【摘要】：前列腺癌是男性中最常見的惡性腫瘤之一,其發(fā)病具有顯著的地區(qū)及種族差異。中國前列腺癌的發(fā)病率也有逐年升高的趨勢。越來越多的證據(jù)表明,年齡、種族和家族史是目前公認的與前列腺癌發(fā)病相關(guān)的三大因素。前列腺癌的發(fā)生、發(fā)展可能是機體遺傳易感性和環(huán)境因素相互作用的結(jié)果,而與此相關(guān)的遺傳變異則決定個體罹患前列腺癌的易感性。迄今為止,在世界上不同種族人群中的全基因組關(guān)聯(lián)性研究已經(jīng)揭示了超過30個易感位點與前列腺癌的發(fā)生風(fēng)險有關(guān)。以候選基因為策略的前列腺癌遺傳易感性的研究也取得了豐碩的成果。目前認為,前列腺癌的發(fā)生、發(fā)展及其預(yù)后與遺傳物質(zhì)的多態(tài)性相關(guān),而且這種相關(guān)性隨著高危易感基因數(shù)目的增加而明顯增高。研究還發(fā)現(xiàn),盡管基因多態(tài)性與前列腺癌發(fā)病風(fēng)險相關(guān),但這種影響的效應(yīng)度往往較低,且受環(huán)境因素的影響(包括內(nèi)、外環(huán)境)。目前尚無經(jīng)多中心驗證的、較為可靠的與前列腺癌發(fā)病風(fēng)險及預(yù)后相關(guān)的遺傳多態(tài)位點。PI3K/AKT/mTOR信號傳導(dǎo)通路主要參與蛋白質(zhì)的合成。近年來,國內(nèi)外陸續(xù)有關(guān)該信號轉(zhuǎn)導(dǎo)通路基因單核苷酸多態(tài)性與前列腺癌易感性關(guān)系的研究報道,但結(jié)果不盡一致。研究發(fā)現(xiàn),該信號通路的活性主要與通路下游的哺乳動物雷帕霉素靶蛋白復(fù)合物1(mammalian target of rapamycin complex 1, mTORC1)的生物學(xué)活性相關(guān),體內(nèi)體外研究證實mTORC的異常與癌癥的發(fā)生相關(guān),并且是雷帕霉素作用的靶點。因此,有理由認為該復(fù)合物相關(guān)基因的多態(tài)性位點與前列腺癌發(fā)病風(fēng)險可能相關(guān)。mTORC1由一系列直接或間接作用于nTOR的關(guān)鍵分子共同組成,這些分子包括Raptor、mLST8、DEPTOR、PRAS40和]mTOR。其中,Raptor與mLST8起正向調(diào)節(jié)nTOR的作用,而DEPTOR和PRAS40起負向調(diào)節(jié)mTOR的作用。本研究將以mTORC1相關(guān)基因為候選基因,結(jié)合mTOR分子]mRNA及蛋白質(zhì)的表達及下游靶基因p70S6K和4EBP1的表達,在前列腺癌細胞株和前列腺癌組織標本上進行系統(tǒng)性的研究,有助于發(fā)現(xiàn)mTORC1在中國人群中與前列腺癌發(fā)病風(fēng)險有關(guān)的遺傳易感位點,從而為揭示PI3K/AKT/mTOR信號通路相關(guān)基因多態(tài)性在前列腺發(fā)病中的作用提供可能的遺傳學(xué)證據(jù),并為前列腺癌術(shù)后生化復(fù)發(fā)的預(yù)測提供可能的新的生物學(xué)標記。第一部分mTORC1相關(guān)基因遺傳多態(tài)性與前列腺癌易感性的關(guān)聯(lián)性研究為了研究mTORC1相關(guān)基因的、具有潛在功能性的SNPs與前列腺癌發(fā)病風(fēng)險的關(guān)聯(lián)性,我們在中國華東地區(qū)漢族人群中選擇1015例前列腺癌病例和1093例健康男性,開展以醫(yī)院為基礎(chǔ)的病例-對照關(guān)聯(lián)性研究,并采用Taqman探針方法對SNPs進行基因分型檢測,運用SAS9.1.3軟件包進行統(tǒng)計學(xué)描述及關(guān)聯(lián)分析。結(jié)果發(fā)現(xiàn),病例組以進展期前列腺癌為主,其中PSA大于20ng/ml的患者占54.4%,小于lOng/ml的患者占17.7%；而Gleason分級≤7(3+4)的患者占31.2%,≥7(3+4)的患者占59.7%；臨床分期Ⅰ+Ⅱ期患者占43.3%,Ⅲ+Ⅳ期患者占49.1%。本研究共檢測了mTORC1中5個基因的16個SNP位點,結(jié)果表明mTOR-rs2536 [加性模型：1.34(1.08-1.66),P=0.008；顯性模型：1.42(1.13-1.78),P=0.003],mTOR-rs1034528[加性模型：1.21(1.03-1.42),P=0.019；顯性模型：1.29(1.07-1.55), P=0.007], mTOR-rs2295080 [加性模型：0.80(0.69-0.94),P=0.005；顯性模型：0.76(0.64-0.92),P=0.003],及RPTOR-rs1062935[加性模型：1.14(1.01-1.29),P=0.0341；顯性模型：1.28(1.06-1.56),P=0.0127]與前列腺癌的發(fā)病風(fēng)險顯著關(guān)聯(lián)。而亞組分析的結(jié)果也表明,mTORC1中mTOR-rs536,-134528,-2295080,以及RPTOR-rs1062935與前列腺癌的發(fā)病風(fēng)險相關(guān),而患前列腺癌的風(fēng)險也隨著復(fù)合物中相關(guān)風(fēng)險基因數(shù)量的增加而明顯提高。本研究中的基因內(nèi)多位點單倍體分析、基因內(nèi)多位點聯(lián)合效應(yīng)分析、以及基因間的聯(lián)合效應(yīng)分析中都發(fā)現(xiàn)與前列腺癌發(fā)病風(fēng)險的關(guān)聯(lián)性。在logistic顯性模型中我們發(fā)現(xiàn)隨著風(fēng)險基因型數(shù)目的增加,患前列腺癌的風(fēng)險也逐漸提高,這種等位基因劑量相關(guān)的效應(yīng)在mTOR多位點的聯(lián)合分析中顯示明顯的前列腺癌發(fā)病關(guān)聯(lián)性,在小于69歲,BMI體重指數(shù)小于24kg/m2,曾經(jīng)吸煙,Gleason評分≤7(3+4),和臨床分期Ⅲ+Ⅳ的人群中尤其明顯。在進一步的交互作用分析中,我們發(fā)現(xiàn)BMI和mTOR-rs2536 (P=0.0031), BMI和AKTlS1-rs2290774 (P=0.0496),以及DEPTOR-rs4871827與RPTOR-rs1062935 (P=0.0224)之間在前列腺癌發(fā)病方面均存在交互作用。綜上所述,本研究運用較大的樣本量分析了mTORC1中SNPs與前列腺癌發(fā)病風(fēng)險的關(guān)連性,并獲得了有價值的信息。但是本研究還是存在一些局限性。尚需要多中心、更大樣本量及設(shè)計更加合理的研究來證實我們的研究結(jié)果。