長(zhǎng)鏈非編碼RNA與結(jié)直腸癌易感性及預(yù)后相關(guān)研究
發(fā)布時(shí)間：2018-08-13 12:23
【摘要】：目的：1.探討lncRNA基因遺傳變異與結(jié)直腸癌發(fā)病風(fēng)險(xiǎn)的關(guān)聯(lián)；2.探討lncRNA基因遺傳變異對(duì)結(jié)直腸癌患者預(yù)后的影響；3.從lncRNA表達(dá)譜入手,篩選出與結(jié)直腸癌相關(guān)的差異表達(dá)lncRNA;4.在表達(dá)水平上對(duì)部分篩選的lncRNA進(jìn)行驗(yàn)證,并分析其異常表達(dá)對(duì)結(jié)直腸癌患者預(yù)后的影響。方法：首先采用病例-對(duì)照研究設(shè)計(jì)分析lncRNA基因遺傳變異對(duì)結(jié)直腸癌易感性的影響。運(yùn)用中通量MassARRAY基因分型技術(shù)對(duì)腫瘤相關(guān)關(guān)鍵lncRNA基因的候選SNPs進(jìn)行分型。應(yīng)用Pearsonχ2檢驗(yàn)、Cochran-Armitage趨勢(shì)檢驗(yàn)及非條件Logistic回歸模型分析候選SNPs與結(jié)直腸癌發(fā)生風(fēng)險(xiǎn)的關(guān)聯(lián),并對(duì)基因-基因、基因-環(huán)境交互作用進(jìn)行探討。采用Log-Rank檢驗(yàn)和Cox比例風(fēng)險(xiǎn)模型分析候選SNPs對(duì)結(jié)直腸癌患者術(shù)后總體生存時(shí)間的影響。利用公共生物信息數(shù)據(jù)庫(kù)GEO,通過(guò)對(duì)結(jié)直腸癌lncRNA表達(dá)譜數(shù)據(jù)集的分析,篩選出與結(jié)直腸癌相關(guān)的差異表達(dá)lncRNA。通過(guò)GO和KEGG分析,對(duì)差異表達(dá)lncRNA的鄰近差異表達(dá)基因的功能進(jìn)行聚類富集分析,預(yù)測(cè)差異表達(dá)lncRNA可能涉及的生物過(guò)程及參與的信號(hào)通路。采用實(shí)時(shí)熒光定量PCR方法對(duì)部分篩選的差異表達(dá)lncRNA進(jìn)行檢測(cè)和驗(yàn)證。通過(guò)ROC曲線,評(píng)估陽(yáng)性lncRNA鑒別結(jié)直腸癌組織的能力。結(jié)合臨床病理信息及隨訪資料,分析差異表達(dá)lncRNA與結(jié)直腸癌患者不同臨床病理特征及術(shù)后總體生存時(shí)間的關(guān)系。結(jié)果：1.病例-對(duì)照研究中,我們共納入695例結(jié)直腸癌患者和701例健康對(duì)照。在環(huán)境危險(xiǎn)因素分析中,發(fā)現(xiàn)吸煙、飲酒及腫瘤家族史是結(jié)直腸癌發(fā)病的危險(xiǎn)因素。吸煙者結(jié)直腸癌的發(fā)生風(fēng)險(xiǎn)是不吸煙者的1.42倍(95%CI=1.14-1.81)；飲酒者結(jié)直腸癌的發(fā)生風(fēng)險(xiǎn)是不飲酒者的1.93倍(95%CI=1.43-2.59)；腫瘤家族史能夠顯著增加結(jié)直腸癌的發(fā)病風(fēng)險(xiǎn)(OR=1.73,95%CI=1.27-2.34)。吸煙、飲酒、肥胖及腫瘤家族史與結(jié)直腸癌患者術(shù)后的總體生存時(shí)間無(wú)顯著關(guān)聯(lián)(P0.05)。2.在lncRNA遺傳變異與結(jié)直腸癌發(fā)生風(fēng)險(xiǎn)及預(yù)后研究中,我們對(duì)IncRNA基因的16個(gè)關(guān)鍵SNPs進(jìn)行了分析。研究發(fā)現(xiàn),所納入的16個(gè)遺傳位點(diǎn)與結(jié)直腸癌的易感性未見(jiàn)顯著關(guān)聯(lián)。但分層分析發(fā)現(xiàn),在58歲人群中,PRNCR1 rs 13252298 GG基因型與結(jié)直腸癌的發(fā)病風(fēng)險(xiǎn)為臨界相關(guān),調(diào)整OR值為0.52(95%CI=0.26-1.04)。在≥58歲人群中,UCA1 rs 12462414每增加一個(gè)T等位基因,結(jié)直腸癌的發(fā)病風(fēng)險(xiǎn)增加33%(OR=1.33,95%CI=1.03-1.71).在女性人群中,UCA1 rs 12462414每增加一個(gè)T等位基因,結(jié)直腸癌的發(fā)病風(fēng)險(xiǎn)增加40%(OR=1.40,95%CI=1.04-1.87).生存分析發(fā)現(xiàn)H19上的rs2107425 CT基因型的結(jié)直腸癌患者相比攜帶CC基因型的患者,結(jié)直腸癌術(shù)后死亡的風(fēng)險(xiǎn)增加,結(jié)果具有邊緣顯著性(HR=1.55,95%CI= 0.99-2.41)。H19 rs217727每增加一個(gè)危險(xiǎn)等位基因A,結(jié)直腸癌患者術(shù)后死亡的風(fēng)險(xiǎn)增加35%(HR=1.35, 95%CI=1.02-1.77)。MALAT1 rs619586 GG基因型攜帶者結(jié)直腸癌術(shù)后死亡風(fēng)險(xiǎn)為AA基因型攜帶者的10.31倍(95%CI=1.32-80.59)。3.通過(guò)GEO中結(jié)直腸癌IncRNA表達(dá)譜芯片分析發(fā)現(xiàn),在結(jié)直腸腫瘤細(xì)胞中有176個(gè)IncRNA的表達(dá)水平存在差異,其中98個(gè)表達(dá)上調(diào)。在結(jié)直腸癌組織中有2474個(gè)IncRNA的表達(dá)水平存在差異,其中1613個(gè)表達(dá)上調(diào)。GO分析發(fā)現(xiàn)差異表達(dá)IncRNA可能涉及的生物過(guò)程有：白介素6受體結(jié)合、生長(zhǎng)因子受體結(jié)合、蛋白結(jié)合、細(xì)胞生長(zhǎng)調(diào)節(jié)、胰島素樣生長(zhǎng)因子結(jié)合等。KEGG分析發(fā)現(xiàn)差異表達(dá)的IncRNA可能參與的信號(hào)通路有：脂肪的消化和吸收、甘油酯代謝、腸道免疫網(wǎng)絡(luò)的IgA產(chǎn)生、NOD樣受體信號(hào)通路炎癥性腸病等。4.差異表達(dá)IncRNA的驗(yàn)證分析發(fā)現(xiàn),IncRNA AK022914、AP000525.8、UCA1在結(jié)直腸癌組織中的相對(duì)表達(dá)量高于正常組織,表達(dá)上調(diào),C17orf91表達(dá)下調(diào)(P0.05)。qPCR驗(yàn)證的四種IncRNA差異表達(dá)方向與芯片的結(jié)果一致。ROC曲線分析發(fā)現(xiàn),AK022914、AP000525.8、UCA1、C17orf91的曲線下面積分別為70.5%、67.1%、68.5%和59.8%。