本文選題：3 + 6-DHF��； 參考：《第三軍醫(yī)大學(xué)》2015年博士論文

【摘要】：乳腺癌是全世界女性腫瘤中最常見的類型,乳腺癌的防治研究十分重要。常見黃酮類化合物抗腫瘤活性的藥效學(xué)篩選實驗中發(fā)現(xiàn)3,6-二羥黃酮(3,6-dihydroxyflavone,3,6-DHF)有較強的抗腫瘤活性,能抑制乳腺癌發(fā)生。進一步研究發(fā)現(xiàn)3,6-DHF在發(fā)揮抗乳腺癌發(fā)生效應(yīng)的同時改變了乳腺組織mi RNA的表達譜,其中以miR-34a和mi R-21的變化最為顯著,3,6-DHF可能具有調(diào)控mi R-34a和miR-21的作用。越來越多的證據(jù)表明食物中的營養(yǎng)成分或植物化學(xué)物可以調(diào)節(jié)特定轉(zhuǎn)錄位點的表遺傳變異,這些營養(yǎng)成分或植物化學(xué)物可通過DNA甲基化和組蛋白修飾調(diào)控某些特定基因表達,進而抑制腫瘤的發(fā)生和發(fā)展。miR-34a表達在腫瘤發(fā)生過程中可因其基因啟動子區(qū)DNA甲基化升高而沉默其表達,miR-21的表達水平也受組蛋白乙�；揎椀恼{(diào)節(jié)�；谝陨戏治�,我們提出:3,6-DHF可通過表遺傳修飾調(diào)控乳腺癌發(fā)生過程中mi R-34a和miR-21的表達,抑制乳腺癌的發(fā)生。mi R-34a和miR-21對PI3K/Akt/m TOR信號通路有重要的調(diào)節(jié)作用,10號染色體上缺失與張力蛋白同源的磷酸酶基因(phosphatase and tensin homologue deleted from chromosome 10,PTEN)是PI3K/Akt/mTOR信號通路的抑制劑,而Notch-1是該信號通路的激活劑,PTEN和Notch-1分別是mi R-34a和miR-21的作用靶點,3,6-DHF可能通過mi R-34a和mi R-21調(diào)節(jié)PI3K/Akt/mTOR信號通路。因此,本研究從表遺傳角度出發(fā),探討3,6-DHF通過表遺傳修飾調(diào)控mi R-34a和mi R-21表達的可能機制,并闡明3,6-DHF通過調(diào)控mi R-34a和mi R-21抑制PI3K/Akt/m TOR信號通路,進而抑制乳腺癌發(fā)生的機制。本課題采用甲基亞硝基脲(1-methyl-1-nitrosourea,MNU)誘導(dǎo)大鼠乳腺癌發(fā)生模型,取實驗0、2、18w大鼠乳腺和腫瘤組織分析p-Akt和人甲基胞嘧啶雙加氧酶1(The ten-eleven translocation methylcytosine dioxygenase,TET)的表達;采用生長因子依賴性降低(reduced dependence on growth factors,RDGF)、錨定非依賴性生長特性(anchorage independent growth,AIG)和細胞劃痕實驗評價致癌物4-甲基亞硝胺-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)誘導(dǎo)乳腺上皮細胞mcf10a癌性轉(zhuǎn)化特性;其次,采用甲基化特異性pcr(methylation-specificpcr,msp)、染色體免疫共沉淀(chromatinimmunoprecipitation,chip)、實時熒光定量pcr(quantitativereal-timepcr,qrt-pcr)和sirna轉(zhuǎn)染等方法明確3,6-dhf是否具有調(diào)節(jié)乳腺癌(breastcancer,bc)細胞mir-34a和mir-21基因表遺傳修飾的作用,利用質(zhì)粒轉(zhuǎn)染、dna斑點印跡和蛋白免疫印跡(westernblot,wb)等技術(shù),分析dna甲基轉(zhuǎn)移酶(dnamethyltransferase-1,dnmt)1和tet1的甲基化調(diào)節(jié)機制在3,6-dhf調(diào)控mir-34a表達中的作用;采用wb和轉(zhuǎn)染等技術(shù)進一步明確3,6-dhf通過調(diào)控mir-34a和mir-21抑制pi3k/akt/mtor信號通路及下游蛋白s6蛋白激酶(ribosomalp70-s6kinase,s6k70)、核糖體蛋白s6(ribosomalproteins6,rps6)、真核翻譯起始因子4b(eukaryotictranslationinitiationfactors,eif4b)和真核生物啟動因子4e結(jié)合蛋白(4ebindingproteinl,4ebp)1的表達,進而抑制乳腺癌發(fā)生的作用。