發(fā)布時間：2018-04-04 13:24

  本文選題：結直腸癌　切入點：長鏈非編碼RNA　出處：《浙江大學》2015年博士論文

【摘要】：研究背景 結直腸癌是最常見的消化系統(tǒng)惡性腫瘤之一。2012年全世界新發(fā)結直腸癌病例數(shù)為136萬,因結直腸癌死亡的人數(shù)約69萬人。隨著社會經(jīng)濟的發(fā)展,人們生活方式的改變,我國結直腸癌的發(fā)病率較以前顯著上升。近年來,結直腸癌發(fā)病率的上升趨勢有所放緩,但是由于其發(fā)病隱匿、預后不良,給人民生活造成了較大的負擔。因此,結直腸癌仍然是嚴重威脅我國居民健康的重大問題。 長鏈非編碼RNA是一類轉(zhuǎn)錄本長度介于200nt-100kb之間的不編碼或很少編碼蛋白質(zhì)的RNA分子。研究發(fā)現(xiàn),多種腫瘤中存在lncRNAs的異常表達,而IncRNAs中特定的超保守元件在人類腫瘤細胞中存在著廣泛表達。已有較多的研究者利用lncRNA表達譜芯片,對腫瘤當中的lncRNA表達譜進行了分析,發(fā)現(xiàn)了一些與腫瘤密切相關的lncRNA.但是,目前此類研究仍存在著一些不足之處。首先,現(xiàn)有關于結直腸癌中l(wèi)ncRNA的表達譜分析研究數(shù)量較少。其次當前研究單獨利用lncRNA表達水平來篩選的lncRNA,作用機理的闡述往往無從下手。最后,當前的腫瘤相關長鏈非編碼RNA篩選策略并未考慮到不同基因之間的相互作用。 本研究擬從結直腸癌的lncRNA表達譜入手,利用表達差異分析和共表達網(wǎng)絡分析兩種篩選策略,分別篩選與結直腸癌相關的lncRNA,并在表達水平上對篩選出的結直腸癌相關lncRNA進行驗證,以期望篩選出在結直腸癌中具有生物學功能的lncRNA. 材料與方法 收集2013年5月到2013年12月期間確診為結直腸腺癌96例臨床病例,采集其手術切除的結直腸癌組織和對應癌旁正常組織標本,并從中選擇6對組織進行l(wèi)ncRNA表達譜和mRNA表達譜芯片檢測。在差異表達的lncRNA當中,選取表達差異穩(wěn)定(在6對lncRNA當中表達差異均大于1.5)倍數(shù)最大的10個lncRNA(上調(diào)/下調(diào)各5個)作為差異表達lncRNA。利用lncRNA與mRNA的表達水平信息構建共表達網(wǎng)絡,構建共表達模塊,并通過生物信息學數(shù)據(jù)庫GO和KEGG對構建的共表達模塊進行功能預測,選擇具有一定功能的共表達模塊,然后篩選出模塊中與結直腸癌相關的關鍵lncRNA分子。根據(jù)篩選結果,采用定量RT-PCR手段,在另外的90對結直腸癌組織和癌旁組織當中對篩選出的結直腸癌相關lncRNA分子的表達水平進行檢測。采用配對t檢驗分析這些lncRNA相對表達變化與結直腸癌的發(fā)生、結直腸癌的部位、大小、臨床分期以及分化程度等臨床特征之間的關聯(lián)情況,并通過ROC曲線分析比較各個lncRNA用于判斷組織是否癌變時的準確性。 結果 lncRNA表達譜芯片檢測的30586個lncRNA中,在癌組織當中出現(xiàn)表達上調(diào)的lncRNA有556個,表達下調(diào)的lncRNA有1040個。26109個mRNA當中在結直腸癌組織當中表達上調(diào)的mRNA為889個,表達下調(diào)的977個。在這些差異表達lncRNA當中,表達差異穩(wěn)定且變化倍數(shù)最大的10個lncRNA分別是：上調(diào)CCAT1、UCA1、RP5-881L22.5、NOS2P3、BC005081;下調(diào)AK055386、AC078941.1、 RP4-800J21.3、RP11-628E19.3、RP11-384P7.7. 