AK022914鑒別結(jié)直腸腫瘤組織和正常組織的能力最佳,其靈敏度、特異度可分別達(dá)到71.2%、67.3%。分析發(fā)現(xiàn)UCA1rs12462414遺傳變異與IncRNA UCA1表達(dá)量之間未見(jiàn)顯著關(guān)聯(lián)。生存分析發(fā)現(xiàn),lncRNA C17orf91低表達(dá)可顯著降低結(jié)直腸癌患者術(shù)后死亡風(fēng)險(xiǎn)(HR=0.21,95%CI=0.06-0.75)。結(jié)論：1.吸煙、飲酒及腫瘤家族史是結(jié)直腸癌發(fā)病的危險(xiǎn)因素；2.所納入的lncRNA遺傳變異與結(jié)直腸癌的發(fā)病風(fēng)險(xiǎn)無(wú)明顯關(guān)聯(lián)。但在≥58歲人群和女性人群中,UCA1 rs 12462414遺傳變異可增加結(jié)直腸癌的發(fā)病風(fēng)險(xiǎn)。3. lncRNA H19 rs217727和MALAT1 rs619586遺傳變異與結(jié)直腸癌患者術(shù)后的總體生存時(shí)間相關(guān)。4. lncRNA AK022914、AP000525.8和UCA1在結(jié)直腸癌組織中的表達(dá)上調(diào),C17orf91表達(dá)下調(diào),可能與結(jié)直腸癌的發(fā)生存在關(guān)聯(lián)。5. lncRNA C17orf91表達(dá)下調(diào)有利于提高結(jié)直腸癌患者術(shù)后總體生存時(shí)間,降低死亡風(fēng)險(xiǎn)。以上研究結(jié)論仍需要在大樣本人群中進(jìn)行驗(yàn)證,且需要更多的生物學(xué)研究以明確與結(jié)直腸癌發(fā)生發(fā)展相關(guān)lncRNA的功能及作用機(jī)制。
[Abstract]:Objective: 1. To explore the relationship between genetic variation of lncRNA gene and the risk of colorectal cancer; 2. To explore the effect of genetic variation of lncRNA gene on prognosis of colorectal cancer patients; 3. To screen differentially expressed lncRNA associated with colorectal cancer from the expression profiles of lncRNA; 4. To verify and analyze some selected lncRNA at the expression level. Methods: Case-control study design was used to analyze the effect of genetic variation of lncRNA gene on the susceptibility of colorectal cancer patients. The candidate SNPs of key lncRNA genes related to cancer were typed by mid-flux Mass ARRAY genotyping technique. Pearson_2 test and Cochran-Armitage genotyping were used. Trend test and unconditional logistic regression model were used to analyze the association between candidate SNPs and the risk of colorectal cancer, and gene-gene and gene-environment interactions were discussed. The differential expression of lncRNA associated with colorectal cancer was screened by analyzing the data set of lncRNA expression profiles of colorectal cancer based on GEO database. The clustering enrichment analysis of the functions of adjacent differentially expressed genes differentially expressing lncRNA was performed by GO and KEGG analysis to predict the biological processes involved in differential expression of lncRNA and the signaling pathways involved. Real-time fluorescence quantitative PCR was used to detect and validate the differentially expressed lncRNA. ROC curve was used to evaluate the ability of positive lncRNA to differentiate colorectal cancer tissues. The clinicopathological features and overall survival time of patients with differentially expressed lncRNA and colorectal cancer were analyzed by combining clinicopathological information and follow-up data. Results: 1. In the case-control study, 695 patients with colorectal cancer and 701 healthy controls were enrolled. Smoking, alcohol consumption and family history of cancer were found to be risk factors for colorectal cancer. The risk of colorectal cancer in smokers was 1.42 times higher than that in non-smokers (95% CI = 1.14-1.81); and in drinkers (95% CI = 1.14-1.81). The risk of colorectal cancer was 1.93 times higher than that of