基于组织表达谱的结直肠癌相关长链非编码RNA的筛选和验证

发布时间：2018-04-04 13:24

  本文选题：结直肠癌　切入点：长链非编码RNA　出处：《浙江大学》2015年博士论文

【摘要】：研究背景 结直肠癌是最常见的消化系统恶性肿瘤之一。2012年全世界新发结直肠癌病例数为136万,因结直肠癌死亡的人数约69万人。随着社会经济的发展,人们生活方式的改变,我国结直肠癌的发病率较以前显著上升。近年来,结直肠癌发病率的上升趋势有所放缓,但是由于其发病隐匿、预后不良,给人民生活造成了较大的负担。因此,结直肠癌仍然是严重威胁我国居民健康的重大问题。 长链非编码RNA是一类转录本长度介于200nt-100kb之间的不编码或很少编码蛋白质的RNA分子。研究发现,多种肿瘤中存在lncRNAs的异常表达,而IncRNAs中特定的超保守元件在人类肿瘤细胞中存在着广泛表达。已有较多的研究者利用lncRNA表达谱芯片,对肿瘤当中的lncRNA表达谱进行了分析,发现了一些与肿瘤密切相关的lncRNA.但是,目前此类研究仍存在着一些不足之处。首先,现有关于结直肠癌中lncRNA的表达谱分析研究数量较少。其次当前研究单独利用lncRNA表达水平来筛选的lncRNA,作用机理的阐述往往无从下手。最后,当前的肿瘤相关长链非编码RNA筛选策略并未考虑到不同基因之间的相互作用。 本研究拟从结直肠癌的lncRNA表达谱入手,利用表达差异分析和共表达网络分析两种筛选策略,分别筛选与结直肠癌相关的lncRNA,并在表达水平上对筛选出的结直肠癌相关lncRNA进行验证,以期望筛选出在结直肠癌中具有生物学功能的lncRNA. 材料与方法 收集2013年5月到2013年12月期间确诊为结直肠腺癌96例临床病例,采集其手术切除的结直肠癌组织和对应癌旁正常组织标本,并从中选择6对组织进行lncRNA表达谱和mRNA表达谱芯片检测。在差异表达的lncRNA当中,选取表达差异稳定(在6对lncRNA当中表达差异均大于1.5)倍数最大的10个lncRNA(上调/下调各5个)作为差异表达lncRNA。利用lncRNA与mRNA的表达水平信息构建共表达网络,构建共表达模块,并通过生物信息学数据库GO和KEGG对构建的共表达模块进行功能预测,选择具有一定功能的共表达模块,然后筛选出模块中与结直肠癌相关的关键lncRNA分子。根据筛选结果,采用定量RT-PCR手段,在另外的90对结直肠癌组织和癌旁组织当中对筛选出的结直肠癌相关lncRNA分子的表达水平进行检测。采用配对t检验分析这些lncRNA相对表达变化与结直肠癌的发生、结直肠癌的部位、大小、临床分期以及分化程度等临床特征之间的关联情况,并通过ROC曲线分析比较各个lncRNA用于判断组织是否癌变时的准确性。 结果 lncRNA表达谱芯片检测的30586个lncRNA中,在癌组织当中出现表达上调的lncRNA有556个,表达下调的lncRNA有1040个。26109个mRNA当中在结直肠癌组织当中表达上调的mRNA为889个,表达下调的977个。在这些差异表达lncRNA当中,表达差异稳定且变化倍数最大的10个lncRNA分别是：上调CCAT1、UCA1、RP5-881L22.5、NOS2P3、BC005081;下调AK055386、AC078941.1、 RP4-800J21.3、RP11-628E19.3、RP11-384P7.7. 用lncRNA与mRNA的表达水平信息构建共表达网络与无尺度网络的拟合度为0.813。在该网络当中,通过聚类方法筛选到了9个共表达模块,其中的4个模块在功能预测分析的时候表现出基因功能的富集,所富集的基因功能为细胞周期调节、细胞运动、染色体组分、收缩性纤维成分、细胞骨架成分等；2个模块在3条信号传导通路上发生富集,分别为卵母细胞减数分裂相关通路、细胞周期相关通路以及黏着斑信号传导相关通路。这四个共表达模块当中分别包含了54、19、14、21个关键基因,最后从中筛选出在模块内连接度最大的lncRNA作为模块的关键lncRNA纳入后续的验证研究。最终确定的关键lncRNA为RP11-58A12.3. UBE2Q2P1、SENP3-EIF4A1、AC010226.4-2、CTC-454M9.1. 通过两种筛选策略筛选到的共15个lncRNA在90对结直肠癌组织和癌旁组织样本当中被验证。定量RT-PCR检测显示CCAT1、UCA1、RP5-881L22.5在结直肠癌组织当中的表达水平显著高于配对的癌旁组织(p0.05), AK055386、 AC078941.1、RP4-800J21.3、RP11-628E19.3、RP11-384P7.7、RP11-58A12.3、 UBE2Q2P1、SENP3-EIF4A1、AC010226.4-2、CTC-454M9.1在结直肠癌组织当中的表达水平显著低于配对的癌旁组织(p0.05),与表达谱结果一致；CCAT1以及RP5-881L22.5在结肠癌组织当中特异性高表达(p0.05),而在直肠癌组织当中表达水平与配对的癌旁组织之间并无显著差异。UCA1在直肠癌组织当中特异性高表达(p0.05),而在结肠癌组织当中的表达水平与配对的癌旁组织之间并无显著差异。CCAT1在早中期结直肠癌组织表达显著高于癌旁组织(p0.05),而在晚期结直肠癌当中的表达与癌旁组织无显著差异。RP5-881L22.5在早中期结直肠癌组织表达与癌旁组织无显著差异,而在晚期结直肠癌当中的表达显著高于癌旁组织(p0.05)。UCA1和RP5-881L22.5在中高分化腺癌组织当中表达水平显著高于配对的癌旁组织,而在低分化腺癌当中的表达与癌旁组织无显著差异。ROC比较分析显示,在癌组织中下调的lncRNA用于判断组织是否癌变时,ROC曲线下面积显著大于表达上调的lncRNA (p0.001)。两种筛选策略筛选到的lncRNA第一主成分的ROC曲线下面积并无显著差异。 结论 (1)长链非编码RNA CCAT1、UCA1、RP5-881L22.5在结直肠癌组织当中表达显著上调,AK055386、AC078941.1、RP4-800J21.3、RP11-628E19.3、 RP11-384P7.7、RP11-58A12.3、UBE2Q2P1、SENP3-EIF4A1、AC010226.4-2、 CTC-454M9.1在结直肠癌组织当中表达显著下调,其表达水平可能与结直肠癌的发生存在关联。 (2)CCAT1在结肠癌和早中期结直肠癌当中特异高表达；UCAl在直肠癌和中高分化腺癌及中晚期结直肠癌组织中高表达；RP5-881L22.5在结肠癌和中高分化腺癌及晚期结直肠癌组织当中高表达。 (3)上调的lncRNA在诊断结直肠癌时其诊断效能显著低于下调的lncRNA;而两种筛选lncRNA的策略筛选出来的lncRNA总体上诊断结直肠癌的效能没有显著差异。
[Abstract]:Background of the study