用lncRNA與mRNA的表達水平信息構建共表達網(wǎng)絡與無尺度網(wǎng)絡的擬合度為0.813。在該網(wǎng)絡當中,通過聚類方法篩選到了9個共表達模塊,其中的4個模塊在功能預測分析的時候表現(xiàn)出基因功能的富集,所富集的基因功能為細胞周期調(diào)節(jié)、細胞運動、染色體組分、收縮性纖維成分、細胞骨架成分等；2個模塊在3條信號傳導通路上發(fā)生富集,分別為卵母細胞減數(shù)分裂相關通路、細胞周期相關通路以及黏著斑信號傳導相關通路。這四個共表達模塊當中分別包含了54、19、14、21個關鍵基因,最后從中篩選出在模塊內(nèi)連接度最大的lncRNA作為模塊的關鍵lncRNA納入后續(xù)的驗證研究。最終確定的關鍵lncRNA為RP11-58A12.3. UBE2Q2P1、SENP3-EIF4A1、AC010226.4-2、CTC-454M9.1. 通過兩種篩選策略篩選到的共15個lncRNA在90對結直腸癌組織和癌旁組織樣本當中被驗證。定量RT-PCR檢測顯示CCAT1、UCA1、RP5-881L22.5在結直腸癌組織當中的表達水平顯著高于配對的癌旁組織(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在結直腸癌組織當中的表達水平顯著低于配對的癌旁組織(p0.05),與表達譜結果一致；CCAT1以及RP5-881L22.5在結腸癌組織當中特異性高表達(p0.05),而在直腸癌組織當中表達水平與配對的癌旁組織之間并無顯著差異。UCA1在直腸癌組織當中特異性高表達(p0.05),而在結腸癌組織當中的表達水平與配對的癌旁組織之間并無顯著差異。CCAT1在早中期結直腸癌組織表達顯著高于癌旁組織(p0.05),而在晚期結直腸癌當中的表達與癌旁組織無顯著差異。RP5-881L22.5在早中期結直腸癌組織表達與癌旁組織無顯著差異,而在晚期結直腸癌當中的表達顯著高于癌旁組織(p0.05)。UCA1和RP5-881L22.5在中高分化腺癌組織當中表達水平顯著高于配對的癌旁組織,而在低分化腺癌當中的表達與癌旁組織無顯著差異。ROC比較分析顯示,在癌組織中下調(diào)的lncRNA用于判斷組織是否癌變時,ROC曲線下面積顯著大于表達上調(diào)的lncRNA (p0.001)。兩種篩選策略篩選到的lncRNA第一主成分的ROC曲線下面積并無顯著差異。 結論 (1)長鏈非編碼RNA CCAT1、UCA1、RP5-881L22.5在結直腸癌組織當中表達顯著上調(diào),AK055386、AC078941.1、RP4-800J21.3、RP11-628E19.3、 RP11-384P7.7、RP11-58A12.3、UBE2Q2P1、SENP3-EIF4A1、AC010226.4-2、 CTC-454M9.1在結直腸癌組織當中表達顯著下調(diào),其表達水平可能與結直腸癌的發(fā)生存在關聯(lián)。 (2)CCAT1在結腸癌和早中期結直腸癌當中特異高表達；UCAl在直腸癌和中高分化腺癌及中晚期結直腸癌組織中高表達；RP5-881L22.5在結腸癌和中高分化腺癌及晚期結直腸癌組織當中高表達。 (3)上調(diào)的lncRNA在診斷結直腸癌時其診斷效能顯著低于下調(diào)的lncRNA;而兩種篩選lncRNA的策略篩選出來的lncRNA總體上診斷結直腸癌的效能沒有顯著差異。
[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 .

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.34

【參考文獻】

相關期刊論文 前1條

1 肖炳q，

本文編號：1710138