第二部分mTOR基因3,非編碼區(qū)陽性關(guān)聯(lián)位點的功能學(xué)研究在第一部分的關(guān)聯(lián)性研究中,我們發(fā)現(xiàn)多態(tài)位點mTOR-rs2536,-rs1034528,-rs2295080,和RPTOR-rs1062935與前列腺癌的發(fā)病風(fēng)險關(guān)聯(lián)。通過在線SNP分析平臺(http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm),我們進一步發(fā)現(xiàn)這些位點分別位于相關(guān)基因的3'UTR,內(nèi)含子,基因旁區(qū)和內(nèi)含子區(qū)。鑒于3'UTR區(qū)是調(diào)節(jié)基因活性的關(guān)鍵區(qū)域,并和腫瘤的發(fā)生發(fā)展密切關(guān)聯(lián),因此本部分對mTOR-rs2536的生物學(xué)意義進行了功能學(xué)研究,探尋其可能的生物學(xué)調(diào)控機制。在本研究中,我們構(gòu)建并克隆了含有mTOR 3'UTR全序列的PGL3-Promoter-mTOR3'UTR質(zhì)粒,質(zhì)粒中分別導(dǎo)入mTOR-rs2536野生型及突變型堿基。雙熒光素報告基因法檢測結(jié)果顯示,瞬時轉(zhuǎn)染miRNA-767-3p類似物48小時后rs2536突變型的熒光相對比值要低于野生型,而該miRNA抑制劑的作用剛好相反,提示miRNA-767-3p對野生型堿基的抑制作用較明顯,結(jié)果與前期相關(guān)性分析結(jié)果一致,提示該位點可能通過與相應(yīng)miRNA結(jié)合進而影響mTOR的表達。在隨后的SNPexp在線分析平臺上做出的基因型-免疫表型相關(guān)性分析結(jié)果提示,mTOR-rs2536位點影響mTOR基因的表達水平,而且這種影響可能具有種族相關(guān)性。在本研究中,我們沒有發(fā)現(xiàn)mTOR及其下游基因4EBP1和p70S6K的表達與mTOR-rs2536基因型之間的相關(guān)性；但我們發(fā)現(xiàn),在癌組織中mTOR的表達水平(35.7%)要高于癌旁組織(28.8%)；而4EBP1在癌組織及癌旁組織中的高表達率分別為32.1%和13.3%；p70S6K在癌組織及癌旁組織中的高表達率分別達到71.4%和39.4%。提示mTOR、4EBP1及p70S6K的活性與前列腺癌的發(fā)生或發(fā)展相關(guān)。綜上所述,mTOR-rs2536可能通過影響mTOR 3'UTR與相應(yīng)miRNA的結(jié)合達到調(diào)控mTOR的作用。但目前的實驗證據(jù)還是比較輕微,只能作為一種參考,要證實mTOR3'UTR點突變對mTOR本身表達的實際影響,還需要找到本身帶有mTOR該SNP的細胞系進行mRNA和蛋白質(zhì)水平的研究,才具有確實的說服力。這是本論文的主要不足以及今后研究的方向。第三部分mTORC1相關(guān)基因遺傳多態(tài)位點對前列腺癌臨床結(jié)局的風(fēng)險評估在mTORC1相關(guān)基因的遺傳變異因素與前列腺癌預(yù)后的相關(guān)性研究中,我們選取423例有較完善臨床隨訪資料的前列腺腺泡腺癌患者,結(jié)合前期發(fā)病風(fēng)險關(guān)聯(lián)的易感位點mTOR-rs2536、-rs1034528和-rs2295080以及RPTOR-rs1062935不同基因型進行前列腺癌根治術(shù)后的生存預(yù)后風(fēng)險評估。結(jié)果發(fā)現(xiàn),在0.1的檢驗標準下,相對于mTOR-rs1062935TT基因型,攜帶CT/CC基因型的患者遠期生化復(fù)發(fā)的幾率要高(-Plog-rank=0.067);而在該標準下相對于mTOR-rs2536AA基因型,攜帶GG/AG基因型的患者近期生化復(fù)發(fā)的幾率要高(-PWilcoxon=0.056)。在56例前列腺癌蛋白質(zhì)表達和預(yù)后相關(guān)性分析中我們發(fā)現(xiàn),p70s6k的高表達能明顯增加前列腺癌術(shù)后生化復(fù)發(fā)的幾率,但是本研究中蛋白質(zhì)表達與隨訪結(jié)果的例數(shù)非常有限,結(jié)果尚需加大樣本量進一步驗證。
[Abstract]:Prostate cancer is one of the most common malignant tumors in men, with significant regional and racial differences. The incidence of prostate cancer in China is also increasing year by year. More and more evidence shows that age, race and family history are the three major factors associated with prostate cancer. This may be the result of the interaction of genetic predisposition and environmental factors, and the genetic variation associated with it determines the susceptibility of individuals to prostate cancer. So far, more than 30 susceptible loci in different ethnic groups in the world have been revealed to be associated with the risk of prostate cancer. The study of genetic susceptibility to prostate cancer with candidate genes has also been fruitful. It is now believed that the occurrence, development and prognosis of prostate cancer are associated with genetic polymorphisms, and this correlation increases with the increase in the number of Gao Weiyi genes. The risk of adenocarcinoma is related, but the effect of this effect is often low and is affected by