Colorectal cancer is one of the most common malignant tumors in the digestive system . In 2012 , the number of new colorectal cancer in the world was 136 million . With the development of social economy , the incidence of colorectal cancer increased significantly .

Long - chain non - coding RNA is a kind of RNA molecule whose length is between 200 nt and 100 kb . It has been found that there is an abnormal expression of lncRNA in various tumors , and the expression of lncRNA in human tumor cells is less .

In order to screen the lncRNA associated with colorectal cancer , lncRNA associated with colorectal cancer was screened from the expression profile of lncRNA in colorectal cancer , and lncRNA associated with colorectal cancer was verified at the level of expression , in order to screen the lncRNA with biological function in colorectal cancer .

Materials and Methods

The expression level of lncRNA was detected by means of quantitative RT - PCR . The expression level of lncRNA and mRNA was used to determine the expression level of lncRNA in colorectal cancer .

Results

Among the 30586 lncRNAs detected by lncRNA expression profiling chip , there were 556 up - regulated lncRNA in the carcinoma tissues , and the down - regulated expression of lncRNA was raised up - regulated in the tissues of colorectal cancer . The 10 lncRNAs were up - regulated CCAT1 , UCA1 , RP5 - 881L22.5 , NOS2P3 , BC005081 ; downregulated AK055386 , AC078941.1 , RP4 - 800J21.3 , RP11 - 628E19.3 , RP11 - 3847.7 .

The expression level information of lncRNA and mRNA was used to construct the fitting degree of the co - expression network and the non - scale network . In this network , nine co - expression modules were screened by cluster method . Four of them showed the enrichment of gene function during functional prediction analysis , and the enriched gene function was cell cycle regulation , cell movement , chromosome component , contractile fiber component , cytoskeletal component , etc .
Two modules were enriched in three signal transduction pathways , which were associated with meiosis - related pathways , cell cycle - related pathways and adhesion - plaque signaling pathways , respectively . Among these four co - expression modules , 54 , 19 , 14 , 21 key genes were included . Finally , we screened lncRNA as the key lncRNA of the module to be integrated into subsequent validation studies . The key lncRNA was RP11 - 58A12 . 3 . UBE2Q2P1 , SENP3 - EIF4A1 , AC010226.4 -2 , CTC - 4549.1 .

The expression levels of CCAT1 , UCA1 , RP5 - 881L22.5 in colorectal cancer tissues were significantly higher than those of paired adjacent tissues ( p0.05 ) , AK055386 , AC078941.1 , RP4 - 800J21.3 , RP11 - 628E19.3 , RP11 - 3847.7 , RP11 - 58A12 . 3 , UBE2Q2P1 , SENP3 - EIF4A1 , AC010226.4 -2 , CTC - 4549.1 .
There was no significant difference in the expression of CCAT1 and RP5 - 881L22.5 in colorectal cancer tissues .

Conclusion

( 1 ) The expression of long - chain non - coding RNA CCAT1 , UCA1 , RP5 - 881L22.5 in colorectal cancer tissue was significantly up - regulated , AK055386 , AC078941.1 , RP4 - 800J21.3 , RP11 - 628E19.3 , RP11 - 3847.7 , RP11 - 58A12 . 3 , UBE2Q2P1 , SENP3 - EIF4A1 , AC010226.4 -2 , CTC - 4549.1 9.1 were downregulated in colorectal cancer tissue , and their expression level could be associated with the occurrence of colorectal cancer .

( 2 ) CCAT1 was highly expressed in colon cancer and early stage colorectal cancer ;
High expression of UCAl in rectal cancer and middle and advanced colorectal cancer tissues
RP5 - 881L22.5 was highly expressed in colon cancer and moderately high differentiated adenocarcinoma and advanced colorectal cancer tissue .

( 3 ) Up - regulated lncRNA was significantly lower in the diagnosis of colorectal cancer than down - regulated lncRNA , while the efficacy of the two methods for screening lncRNA was not significantly different in the diagnosis of colorectal cancer .

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

【参考文献】

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