3,6-二羟黄酮表遗传调控miR-34a和miR-21抑制乳腺癌发生分子机制研究

发布时间：2018-05-13 14:52

本文选题：3 + 6-DHF　；参考：《第三军医大学》2015年博士论文

【摘要】：乳腺癌是全世界女性肿瘤中最常见的类型,乳腺癌的防治研究十分重要。常见黄酮类化合物抗肿瘤活性的药效学筛选实验中发现3,6-二羟黄酮(3,6-dihydroxyflavone,3,6-DHF)有较强的抗肿瘤活性,能抑制乳腺癌发生。进一步研究发现3,6-DHF在发挥抗乳腺癌发生效应的同时改变了乳腺组织mi RNA的表达谱,其中以miR-34a和mi R-21的变化最为显著,3,6-DHF可能具有调控mi R-34a和miR-21的作用。越来越多的证据表明食物中的营养成分或植物化学物可以调节特定转录位点的表遗传变异,这些营养成分或植物化学物可通过DNA甲基化和组蛋白修饰调控某些特定基因表达,进而抑制肿瘤的发生和发展。miR-34a表达在肿瘤发生过程中可因其基因启动子区DNA甲基化升高而沉默其表达,miR-21的表达水平也受组蛋白乙酰化修饰的调节。基于以上分析,我们提出:3,6-DHF可通过表遗传修饰调控乳腺癌发生过程中mi R-34a和miR-21的表达,抑制乳腺癌的发生。mi R-34a和miR-21对PI3K/Akt/m TOR信号通路有重要的调节作用,10号染色体上缺失与张力蛋白同源的磷酸酶基因(phosphatase and tensin homologue deleted from chromosome 10,PTEN)是PI3K/Akt/mTOR信号通路的抑制剂,而Notch-1是该信号通路的激活剂,PTEN和Notch-1分别是mi R-34a和miR-21的作用靶点,3,6-DHF可能通过mi R-34a和mi R-21调节PI3K/Akt/mTOR信号通路。因此,本研究从表遗传角度出发,探讨3,6-DHF通过表遗传修饰调控mi R-34a和mi R-21表达的可能机制,并阐明3,6-DHF通过调控mi R-34a和mi R-21抑制PI3K/Akt/m TOR信号通路,进而抑制乳腺癌发生的机制。本课题采用甲基亚硝基脲(1-methyl-1-nitrosourea,MNU)诱导大鼠乳腺癌发生模型,取实验0、2、18w大鼠乳腺和肿瘤组织分析p-Akt和人甲基胞嘧啶双加氧酶1(The ten-eleven translocation methylcytosine dioxygenase,TET)的表达;采用生长因子依赖性降低(reduced dependence on growth factors,RDGF)、锚定非依赖性生长特性(anchorage independent growth,AIG)和细胞划痕实验评价致癌物4-甲基亚硝胺-1-(3-吡啶)-1-丁酮(4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,nnk)和苯并芘(benzo[a]pyrene,b[a]p)诱导乳腺上皮细胞mcf10a癌性转化特性;其次,采用甲基化特异性pcr(methylation-specificpcr,msp)、染色体免疫共沉淀(chromatinimmunoprecipitation,chip)、实时荧光定量pcr(quantitativereal-timepcr,qrt-pcr)和sirna转染等方法明确3,6-dhf是否具有调节乳腺癌(breastcancer,bc)细胞mir-34a和mir-21基因表遗传修饰的作用,利用质粒转染、dna斑点印迹和蛋白免疫印迹(westernblot,wb)等技术,分析dna甲基转移酶(dnamethyltransferase-1,dnmt)1和tet1的甲基化调节机制在3,6-dhf调控mir-34a表达中的作用;采用wb和转染等技术进一步明确3,6-dhf通过调控mir-34a和mir-21抑制pi3k/akt/mtor信号通路及下游蛋白s6蛋白激酶(ribosomalp70-s6kinase,s6k70)、核糖体蛋白s6(ribosomalproteins6,rps6)、真核翻译起始因子4b(eukaryotictranslationinitiationfactors,eif4b)和真核生物启动因子4e结合蛋白(4ebindingproteinl,4ebp)1的表达,进而抑制乳腺癌发生的作用。实验结果:(1)mnu诱导大鼠乳腺癌发生过程中取0、2、18w实验大鼠乳腺和肿瘤组织,免疫组化染色表明3,6-dhf能抑制大鼠乳腺和肿瘤组织p-akt的表达,提高tet1的表达水平。致癌物nnk和b[a]p处理乳腺上皮细胞mcf10a(carcinogens-treatedcell,cart)30d,cart细胞表现出显著的rdgf和aig癌细胞特性;与30-dcart细胞相比,3,6-dhf与致癌物联合作用(c-dhf)能显著降低细胞rdgf和aig能力;细胞划痕实验也显示,30-dcart细胞迁移和增殖能力显著增强,c-dhf细胞迁移和增殖能力明显降低。qrt-pcr检测结果显示cart细胞mir-34a表达水平显著下降,mir-21表达水平显著升高,3,6-dhf共同作用可显著抑制致癌剂诱导的mir-34a下调和mir-21上调。此外,3,6-dhf作用乳腺癌(breastcancer,bc)细胞株,能显著上调mda-mb-231(m231)、mcf-7(m7)和mda-mb-453(m453)细胞mir-34a的水平,下调其mir-21水平,anti-mir-34a寡核苷酸或pcdna6.2-gw/mir-21质粒转染能抑制3,6-dhf对m231细胞mir-34a和mir-21的调节。(2)msp检测致癌物作用mcf10a细胞和bc细胞mir-34a基因启动子区dna甲基化。结果显示,30-dc-dhf细胞mir-34a基因启动子区dna甲基化显著降低;同样,3,6-dhf可明显降低bc细胞mir-34a基因启动子区dna甲基化;chip-qpcr检测m231细胞mir-21基因启动子区组蛋白修饰h3k9-14ac、h3k27ac(转录激活的组蛋白修饰)和h3k27me3(转录抑制的组蛋白修饰)水平,结果显示3,6-dhf显著降低h3k9-14ac修饰水平。(3)wb结果显示cart细胞tet1蛋白表达降低,c-dhf细胞tet1蛋白表达高于cart细胞,免疫组化染色显示在mnu诱导的大鼠乳腺癌发生过程中给予3,6-dhf(20mg/kg,i.g.)能提高大鼠乳腺和肿瘤组织tet1蛋白表达;qrt-pcr、wb和dna斑点杂交检测m231细胞tet1和dna甲基化中间产物5羟甲基胞嘧啶(5-hydroxymethylcytosion,5hmc)的表达,结果显示3,6-dhf可显著提高细胞tet1和5hmc的表达水平,免疫组化染色显示3,6-dhf能提高m231细胞裸鼠移殖瘤组织tet1和5hmc的表达;msp检测结果显示3,6-dhf能降低m231细胞tet1基因启动子区dna甲基化。