本文選題：下咽鱗狀細(xì)胞癌 + lncRNA　； 參考：《山東大學(xué)》2016年博士論文

【摘要】：第一部分：下咽鱗狀細(xì)胞癌患者lncRNA和mRNA表達(dá)譜的基因芯片分析以及生物信息學(xué)研究研究背景：長鏈非編碼RNA (Long non-coding RNA, lncRNA)是一種序列長度超過200個(gè)核苷酸(Nucleotide, nt)的非編碼RNA (non-coding RNA, ncRNA)的總稱。大量的研究表明,lncRNA數(shù)量龐大,調(diào)控方式多樣,它們可以通過與DNA、RNA或者蛋白質(zhì)的相互作用,以RNA的形式在生命活動(dòng)調(diào)控網(wǎng)絡(luò)的多個(gè)層面(表觀遺傳調(diào)控、轉(zhuǎn)錄調(diào)控以及轉(zhuǎn)錄后調(diào)控等)中調(diào)控基因的表達(dá),從而參與X染色體沉默、基因組印記、染色質(zhì)修飾、轉(zhuǎn)錄激活、轉(zhuǎn)錄干擾,以及核內(nèi)運(yùn)輸?shù)榷喾N重要生命活動(dòng)的調(diào)控過程。有研究表明,lncRNA參與了多種腫瘤的發(fā)生發(fā)展、侵襲轉(zhuǎn)移、放化療抵抗等。然而,lncRNA在下咽鱗狀細(xì)胞癌(Hypopharyngeal Squamous Cell Carcinoma, HSCC)發(fā)生、發(fā)展中所起的作用,以及其作用形式和機(jī)制等方面目前仍尚不清楚。實(shí)驗(yàn)方法：我們選取23例初次接受手術(shù)治療的HSCC患者作為研究對(duì)象,于術(shù)中收集HSCC組織樣本和對(duì)應(yīng)的癌旁非腫瘤粘膜組織樣本。其中3對(duì)組織利用Arraystar lncRNA芯片檢測(cè)了HSCC組織和癌旁非腫瘤粘膜組織中l(wèi)ncRNA和mRNA的表達(dá)譜,并分析了其差異表達(dá)；其余20對(duì)組織利用實(shí)時(shí)定量PCR (Quantitative Real-time Polymerase Chain Reaction, qRT-PCR)方法對(duì)芯片結(jié)果進(jìn)行驗(yàn)證；進(jìn)一步利用生物信息學(xué)手段如：京都基因與基因組百科全書(Kyoto Encyclopedia of Genes and Genomes, KEGG)分析、基因本體(Gene Ontology, GO)分析、類增強(qiáng)子功能lncRNA譜(Enhancer lncRNA profiling)、基因間lncRNA譜(Intergenic lncRNA profi ling)、同源盒聚類譜(HOXcluster profiling)等分析方法,分析差異表達(dá)lncRNAs和mRNAs可能的生物學(xué)功能：篩選出一批針對(duì)HSCC有進(jìn)一步研究價(jià)值的候選lncRNAs和mRNAs。實(shí)驗(yàn)結(jié)果：1.與癌旁非腫瘤粘膜組織相比,669條lncRNAs在HSCC組織中表達(dá)顯著增高,630條lncRNAs在HSCC組織中表達(dá)顯著降低；684條mRNAs在HSCC組織中表達(dá)顯著增高,748條mRNAs在HSCC組織中表達(dá)顯著降低。2.隨機(jī)選取2條差異表達(dá)的lncRNAs (ab209630, ab019562)和2條差異表達(dá)的mRNAs (SPP1,TJP2),應(yīng)用qRT-PCR方法在20例HSCC患者的HSCC組織和癌旁非腫瘤粘膜組織中對(duì)基因芯片結(jié)果進(jìn)行了驗(yàn)證。qRT-PCR驗(yàn)證表明,基因芯片結(jié)果準(zhǔn)確、可靠。3.差異表達(dá)的lncRNAs和mRNAs分布在包括X和Y染色體的所有染色體中。KEGG路徑分析表明,差異表達(dá)mRNAs的生物學(xué)功能與48條細(xì)胞通路相關(guān),而這些細(xì)胞通路可能與HSCC的發(fā)生發(fā)展相關(guān)。GO分析表明,593條與生物進(jìn)程相關(guān)的mRNAs,50條與細(xì)胞組成相關(guān)的uRNAs,以及46條與分子功能相關(guān)的mRNAs在HSCC中高表達(dá)；280條與生物進(jìn)程相關(guān)的mRNAs,58條與細(xì)胞組成相關(guān)的mRNAs,以及71條與分子功能相關(guān)的mRNAs在HSCC中低表達(dá)。4.此外,我們發(fā)現(xiàn)有8條增強(qiáng)型lncRNAs (Enhancer-like lncRNAs)和21條基因間lncRNAs (Intergenic lncRNAs)與其相鄰的mRNA間有協(xié)同調(diào)節(jié)轉(zhuǎn)錄關(guān)系。實(shí)驗(yàn)結(jié)論：這些發(fā)現(xiàn)將有助于我們進(jìn)一步闡明HSCC的發(fā)生機(jī)制,并為HSCC新的治療靶點(diǎn)的提出和腫瘤生物標(biāo)志物的發(fā)現(xiàn)提供參考。第二部分：PHLPP1和PHLPP2在下咽鱗狀細(xì)胞癌患者中的表達(dá)水平及其臨床意義研究背景：PHLPP (Pleckstrin Homology [PH] Domain Leucine-rich Repeat [LRR] Protein Phosphatase)家族是新發(fā)現(xiàn)的一類絲氨酸/蘇氨酸蛋白磷酸酶(Ser/Thr Protein Phosphatases),該家族包含了PHLPP 1和PHLPP2兩個(gè)成員。