non-drinkers (95% CI = 1.43-2.59); family history of cancer significantly increased the risk of colorectal cancer (OR = 1.73, 95% CI = 1.27-2.34). Smoking, drinking, obesity, and family history of cancer were not significantly associated with overall survival time after colorectal cancer surgery (P 0.05). In the study of risk and prognosis of colorectal cancer, we analyzed 16 key SNPs of the IncRNA gene and found that there was no significant association between the 16 loci included and the susceptibility to colorectal cancer. The overall OR value was 0.52 (95% CI = 0.26-1.04). For each T allele added to UCA1 RS 12462414, the risk of colorectal cancer increased by 33% (OR = 1.33, 95% CI = 1.03-1.71). For each T allele added to UCA1 RS 12462414, the risk of colorectal cancer increased by 40% (OR = 1.40, 95% CI = 1.04-1.87) in women. Patients with rs2107425 CT genotype on H19 had an increased risk of postoperative mortality, with marginal significance (HR = 1.55, 95% CI = 0.99-2.41). Each addition of a risk allele A to H19 rs217727 increased the risk of postoperative mortality by 35% (HR = 1.35, 95% CI = 1.02). Postoperative mortality risk of colorectal cancer in MALAT1 rs619586 GG genotype carriers was 10.31 times higher than that in AA genotype carriers (95% CI = 1.32-80.59). 3. IncRNA expression profiles of colorectal cancer in GEO showed that there were 176 differences in the expression level of IncRNA in colorectal cancer cells, 98 of which were up-regulated. GO analysis revealed that the differential expression of IncRNA may involve biological processes such as interleukin-6 receptor binding, growth factor receptor binding, protein binding, cell growth regulation, insulin-like growth factor binding and so on. Involved in the signaling pathways are: fat digestion and absorption, glyceride metabolism, intestinal immune network IgA production, NOD-like receptor signaling pathway inflammatory bowel disease and so on. 4. Differential expression of IncRNA AK022914, AP000525.8, UCA1 in colorectal cancer tissue relative expression is higher than normal tissue, up-regulated expression, C17 orf91. The results of ROC curve analysis showed that the under-curve areas of AK022914, AP000525.8, UCA1, C17orf91 were 70.5%, 67.1%, 68.5% and 59.8%, respectively. AK022914 had the best ability to differentiate colorectal tumor tissue from normal tissue, and its sensitivity and specificity could be achieved respectively. 71.2%, 67.3%. There was no significant correlation between the genetic variation of UCA1rs12462414 and the expression of IncRNA UCA1. 2. There was no significant correlation between the genetic variation of lncRNA and the risk of colorectal cancer. However, the genetic variation of UCA1 RS 12462414 increased the risk of colorectal cancer in women aged 58 or older. 3. The genetic variation of lncRNA H19 rs217727 and MALAT 1 rs619586 correlated with the overall survival time of colorectal cancer patients after surgery. AK022914, AP000525.8 and UCA1 were up-regulated and C17orf91 was down-regulated in colorectal cancer tissues, which may be related to the occurrence of colorectal cancer. 5. The down-regulated expression of lncRNA C17orf91 is helpful to improve the overall survival time and reduce the risk of death in colorectal cancer patients. More biological studies are needed to clarify the function and mechanism of lncRNA associated with the development of colorectal cancer.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34