environmental factors (including internal and external environment). There is no multicenter, more reliable genetic polymorphic loci.PI3K/AKT/mTOR signaling pathway related to the risk and prognosis of prostate cancer, which is mainly involved in the synthesis of protein. The study on the relationship between the single nucleotide polymorphism of the signal transduction pathway and the susceptibility to prostate cancer has been reported at home and abroad, but the results are not consistent. The study found that the activity of the signal pathway is mainly with the mammalian rapamycin target protein complex 1 (mammalian target of rapamycin complex 1, mTORC1) downstream. Biological activity is related, in vivo and in vitro studies have confirmed that the abnormality of mTORC is associated with the occurrence of cancer, and is a target for the effect of rapamycin. Therefore, it is reasonable to think that the polymorphic loci of the complex gene may be associated with the risk of prostate cancer and the.MTORC1 is composed of a series of key molecules that direct or indirectly act on nTOR. These molecules include Raptor, mLST8, DEPTOR, PRAS40 and]mTOR., and Raptor and mLST8 play a positive role in regulating nTOR, while DEPTOR and PRAS40 play a negative role in regulating mTOR. Systematic studies on cell lines and prostate cancer specimens help to find mTORC1 genetic susceptibility loci associated with the risk of prostate cancer in Chinese population, thus providing possible genetic evidence to reveal the role of PI3K/AKT/mTOR signaling associated gene polymorphism in the pathogenesis of prostate and for prostate cancer. The prediction of postoperative biochemical recurrence provides possible new biological markers. Part 1 Association of genetic polymorphisms of mTORC1 related genes and prostate cancer susceptibility in order to study the association of mTORC1 related genes with potentially functional SNPs and the risk of prostate cancer. We selected the Han population in eastern China. In 1015 cases of prostate cancer and 1093 healthy men, a case control association study based on the hospital was carried out, and the Taqman probe method was used to detect SNPs genotyping, and the SAS9.1.3 software package was used to carry out statistical description and correlation analysis. The results showed that the case group was mainly the advanced prostate cancer, of which PSA was greater than 20ng/ The patients in ML accounted for 54.4% and less than lOng/ml, while those with Gleason < 7 (3+4) accounted for 31.2% and 7 (3+4) accounted for 59.7%; clinical stage I + II patients accounted for 43.3%, and the patients in stage III + IV accounted for 16 SNP sites of the 5 genes in mTORC1, and the results showed that mTOR-rs2536 [additive model: 1.34 (1.08-1.66) (1.08-1.66). ), P=0.008; explicit model: 1.42 (1.13-1.78), P=0.003], mTOR-rs1034528[additive model: 1.21 (1.03-1.42), P=0.019; dominant model: 1.29 (1.07-1.55), P=0.007], mTOR-rs2295080 [additive model: 0.80 (0.69-0.94), P=0.005; 