PHLPPs在多種腫瘤的形成和轉(zhuǎn)移中發(fā)揮著重要的抑制作用,然而,PHLPPs在下咽鱗狀細(xì)胞癌(Hypopharyngeal Squamous Cell Carcinoma, HSCC)患者中的表達(dá)水平及其臨床意義目前未有研究報(bào)道。實(shí)驗(yàn)方法：選取138例初次接受手術(shù)治療的HSCC患者作為研究對(duì)象,術(shù)中收集138例HSCC組織樣本和64例癌旁非腫瘤粘膜組織樣本。分別應(yīng)用實(shí)時(shí)定量PCR (Quantitative Real-time Polymerase Chain Reaction, qRT-PCR)和免疫組織化學(xué)染色(Immunohistochemistry Staining, IHC)檢測(cè)HSCC組織和癌旁組織中PHLPP1和PHLPP2的mRNA和蛋白相對(duì)表達(dá)水平。HSCC組織與配對(duì)癌旁組織PHLPP 1和PHLPP2的mRNA相對(duì)表達(dá)量分析采用配對(duì)Wilcoxon秩和檢驗(yàn)；采用獨(dú)立樣本Mann-Whitney雙尾檢驗(yàn)比較兩組樣本間的蛋白表達(dá)情況；PHLPP 1與PHLPP2兩者的相關(guān)性采用Spearman檢驗(yàn)；采用Pearson卡方檢驗(yàn)或Fisher精確檢驗(yàn)分析PHLPP1和PHLPP2的表達(dá)情況與患者臨床病理特征之間的關(guān)系；生存曲線的繪制采用Kaplan-Meier分析法,應(yīng)用log-rank檢驗(yàn)分析曲線間的差異；應(yīng)用Cox單因素和多因素回歸模型分析影響HSCC患者生存時(shí)間的危險(xiǎn)因素。實(shí)驗(yàn)結(jié)果：1.在配對(duì)的64例HSCC組織和癌旁組織中,PHLPP 1-mRNA在HSCC組織中相對(duì)表達(dá)量為0.0037±0.0024,顯著低于配對(duì)的癌旁組織的相對(duì)表達(dá)量(0.0060±0.0035),差異有統(tǒng)計(jì)學(xué)意義(P0.0001)。PHLPP2-mRNA在HSCC組織中相對(duì)表達(dá)量為0.0030±0.0023,而在配對(duì)的癌旁組織中的相對(duì)表達(dá)量為0.0054±0.0036,兩者差異有統(tǒng)計(jì)學(xué)意義(P0.0001)。Spearman相關(guān)分析結(jié)果表明：PHLPP 1-mRNA和PHLPP2-mRNA無論在HSCC組織中(相關(guān)系數(shù)r=0.678,P0.001)還是在癌旁組織中(相關(guān)系數(shù)r=0.460,P0.001)均存在著正相關(guān)。2. PHLPP 1和PHLPP2蛋白在腫瘤細(xì)胞和癌旁非腫瘤組織上皮細(xì)胞中均主要表達(dá)于細(xì)胞膜和細(xì)胞質(zhì)。PHLPP1在87.5%(56/64)癌旁組織中,PHLPP2在85.9%(55/64)癌旁組織中呈現(xiàn)出高表達(dá)。與之形成鮮明對(duì)比的是,PHLPP1在83.3%(115/138)的HSCC組織中,PHLPP2在82.6%(114/138)的HSCC組織中呈現(xiàn)出低表達(dá)。兩種PHLPP亞型在HSCC組織中表達(dá)均明顯降低(P0.0001)。兩種PHLPP亞型的染色強(qiáng)度在HSCC組織中呈明顯的正相關(guān)(相關(guān)系數(shù)r=0.873,P0.001),說明在同一患者的HSCC組織中,兩種PHLPP亞型表達(dá)常同時(shí)降低。3. Pearson卡方檢驗(yàn)或Fisher精確檢驗(yàn)分析PHLPP1和PHLPP2的蛋白表達(dá)情況與患者臨床病理特征之間的關(guān)系表明：兩種PHLPP亞型蛋白表達(dá)水平與HSCC臨床分期(P0.05)、病理分化(P0.05)以及頸部淋巴結(jié)轉(zhuǎn)移(P0.05)明顯相關(guān)。PHLPP1與腫瘤T分期(P=0.047)明顯相關(guān)。4. Kaplan-Meier分析表明,PHLPP1和PHLPP2低表達(dá)患者的總體生存(overall survival, OS)率低于PHLPP1和PHLPP2高表達(dá)患者(兩者的P值分別為0.004和0.008)。低表達(dá)PHLPP 1患者的三年生存率為46.2%,高表達(dá)PHLPP1患者的三年生存率為82.2%；低表達(dá)PHLPP2患者的三年生存率為46.7%,高表達(dá)PHLPP1患者的三年生存率為78.8%。單因素Cox回歸分析結(jié)果表明頸部淋巴結(jié)轉(zhuǎn)移、T分期、病理分化、臨床分期以及PHLPPK PHLPP2蛋白表達(dá)均和患者的預(yù)后有關(guān)。進(jìn)一步的多因素Cox回歸分析表明,頸部淋巴結(jié)轉(zhuǎn)移(P=0.042,危險(xiǎn)比[Hazard Ratio, HR]=1.617,95%可信區(qū)間[Confidence Interval, CI]:1.018-2.567), T分期(P=0.035, HR=1.665,95%CI:1.035-2.679)和PHLPP 1蛋白表達(dá)(P=0.018,HR=0.402,95%CI:0.189-0.854)是HSCC患者預(yù)后的獨(dú)立危險(xiǎn)因素,而病理分化、臨床分期和PHLPP2均不能作為獨(dú)立預(yù)后因素。實(shí)驗(yàn)結(jié)論：PHLPPs在HSCC中的表達(dá)較癌旁非腫瘤粘膜明顯降低,其異常表達(dá)與腫瘤分化、臨床分期和頸部淋巴結(jié)轉(zhuǎn)移明顯相關(guān)。PHLPPs的表達(dá)異常與HSCC患者的總體生存率顯著降低相關(guān)。此外,PHLPP 1是HSCC患者術(shù)后生存率的獨(dú)立預(yù)后因素。這些結(jié)果表明,PHLPPs可能為未來HSCC的治療提供新選擇。