，
本文編號(hào)：2180980
[Abstract]:Objective: 1. To explore the relationship between genetic variation of lncRNA gene and the risk of colorectal cancer; 2. To explore the effect of genetic variation of lncRNA gene on prognosis of colorectal cancer patients; 3. To screen differentially expressed lncRNA associated with colorectal cancer from the expression profiles of lncRNA; 4. To verify and analyze some selected lncRNA at the expression level. Methods: Case-control study design was used to analyze the effect of genetic variation of lncRNA gene on the susceptibility of colorectal cancer patients. The candidate SNPs of key lncRNA genes related to cancer were typed by mid-flux Mass ARRAY genotyping technique. Pearson_2 test and Cochran-Armitage genotyping were used. Trend test and unconditional logistic regression model were used to analyze the association between candidate SNPs and the risk of colorectal cancer, and gene-gene and gene-environment interactions were discussed. The differential expression of lncRNA associated with colorectal cancer was screened by analyzing the data set of lncRNA expression profiles of colorectal cancer based on GEO database. The clustering enrichment analysis of the functions of adjacent differentially expressed genes differentially expressing lncRNA was performed by GO and KEGG analysis to predict the biological processes involved in differential expression of lncRNA and the signaling pathways involved. Real-time fluorescence quantitative PCR was used to detect and validate the differentially expressed lncRNA. ROC curve was used to evaluate the ability of positive lncRNA to differentiate colorectal cancer tissues. The clinicopathological features and overall survival time of patients with differentially expressed lncRNA and colorectal cancer were analyzed by combining clinicopathological information and follow-up data. Results: 1. In the case-control study, 695 patients with colorectal cancer and 701 healthy controls were enrolled. Smoking, alcohol consumption and family history of cancer were found to be risk factors for colorectal cancer. The risk of colorectal cancer in smokers was 1.42 times higher than that in non-smokers (95% CI = 1.14-1.81); and in drinkers (95% CI = 1.14-1.81). The risk of colorectal cancer was 1.93 times higher than that of non-drinkers (95% CI = 1.43-2.59); family history of cancer significantly increased the risk of colorectal cancer (OR = 1.73, 95% CI = 1.27-2.34). Smoking, drinking, obesity, and family history of cancer were not significantly associated with overall survival time after colorectal cancer surgery (P 0.05). In the study of risk and prognosis of colorectal cancer, we analyzed 16 key SNPs of the IncRNA gene and found that there was no