(4)dnmt活性检测结果显示3,6-dhf能有效抑制bc细胞dnmt1的活性,利用autodock-vina软件模拟3,6-dhf和dnmt1蛋白的相互作用,结果显示3,6-dhf与dnmt1天然疏水活性结构域最低的结合能为-7.1kcal/mol;采用pcdna3/myc-dnmt1质粒转染m231细胞,wb结果显示dnmt1蛋白表达明显增高,tet1蛋白表达明显下降,3,6-dhf不能提高dnmt1过表达m231细胞内tet1的蛋白表达;msp结果显示,3,6-dhf不能降低dnmt1过表达细胞内tet1基因启动子区dna甲基化。采用tet1sirna转染m231细胞沉默tet1蛋白表达,3,6-dhf不能提高转染后细胞tet1的蛋白表达;qrt-pcr结果显示,3,6-dhf不能提高转染后细胞mir-34amrna水平;msp结果显示,3,6-dhf不能降低转染后细胞mir-34a基因启动子区dna甲基化。(5)对pi3k/akt/mtor信号通路的研究显示,随着致癌物作用时间的延长,cart细胞pi3k(p85、p110)、p-akt(thr308、ser473)和p-mtor(s2448、s2481)蛋白表达逐渐升高,而c-dhf细胞pi3k、p-akt和p-mtor蛋白表达低于cart细胞;akt和mtor激酶活性检测结果显示,与mcf10a细胞相比,cart细胞akt和mtor的活性升高,而c-dhf细胞akt和mtor的活性低于cart细胞;免疫组化染色显示3,6-dhf(20mg/kg,i.g.)干预的mnu诱导大鼠乳腺和肿瘤组织p-akt表达低于没有干预的大鼠。同样,3,6-dhf降低bc细胞pi3k/akt/mtor信号通路pi3k、p-akt和p-mtor的蛋白表达,降低细胞akt和mtor的活性;免疫组化染色显示y|鼠3,6-dhf(20mg/kg,i.g.)干预能显著降低m231细胞y|鼠移殖瘤组织p-akt(t308)的表达;wb检测pi3k/akt/mtor信号通路下游蛋白的表达,结果显示3,6-dhf能显著降低该通路下游s6k70、rps6、eif4b和4ebp1的蛋白表达水平。(6)WB结果显示,随着致癌物作用时间的延长,Car T细胞PTEN蛋白表达水平逐渐降低,Notch-1和Hes-1的蛋白表达水平升高,而C-DHF细胞PTEN蛋白表达水平高于Car T细胞,Notch-1和Hes-1的蛋白表达水平低于Car T细胞。3,6-DHF作用BC细胞能提高PTEN蛋白表达,降低Notch-1和Hes-1蛋白表达。M231细胞转染mi R-21质粒(TCmiR-21)或转染mi R-34a寡核苷酸(TCanti-34a),WB检测结果显示3,6-DHF不能提高TCmi R-21细胞PTEN蛋白表达水平和降低TCanti-34a细胞Notch-1蛋白表达水平,3,6-DHF对TCmi R-21和TCanti-34a细胞Akt和mTOR的活性没有抑制作用。实验结论:(1)3,6-DHF能抑制大鼠乳腺癌发生,抑制致癌物诱导MCF10A细胞发生癌性转化,并能抑制MCF10A细胞癌性转化过程中mi R-34a水平的降低和mi R-21水平的升高。3,6-DHF作用乳腺癌细胞能升高mi R-34a的水平和降低mi R-21的水平;(2)MSP和ChIP-q PCR分析结果提示3,6-DHF通过DNA甲基化调节mi R-34a水平,通过组蛋白乙酰化调节mi R-21的水平;进一步研究表明3,6-DHF通过抑制DNMT1的活性,促进TET1表达,进而调节miR-34a基因启动子区DNA甲基化,促进miR-34a基因转录。(3)利用质粒或寡核苷酸转染,明确3,6-DHF通过mi R-34a和mi R-21调节PTEN、Notch-1蛋白表达,抑制PI3K/Akt/mTOR信号通路活性和下游蛋白表达,进而发挥抑制乳腺癌发生的作用。
[Abstract]:Breast cancer is the most common type of female tumor in the world. The study of the prevention and control of breast cancer is very important. In the screening experiment of the anti tumor activity of common flavonoids, it is found that 3,6- dihydroxy flavonoids (3,6-dihydroxyflavone, 3,6-DHF) have strong anti-tumor activity and can inhibit the occurrence of breast cancer. Further research found that 3,6-DHF is in the hair. The expression of MI RNA in mammary gland tissues was changed at the same time, with the most significant changes in miR-34a and MI R-21, and 3,6-DHF may have the role of regulating mi R-34a and miR-21. More and more evidence suggests that nutrients or phytochemicals in food can regulate epigenetic variation at specific transcriptional sites. Some nutrients or phytochemicals can regulate the expression of certain genes through DNA methylation and histone modification, and then inhibit the development of tumor and develop.MiR-34a expression in the process of tumor genesis, which can be silenced by the increase of DNA methylation in the promoter region of the gene, and the expression level of miR-21 is also regulated by histone acetylation. Based on the above analysis, we suggest that 3,6-DHF can regulate the expression of MI R-34a and miR-21 during the development of breast cancer by epigenetic