长链非编码RNA与结直肠癌易感性及预后相关研究
发布时间：2018-08-13 12:23
【摘要】：目的：1.探讨lncRNA基因遗传变异与结直肠癌发病风险的关联；2.探讨lncRNA基因遗传变异对结直肠癌患者预后的影响；3.从lncRNA表达谱入手,筛选出与结直肠癌相关的差异表达lncRNA;4.在表达水平上对部分筛选的lncRNA进行验证,并分析其异常表达对结直肠癌患者预后的影响。方法：首先采用病例-对照研究设计分析lncRNA基因遗传变异对结直肠癌易感性的影响。运用中通量MassARRAY基因分型技术对肿瘤相关关键lncRNA基因的候选SNPs进行分型。应用Pearsonχ2检验、Cochran-Armitage趋势检验及非条件Logistic回归模型分析候选SNPs与结直肠癌发生风险的关联,并对基因-基因、基因-环境交互作用进行探讨。采用Log-Rank检验和Cox比例风险模型分析候选SNPs对结直肠癌患者术后总体生存时间的影响。利用公共生物信息数据库GEO,通过对结直肠癌lncRNA表达谱数据集的分析,筛选出与结直肠癌相关的差异表达lncRNA。通过GO和KEGG分析,对差异表达lncRNA的邻近差异表达基因的功能进行聚类富集分析,预测差异表达lncRNA可能涉及的生物过程及参与的信号通路。采用实时荧光定量PCR方法对部分筛选的差异表达lncRNA进行检测和验证。通过ROC曲线,评估阳性lncRNA鉴别结直肠癌组织的能力。结合临床病理信息及随访资料,分析差异表达lncRNA与结直肠癌患者不同临床病理特征及术后总体生存时间的关系。结果：1.病例-对照研究中,我们共纳入695例结直肠癌患者和701例健康对照。在环境危险因素分析中,发现吸烟、饮酒及肿瘤家族史是结直肠癌发病的危险因素。吸烟者结直肠癌的发生风险是不吸烟者的1.42倍(95%CI=1.14-1.81)；饮酒者结直肠癌的发生风险是不饮酒者的1.93倍(95%CI=1.43-2.59)；肿瘤家族史能够显著增加结直肠癌的发病风险(OR=1.73,95%CI=1.27-2.34)。吸烟、饮酒、肥胖及肿瘤家族史与结直肠癌患者术后的总体生存时间无显著关联(P0.05)。2.在lncRNA遗传变异与结直肠癌发生风险及预后研究中,我们对IncRNA基因的16个关键SNPs进行了分析。研究发现,所纳入的16个遗传位点与结直肠癌的易感性未见显著关联。但分层分析发现,在58岁人群中,PRNCR1 rs 13252298 GG基因型与结直肠癌的发病风险为临界相关,调整OR值为0.52(95%CI=0.26-1.04)。在≥58岁人群中,UCA1 rs 12462414每增加一个T等位基因,结直肠癌的发病风险增加33%(OR=1.33,95%CI=1.03-1.71).在女性人群中,UCA1 rs 12462414每增加一个T等位基因,结直肠癌的发病风险增加40%(OR=1.40,95%CI=1.04-1.87).生存分析发现H19上的rs2107425 CT基因型的结直肠癌患者相比携带CC基因型的患者,结直肠癌术后死亡的风险增加,结果具有边缘显著性(HR=1.55,95%CI= 0.99-2.41)。H19 rs217727每增加一个危险等位基因A,结直肠癌患者术后死亡的风险增加35%(HR=1.35, 95%CI=1.02-1.77)。MALAT1 rs619586 GG基因型携带者结直肠癌术后死亡风险为AA基因型携带者的10.31倍(95%CI=1.32-80.59)。3.通过GEO中结直肠癌IncRNA表达谱芯片分析发现,在结直肠肿瘤细胞中有176个IncRNA的表达水平存在差异,其中98个表达上调。在结直肠癌组织中有2474个IncRNA的表达水平存在差异,其中1613个表达上调。GO分析发现差异表达IncRNA可能涉及的生物过程有：白介素6受体结合、生长因子受体结合、蛋白结合、细胞生长调节、胰岛素样生长因子结合等。KEGG分析发现差异表达的IncRNA可能参与的信号通路有：脂肪的消化和吸收、甘油酯代谢、肠道免疫网络的IgA产生、NOD样受体信号通路炎症性肠病等。4.差异表达IncRNA的验证分析发现,IncRNA AK022914、AP000525.8、UCA1在结直肠癌组织中的相对表达量高于正常组织,表达上调,C17orf91表达下调(P0.05)。qPCR验证的四种IncRNA差异表达方向与芯片的结果一致。ROC曲线分析发现,AK022914、AP000525.8、UCA1、C17orf91的曲线下面积分别为70.5%、67.1%、68.5%和59.8%。AK022914鉴别结直肠肿瘤组织和正常组织的能力最佳,其灵敏度、特异度可分别达到71.2%、67.3%。分析发现UCA1rs12462414遗传变异与IncRNA UCA1表达量之间未见显著关联。生存分析发现,lncRNA C17orf91低表达可显著降低结直肠癌患者术后死亡风险(HR=0.21,95%CI=0.06-0.75)。结论：1.吸烟、饮酒及肿瘤家族史是结直肠癌发病的危险因素；2.所纳入的lncRNA遗传变异与结直肠癌的发病风险无明显关联。但在≥58岁人群和女性人群中,UCA1 rs 12462414遗传变异可增加结直肠癌的发病风险。3. lncRNA H19 rs217727和MALAT1 rs619586遗传变异与结直肠癌患者术后的总体生存时间相关。4. lncRNA AK022914、AP000525.8和UCA1在结直肠癌组织中的表达上调,C17orf91表达下调,可能与结直肠癌的发生存在关联。5. lncRNA C17orf91表达下调有利于提高结直肠癌患者术后总体生存时间,降低死亡风险。以上研究结论仍需要在大样本人群中进行验证,且需要更多的生物学研究以明确与结直肠癌发生发展相关lncRNA的功能及作用机制。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34
，
本文编号：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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34
，
本文编号：2180980
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