實驗結(jié)果:(1)mnu誘導(dǎo)大鼠乳腺癌發(fā)生過程中取0、2、18w實驗大鼠乳腺和腫瘤組織,免疫組化染色表明3,6-dhf能抑制大鼠乳腺和腫瘤組織p-akt的表達,提高tet1的表達水平。致癌物nnk和b[a]p處理乳腺上皮細胞mcf10a(carcinogens-treatedcell,cart)30d,cart細胞表現(xiàn)出顯著的rdgf和aig癌細胞特性;與30-dcart細胞相比,3,6-dhf與致癌物聯(lián)合作用(c-dhf)能顯著降低細胞rdgf和aig能力;細胞劃痕實驗也顯示,30-dcart細胞遷移和增殖能力顯著增強,c-dhf細胞遷移和增殖能力明顯降低。qrt-pcr檢測結(jié)果顯示cart細胞mir-34a表達水平顯著下降,mir-21表達水平顯著升高,3,6-dhf共同作用可顯著抑制致癌劑誘導(dǎo)的mir-34a下調(diào)和mir-21上調(diào)。此外,3,6-dhf作用乳腺癌(breastcancer,bc)細胞株,能顯著上調(diào)mda-mb-231(m231)、mcf-7(m7)和mda-mb-453(m453)細胞mir-34a的水平,下調(diào)其mir-21水平,anti-mir-34a寡核苷酸或pcdna6.2-gw/mir-21質(zhì)粒轉(zhuǎn)染能抑制3,6-dhf對m231細胞mir-34a和mir-21的調(diào)節(jié)。(2)msp檢測致癌物作用mcf10a細胞和bc細胞mir-34a基因啟動子區(qū)dna甲基化。結(jié)果顯示,30-dc-dhf細胞mir-34a基因啟動子區(qū)dna甲基化顯著降低;同樣,3,6-dhf可明顯降低bc細胞mir-34a基因啟動子區(qū)dna甲基化;chip-qpcr檢測m231細胞mir-21基因啟動子區(qū)組蛋白修飾h3k9-14ac、h3k27ac(轉(zhuǎn)錄激活的組蛋白修飾)和h3k27me3(轉(zhuǎn)錄抑制的組蛋白修飾)水平,結(jié)果顯示3,6-dhf顯著降低h3k9-14ac修飾水平。(3)wb結(jié)果顯示cart細胞tet1蛋白表達降低,c-dhf細胞tet1蛋白表達高于cart細胞,免疫組化染色顯示在mnu誘導(dǎo)的大鼠乳腺癌發(fā)生過程中給予3,6-dhf(20mg/kg,i.g.)能提高大鼠乳腺和腫瘤組織tet1蛋白表達;qrt-pcr、wb和dna斑點雜交檢測m231細胞tet1和dna甲基化中間產(chǎn)物5羥甲基胞嘧啶(5-hydroxymethylcytosion,5hmc)的表達,結(jié)果顯示3,6-dhf可顯著提高細胞tet1和5hmc的表達水平,免疫組化染色顯示3,6-dhf能提高m231細胞裸鼠移殖瘤組織tet1和5hmc的表達;msp檢測結(jié)果顯示3,6-dhf能降低m231細胞tet1基因啟動子區(qū)dna甲基化。(4)dnmt活性檢測結(jié)果顯示3,6-dhf能有效抑制bc細胞dnmt1的活性,利用autodock-vina軟件模擬3,6-dhf和dnmt1蛋白的相互作用,結(jié)果顯示3,6-dhf與dnmt1天然疏水活性結(jié)構(gòu)域最低的結(jié)合能為-7.1kcal/mol;采用pcdna3/myc-dnmt1質(zhì)粒轉(zhuǎn)染m231細胞,wb結(jié)果顯示dnmt1蛋白表達明顯增高,tet1蛋白表達明顯下降,3,6-dhf不能提高dnmt1過表達m231細胞內(nèi)tet1的蛋白表達;msp結(jié)果顯示,3,6-dhf不能降低dnmt1過表達細胞內(nèi)tet1基因啟動子區(qū)dna甲基化。