modification, and inhibit the occurrence of.Mi R-34a and miR-21 in the occurrence of breast cancer to have an important regulatory effect on the PI3K/Akt/m TOR signaling pathway, and the deletion of the phosphatase gene homologous with tension protein on chromosome 10 (phosphatase and) Tensin homologue deleted from chromosome 10, PTEN) is an inhibitor of the PI3K/Akt/mTOR signaling pathway, and Notch-1 is the activator of the signaling pathway. PTEN and Notch-1 are mi R-34a and acting targets respectively. To explore the possible mechanism of 3,6-DHF regulation of MI R-34a and MI R-21 through epigenetic modification, and to clarify the mechanism that 3,6-DHF can inhibit the occurrence of breast cancer by regulating mi R-34a and MI R-21 to inhibit the occurrence of breast cancer. The expression of p-Akt and human methylcytosine dioxygenase 1 (The ten-eleven translocation methylcytosine dioxygenase, TET) was analyzed in the mammary gland and tumor tissue of 0,2,18w rats, and the growth factor dependence (reduced dependence on growth), and the anchored non dependent growth characteristics were used. 4- methylnitrosamine -1- (3- pyridine) -1- butanone (4- (methylnitrosamino) -1- (3-pyridyl) -1-butanone, NNK) and benzopyrene (benzo[a]pyrene, b[a]p) induced the carcinogenic transformation characteristics of mammary epithelial cells were evaluated by cell scratch test. Omatinimmunoprecipitation, chip), real-time fluorescence quantitative PCR (quantitativereal-timepcr, qRT-PCR) and siRNA transfection methods to determine whether 3,6-dhf has the role of regulating the genetic modification of the miR-34a and miR-21 gene tables of breast cancer (breastcancer, BC) cells, using plasmid transfer, DNA dot blot and protein immunoblotting. To analyze the role of methylation regulation mechanism of DNA methyltransferase (dnamethyltransferase-1, DNMT) 1 and Tet1 in 3,6-dhf regulation of miR-34a expression, and to further clarify 3,6-dhf through regulation of miR-34a and miR-21 to inhibit pi3k/akt/mtor signaling pathway and lower swim protein S6 protein kinase by WB and transfection. Ribosomal protein S6 (ribosomalproteins6, RPS6), eukaryotic translation initiation factor 4B (eukaryotictranslationinitiationfactors, eIF4B) and eukaryotic promoter 4E binding protein (4ebindingproteinl, 4ebp) 1 expression to inhibit the occurrence of breast cancer. The results were as follows: (1) 0,2,18w experiment was taken during MNU induced rat breast cancer The immunohistochemical staining showed that 3,6-dhf could inhibit the expression of p-Akt in the mammary gland and tumor tissue of rats and improve the expression level of Tet1. The carcinogen NNK and b[a]p treated the mcf10a (carcinogens-treatedcell, cart) 30d in the mammary epithelial cells, and the cart cells showed significant rdgf and AIG cell characteristics; 3 The combination of 6-dhf and carcinogens (c-dhf) significantly reduced the ability of cell rdgf and AIG, and the cell scratch test also showed that the migration and proliferation