0.76 (0.64-0.92), 0.76 (0.64-0.92), and additive model: 1.14 0.0341; the dominant model: 1.28 (1.06-1.56), P=0.0127] was associated with the risk of prostate cancer, and the subgroup analysis also showed that mTOR-rs536, -134528, -2295080, and RPTOR-rs1062935 were associated with the risk of prostate cancer, and the risk of prostate cancer also increased with the number of related risk genes in the complex. In this study, the multiple point haploid analysis, intra gene multipoint effect analysis, and the association effect analysis were found to be associated with the risk of prostate cancer. In the logistic dominant model, we found that the risk of prostate cancer is also increased with the increase in the number of risk genotypes. Gradually, the dose dependent effect of this allele was associated with a significant association of prostate cancer in the mTOR multipoint analysis. At the age of less than 69 years, the BMI body mass index was less than 24kg/m2, once smoked, the Gleason score was less than 7 (3+4), and the clinical stage III + IV was especially obvious. In further interaction analysis, we hair BMI and mTOR-rs2536 (P=0.0031), BMI and AKTlS1-rs2290774 (P=0.0496), as well as DEPTOR-rs4871827 and RPTOR-rs1062935 (P=0.0224) have interaction in the pathogenesis of prostate cancer. To sum up, this study used a large sample size to analyze the relationship between SNPs and the risk of prostate cancer in mTORC1 and to obtain the price. However, there are some limitations in this study. We still need multi center, larger sample size and more reasonable design to confirm our research results. Second part of the mTOR gene 3, the functional study of the non coding region positive association loci in the first part of the association study, we found polymorphic loci mTOR-rs2536, -rs 1034528, -rs2295080, and RPTOR-rs1062935 are associated with the risk of prostate cancer. Through the online SNP analysis platform (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm), we further found that these loci are located in the related genes of 3'UTR, intron, para gene and intron. In view of the 3'UTR region is the regulation of gene activity. The key area is closely related to the occurrence and development of the tumor. Therefore, the functional study of the biological significance of mTOR-rs2536 was studied in this part, and the possible mechanism of biological regulation was explored. In this study, we constructed and cloned the PGL3-Promoter-mTOR3'UTR plasmid containing the full sequence of mTOR 3'UTR, and the plasmid was introduced into the wild of mTOR-rs2536 in the wild. The results showed that the relative ratio of the rs2536 mutant was lower than that of the wild type after the transient transfection of miRNA-767-3p analogues for 48 hours, and the effect of the miRNA inhibitor was just the opposite, suggesting that the inhibitory effect of miRNA-767-3p on the wild type base was more obvious, and the results were related to the earlier correlation. The results are