significant association between the 16 loci included and the susceptibility to colorectal cancer. The overall OR value was 0.52 (95% CI = 0.26-1.04). For each T allele added to UCA1 RS 12462414, the risk of colorectal cancer increased by 33% (OR = 1.33, 95% CI = 1.03-1.71). For each T allele added to UCA1 RS 12462414, the risk of colorectal cancer increased by 40% (OR = 1.40, 95% CI = 1.04-1.87) in women. Patients with rs2107425 CT genotype on H19 had an increased risk of postoperative mortality, with marginal significance (HR = 1.55, 95% CI = 0.99-2.41). Each addition of a risk allele A to H19 rs217727 increased the risk of postoperative mortality by 35% (HR = 1.35, 95% CI = 1.02). Postoperative mortality risk of colorectal cancer in MALAT1 rs619586 GG genotype carriers was 10.31 times higher than that in AA genotype carriers (95% CI = 1.32-80.59). 3. IncRNA expression profiles of colorectal cancer in GEO showed that there were 176 differences in the expression level of IncRNA in colorectal cancer cells, 98 of which were up-regulated. GO analysis revealed that the differential expression of IncRNA may involve biological processes such as interleukin-6 receptor binding, growth factor receptor binding, protein binding, cell growth regulation, insulin-like growth factor binding and so on. Involved in the signaling pathways are: fat digestion and absorption, glyceride metabolism, intestinal immune network IgA production, NOD-like receptor signaling pathway inflammatory bowel disease and so on. 4. Differential expression of IncRNA AK022914, AP000525.8, UCA1 in colorectal cancer tissue relative expression is higher than normal tissue, up-regulated expression, C17 orf91. The results of ROC curve analysis showed that the under-curve areas of AK022914, AP000525.8, UCA1, C17orf91 were 70.5%, 67.1%, 68.5% and 59.8%, respectively. AK022914 had the best ability to differentiate colorectal tumor tissue from normal tissue, and its sensitivity and specificity could be achieved respectively. 71.2%, 67.3%. There was no significant correlation between the genetic variation of UCA1rs12462414 and the expression of IncRNA UCA1. 2. There was no significant correlation between the genetic variation of lncRNA and the risk of colorectal cancer. However, the genetic variation of UCA1 RS 12462414 increased the risk of colorectal cancer in women aged 58 or older. 3. The genetic variation of lncRNA H19 rs217727 and MALAT 1 rs619586 correlated with the overall survival time of colorectal cancer patients after surgery. AK022914, AP000525.8 and UCA1 were up-regulated and C17orf91 was down-regulated in colorectal cancer tissues, which may be related to the occurrence of colorectal cancer. 5. The down-regulated expression of lncRNA C17orf91 is helpful to improve the overall survival time and reduce the risk of death in colorectal cancer patients. More biological studies are needed to clarify the function and mechanism of lncRNA associated with the development of colorectal cancer.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.34
，
本文編號(hào)：2180980
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