ability of 30-dcart cells increased significantly, the migration and proliferation ability of c-dhf cells decreased significantly by.Qrt-pcr detection results, and the miR-34a table level of cart cells decreased significantly, the expression level of miR-21 increased significantly and 3,6-d was 3,6-d. 3,6-d The combined effect of HF can significantly inhibit the down regulation of miR-34a induced by carcinogens and the up regulation of miR-21. In addition, the 3,6-dhf action of breastcancer, BC cell lines can significantly increase the level of MDA-MB-231 (m231), MCF-7 (M7) and MDA-MB-453 (m453) cells, down the levels of the oligonucleotides or plasmid transfection energy. Inhibition of 3,6-dhf on the regulation of miR-34a and miR-21 in m231 cells. (2) MSP detected the DNA methylation of the promoter region of mcf10a and BC cells in BC cells. The results showed that the DNA methylation of 30-dc-dhf cell miR-34a gene promoter region decreased significantly. PCR detected the m231 cell miR-21 gene promoter region histone modification h3k9-14ac, h3k27ac (transcriptional activated histone modification) and H3K27me3 (transcriptional inhibition histone modification) level. The results showed that 3,6-dhf significantly reduced the level of h3k9-14ac modification. (3) WB results showed that Tet1 protein expression in cart cells decreased and the expression of c-dhf cell protein was higher than that of h3k9-14ac. The immuno histochemical staining showed that 3,6-dhf (20mg/kg, i.g.) could improve the expression of Tet1 protein in the breast and tumor tissues of rats induced by MNU; qRT-PCR, WB and DNA dot blot hybridization detected the expression of 5 hydroxymethyl cytosine (5-hydroxymethylcytosion,) in m231 cell Tet1 and DNA methylation intermediate. 3,6-dhf could significantly increase the expression level of Tet1 and 5hmc. Immunohistochemical staining showed that 3,6-dhf could improve the expression of Tet1 and 5hmc in the transplanted tumor tissues of m231 cells. The results of MSP detection showed that 3,6-dhf could reduce DNA methylation in the promoter region of m231 cell Tet1 gene. (4) the results of activity detection showed that it could effectively inhibit the proliferation of cells. Autodock-vina software was used to simulate the interaction of 3,6-dhf and DNMT1 protein. The results showed that the lowest binding energy of 3,6-dhf and DNMT1 was -7.1kcal/mol, and m231 cells were transfected with pcdna3/myc-dnmt1 plasmid. WB results showed that DNMT1 protein expression was obviously increased, Tet1 protein decreased obviously, 3,6-dhf was not. DNMT1 overexpressed Tet1 protein expression in m231 cells, and MSP results showed that 3,6-dhf could not reduce the DNA methylation of Tet1 gene promoter region in DNMT1 overexpressed cells. Tet1sirna transfected m231 cells silenced Tet1 protein expression, and 3,6-dhf could not improve the expression of protein in the transfected cells. The results showed that the transfection could not improve the