采用tet1sirna轉(zhuǎn)染m231細胞沉默tet1蛋白表達,3,6-dhf不能提高轉(zhuǎn)染后細胞tet1的蛋白表達;qrt-pcr結(jié)果顯示,3,6-dhf不能提高轉(zhuǎn)染后細胞mir-34amrna水平;msp結(jié)果顯示,3,6-dhf不能降低轉(zhuǎn)染后細胞mir-34a基因啟動子區(qū)dna甲基化。(5)對pi3k/akt/mtor信號通路的研究顯示,隨著致癌物作用時間的延長,cart細胞pi3k(p85、p110)、p-akt(thr308、ser473)和p-mtor(s2448、s2481)蛋白表達逐漸升高,而c-dhf細胞pi3k、p-akt和p-mtor蛋白表達低于cart細胞;akt和mtor激酶活性檢測結(jié)果顯示,與mcf10a細胞相比,cart細胞akt和mtor的活性升高,而c-dhf細胞akt和mtor的活性低于cart細胞;免疫組化染色顯示3,6-dhf(20mg/kg,i.g.)干預(yù)的mnu誘導(dǎo)大鼠乳腺和腫瘤組織p-akt表達低于沒有干預(yù)的大鼠。同樣,3,6-dhf降低bc細胞pi3k/akt/mtor信號通路pi3k、p-akt和p-mtor的蛋白表達,降低細胞akt和mtor的活性;免疫組化染色顯示y|鼠3,6-dhf(20mg/kg,i.g.)干預(yù)能顯著降低m231細胞y|鼠移殖瘤組織p-akt(t308)的表達;wb檢測pi3k/akt/mtor信號通路下游蛋白的表達,結(jié)果顯示3,6-dhf能顯著降低該通路下游s6k70、rps6、eif4b和4ebp1的蛋白表達水平。(6)WB結(jié)果顯示,隨著致癌物作用時間的延長,Car T細胞PTEN蛋白表達水平逐漸降低,Notch-1和Hes-1的蛋白表達水平升高,而C-DHF細胞PTEN蛋白表達水平高于Car T細胞,Notch-1和Hes-1的蛋白表達水平低于Car T細胞。3,6-DHF作用BC細胞能提高PTEN蛋白表達,降低Notch-1和Hes-1蛋白表達。M231細胞轉(zhuǎn)染mi R-21質(zhì)粒(TCmiR-21)或轉(zhuǎn)染mi R-34a寡核苷酸(TCanti-34a),WB檢測結(jié)果顯示3,6-DHF不能提高TCmi R-21細胞PTEN蛋白表達水平和降低TCanti-34a細胞Notch-1蛋白表達水平,3,6-DHF對TCmi R-21和TCanti-34a細胞Akt和mTOR的活性沒有抑制作用。實驗結(jié)論:(1)3,6-DHF能抑制大鼠乳腺癌發(fā)生,抑制致癌物誘導(dǎo)MCF10A細胞發(fā)生癌性轉(zhuǎn)化,并能抑制MCF10A細胞癌性轉(zhuǎn)化過程中mi R-34a水平的降低和mi R-21水平的升高。3,6-DHF作用乳腺癌細胞能升高mi R-34a的水平和降低mi R-21的水平;(2)MSP和ChIP-q PCR分析結(jié)果提示3,6-DHF通過DNA甲基化調(diào)節(jié)mi R-34a水平,通過組蛋白乙酰化調(diào)節(jié)mi R-21的水平;進一步研究表明3,6-DHF通過抑制DNMT1的活性,促進TET1表達,進而調(diào)節(jié)miR-34a基因啟動子區(qū)DNA甲基化,促進miR-34a基因轉(zhuǎn)錄。(3)利用質(zhì)�；蚬押塑账徂D(zhuǎn)染,明確3,6-DHF通過mi R-34a和mi R-21調(diào)節(jié)PTEN、Notch-1蛋白表達,抑制PI3K/Akt/mTOR信號通路活性和下游蛋白表達,進而發(fā)揮抑制乳腺癌發(fā)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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

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相關(guān)期刊論文 前2條

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2 賈振偉;高樹新;張永春;張顯華;;TET蛋白的去甲基化機制及其在調(diào)控小鼠發(fā)育過程中的作用[J];遺傳;2015年01期

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