transduction pathway. The study shows that the expression of activin A is closely related to the occurrence and development of tumor, and participates in the regulation of proliferation and apoptosis of various tumor cells. The levels of peripheral blood activin A in patients with colorectal cancer and osteosarcoma, such as large intestine and ovarian cancer, are high expression of activin A, and activin A can induce lung gland. The apoptosis of cancer cells, the promotion of the metastasis of prostate cancer cells, the abnormal expression of activin A and the formation of.DNA methylation related to the formation of malignant tumor fluid are catalyzed and maintained by DNA methyltransferase DNMTs (DNA methyltransferases, Dnmts). The DNMTs family consists of DNMT1, DNMT2, DNMT3a, DNMT3b, and regulating the weight of epigenetic methylation. DNMT1 mainly acts as a maintainable methylation enzyme. Previous studies have shown that DNMTs is highly expressed in tumor cells, and the expression of DNMTs can also affect the process of embryonic development. It is presumed that the higher the cell nuclear potential is, the closer the relationship between the regulation of DNMTs and the regulation of mesoderm cell development and tumor cell proliferation. Whether or not A can affect the proliferation and apoptosis of tumor cells by regulating the expression of DNMTs and affecting the proliferation and apoptosis of tumor cells is still unclear. Therefore, this study selects the NS-1 cell line of murine myeloma cell line sensitive to activin A as a target cell, and discusses the relationship between activin A regulation of NS-1 cell proliferation and apoptosis and the expression of DNMT1. The subject is divided into four aspects. The first part analyses the biological effect of activin A on a variety of tumor cells, and discusses the relationship between activin A and DNMT1. Second the effect of activin A on the proliferation and apoptosis of murine myeloma cells and its signal mechanism and the effect of Smad3 Knock-down on activin signal protein and the effect of overexpression on DNMT1 are discussed. The third part is connected with the activity of activin protein. DNMT1Knock-down, further explore the effect and mechanism of DNMT1 expression on activin A inhibition of NS-1 cell proliferation and induction of apoptosis. Fourth the model of NS-1 bearing mice was established to evaluate the effect of activin A and DNMT1 Knock-down on the tumor formation of NS-1 cells in mice. The expression of.MTT, CFSE staining, Hochest33342 staining and Annexin V-7AAD were used to evaluate the viability and apoptosis of NS-1 tumor cells. PLVX-U6/Puro-DNMT1 sh RNA expression plasmid was constructed, and NS-1 cells were transfected to evaluate the effect of low DNMT1 gene on cell proliferation and apoptosis. The effect of NMT1 gene Knock-down on the tumor formation of NS-1 cells. The first part of the study was the effect of activin A on the activity of various tumor cells and the expression of DNMT1, DNMT1 was highly expressed in the tumor cells, and the expression of activin A in the normal tissue derived cells showed that the activin A acted upon SW480, Hela, A549, and NS-1 cells. It is suggested that activin A can affect the morphological changes of NS-1 cells, and activin A can affect the morphological changes of NS-1 cells at low concentration of 5ng/ml. At the same time, activin A can significantly inhibit the expression of DNMT1 in myeloma cell line NS-1 cells. Therefore, the experiment uses NS-1 cells as activin A sensitive cells for follow-up study. Second parts Activin A induced the apoptosis of mouse myeloma cell line NS-1 in order to determine the effect of activin A on NS-1 cells. First, the MTT assay was used to detect the activity of NS-1 cells. The results showed that activin A can inhibit the.CFSE staining of NS-1 cell viability further. Activin A inhibits NS-1 cell proliferation.Hocheast staining and the result of NS-1. Activin A can promote NS-1 cell apoptosis,.Western blotting results show that activin A up-regulated NS-1 cells Bax, Cyc C and Caspase3 expression, and inhibits Bcl2 and DNMT1 expression, suggesting that activin may induce apoptosis through mitochondrial apoptosis pathway, and its role may be related to down regulation. The expression of activin signal protein Smad3 was expressed in NS-1 cells, but overexpression and Knock-down Smad3 did not affect the DNMT1 expression in NS-1 cells, suggesting that activin A may regulate DNMT1 expression through non dependent pathway of Smad3 signal. Third part DNMT1 Knock-down on the vitality and apoptosis of NS-1 cells The transfection of NS-1 cells with the interference plasmid successfully knocked down the expression of DNMT1, at the same time, the expression of Cyc C and Caspase3 in NS-1 cells and the Bax/Bcl2 ratio.MTT results showed that the NS-1 cells transfected to 0.5 mu g pLVX-DNMT1 plasmid 24h, compared with the control group plasmids transfected to the cell group, the cell viability was significantly decreased. LVX control group plasmid transfected NS-1 cell cell viability further decreased.Annexin V-7AAD results showed that transfection of pLVX-DNMT1 plasmids could induce apoptosis of NS-1 cells. Fourth the effect of DNMT1 Knock-down on the tumor formation of mice NS-1 cells in order to further determine the effect of DNMT1 Knock-down on the apoptosis of activin A induced cells. First of all, NS-1 cells with different concentrations were inoculated on the back of Balb/c mice to observe the tumor formation. The results showed that the 2.0x106 NS-1 cell /0.1ml was inoculated subcutaneously on the back of the mouse, and the tumor mouse model of NS-1 cells was successfully constructed. The effect of DNMT1 Knock-down on the tumor formation in the mice NS-1 cells was further analyzed. The experiment was carried out by pre transfection of pLVX-NC empty plasmid and transfection pLV. X-DNMT1 sh RNA expression plasmid and untransfected wild type NS-1 were inoculated on the back of Balb/c mice. The volume of tumor volume measured in vivo showed that the tumor formation ability of NS-1 cells in pLVX-DNMT1 group decreased significantly and slowed down compared with pLVX-NC group, suggesting that DNMT1 Knock-down could inhibit the tumorigenesis of NS-1 cells. 18 days after the death of Balb/c mice, the results showed that the volume of NS-1 cells in the group pLVX-DNMT1 was significantly lower than that in the pLVX-NC group, with a statistically significant difference. The result of the extraction of the total protein of the tumor tissue Westernblotting showed that the pLVX-DNMT1 group successfully knocked down the expression of DNMT1, and the results of HE staining and immunohistochemical staining showed pLVX-. Compared with group NC, the proportion of cell nuclear plasma in pLVX-DNMT1 group was obviously increased, arranged in disorder, local abscess and necrosis, suggesting that DNMT1 Knock-down could induce apoptosis of NS-1 cytoma, which might have the value of myeloma treatment with.Tunnel staining, compared with pLVX-NC group treated with activin A, DNMT1 Knock-down NS-1 cell swelling of activin A treatment. More staining positive cells exist in the tumor tissue, suggesting that DNMT1 Knock-down promotes the apoptosis of NS-1 cells induced by activin A. To sum up, DNMT1 is an important molecular protein of DNA methylation epigenetic regulation, which reduces the expression level of DNMT1 in NS-1 cells, and can inhibit the proliferation of NS-1 cells through mitochondrial apoptosis pathway and promote NS-1 cells. Apoptosis, activin A can regulate the expression of DNMT1 in tumor cells, which is of great significance in the treatment of tumor diseases caused by abnormal DNA methylation.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R730.2

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