transfection. MSP results showed that 3,6-dhf could not reduce DNA methylation of miR-34a gene promoter region of cells after transfection. (5) the study of pi3k/akt/mtor signaling pathway showed that the expression of PI3K (p85, P110), p-Akt (thr308, P110), and proteins in cart cells increased gradually with the prolongation of the action time of carcinogens. The expression of PI3K, p-Akt and p-mTOR protein was lower than that of cart cells, and the activity of Akt and mTOR kinase activity showed that the activity of Akt and mTOR of cart cells was higher than that of mcf10a cells, while c-dhf cell Akt and the activity of cart cells were lower than those of the cells; immunohistochemistry staining showed that the mammary and tumor tissues of rats were induced by immunohistochemical staining. Similarly, 3,6-dhf decreased the expression of PI3K, p-Akt and p-mTOR in the pi3k/akt/mtor signaling pathway of BC cells, and reduced the activity of Akt and mTOR in the cells. Immunohistochemistry showed that the intervention of 3,6-dhf (20mg/kg, i.g.) could significantly reduce the expression of the graft. The expression of downstream protein in the signal pathway showed that 3,6-dhf could significantly reduce the protein expression level of s6k70, RPS6, eIF4B and 4ebp1 downstream of the pathway. (6) WB results showed that the expression level of PTEN protein in Car T cells decreased gradually with the prolongation of the action time of carcinogens, and the expression level of Notch-1 and Hes-1 increased, while C-DHF cell proteins were increased. The expression level is higher than that of Car T cells. The protein expression level of Notch-1 and Hes-1 is lower than that of Car T cells. BC cells can improve the expression of PTEN protein, and the expression of Notch-1 and Hes-1 proteins can be reduced. The expression level of PTEN protein and the expression level of Notch-1 protein in TCanti-34a cells, 3,6-DHF has no inhibitory effect on the activity of Akt and mTOR in TCmi R-21 and TCanti-34a cells. The experimental conclusion: (1) 3,6-DHF can inhibit the occurrence of breast cancer in rats, inhibit carcinogenesis induced by carcinogenic substances and inhibit the carcinogenic transformation of MCF10A cells. The decrease of MI R-34a level in the course and the increase of MI R-21 level in.3,6-DHF cells can increase the level of MI R-34a and reduce the level of MI R-21; (2) MSP and ChIP-q PCR analysis results suggest that the level of protein acetylation is regulated by methylation. Inhibit the activity of DNMT1, promote the expression of TET1, and then regulate the DNA methylation of the promoter region of the miR-34a gene and promote the miR-34a gene transcription. (3) the expression of 3,6-DHF through mi R-34a and MI R-21 is used to regulate PTEN, Notch-1 protein expression, inhibition of signaling pathway activity and downstream protein expression, and then play inhibitory milk. The role of adenocarcinoma.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9

【参考文献】