consistent, suggesting that the locus may be associated with the corresponding miRNA and then affect the expression of mTOR. The genotype - immunophenotypic correlation analysis made on a subsequent SNPexp online analysis platform suggests that the mTOR-rs2536 locus affects the expression level of the mTOR gene, and this effect may have a racial correlation. In this study, We did not find the correlation between the expression of mTOR and its downstream genes 4EBP1 and p70S6K with the mTOR-rs2536 genotype, but we found that the expression level of mTOR in the cancer tissues (35.7%) was higher than that of the para cancerous tissue (28.8%), and the high expression rate of 4EBP1 in the cancer tissues and Para cancerous tissues was 32.1% and 13.3%, respectively, and p70S6K in cancer tissue and cancer. The high expression rate in the para tissue is 71.4% and 39.4%., respectively, suggesting that the activity of mTOR, 4EBP1 and p70S6K is related to the occurrence or development of prostate cancer. To sum up, mTOR-rs2536 may be used to regulate the role of mTOR 3'UTR and the corresponding miRNA to regulate mTOR. However, the current experimental evidence is still relatively mild and can only be used as a reference. The actual effect of mTOR3'UTR point mutation on the expression of mTOR itself, and the need to find the mRNA and protein levels of the cell lines with mTOR SNP, is a true persuasive. This is the main deficiency of this paper and the direction of future research. The third part of the genetic polymorphic loci of mTORC1 related genes to prostate cancer. The risk assessment of bed outcome was associated with the correlation between the genetic variation factors of mTORC1 related genes and the prognosis of prostate cancer. We selected 423 cases of prostatic adenocarcinoma with more complete clinical follow-up data, the susceptible locus mTOR-rs2536 associated with the preonset risk, -rs1034528 and -rs2295080, and the different bases of RPTOR-rs1062935. The risk assessment of survival after radical prostatectomy was conducted. The results showed that under the 0.1 test standard, the risk of long-term biochemical recurrence was higher in patients carrying CT/CC genotypes than the mTOR-rs1062935TT genotype (-Plog-rank=0.067); compared to the mTOR-rs2536AA genotype, the patients carrying the GG/AG genotype under this standard were in this standard. The risk of recent biochemical recurrence is high (-PWilcoxon=0.056). In the analysis of the protein expression and prognosis of 56 cases of prostate cancer, we found that high expression of P70S6K can significantly increase the risk of postoperative biochemical recurrence of prostate cancer. However, the number of cases of protein expression and follow-up results in this study is very limited, and the results still need to be increased. Further verification.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25

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相關(guān)期刊論文 前1條

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