[Abstract]:In the first part , the gene chip analysis of lncRNA and mRNA expression profiles in patients with hypopharyngeal squamous cell carcinoma and the research background of bioinformatics are as follows : Long - chain non - coding RNA ( lncRNA ) is a kind of non - coding RNA ( ncRNA ) which has more than 200 nucleotides in length .
The remaining 20 pairs were verified by real - time quantitative PCR ( qRT - PCR ) method .
The possible biological functions of lncRNAs and mRNAs were analyzed by means of bioinformatics methods such as Kyoto Encyclopedia of Genes and Genomes , KINS , Gene Ontology , GO , Intergenic lncRNA profiling , Intergenic lncRNA profi ling and HOXcluster profiling .
The expression of mRNA in HSCC was significantly lower than that in HSCC .
In addition , we found that there were 8 enhanced lncRNAs ( Enhanced cer - like lncRNAs ) and 21 intergenic lncRNAs ( Intergenic lncRNAs ) .
The expression of protein was compared between the two groups by Mann - Whitney test .
The correlation between PHl1 and PHLpp2 was determined by the man - man test .
Pearson chi - square test or Fisher ' s exact test was used to analyze the relationship between PHLpp1 and PHLpp2 expression and the clinical pathological characteristics of the patients .
Kaplan - Meier analysis was used to plot survival curves and log - rank test was applied to analyze the difference between curves .
Results : 1 . The relative expression of PHPLC 1 - mRNA in HSCC was 0.0037 鹵 0.0024 , which was significantly lower than that of paired adjacent tissues ( 0.0054 鹵 0.0023 ) . In the HSCC tissue of 85.3 % ( 115 / 138 ) , PHLpp1 and PHLpp2 were expressed in 85.9 % ( 114 / 138 ) of HSCC tissues . The expression of PHlpp2 was significantly lower in HSCC tissue ( r = 0.873 , P0.001 ) . The expression of PHlpp1 and PHLpp2 in HSCC was significantly lower than that in HSCC ( r = 0.873 , P0.001 ) . Pearson chi - square test or Fisher ' s exact test showed that the relationship between the expression of PHL pp1 and PHLpp2 and the clinical pathological characteristics of the patients showed that the expression level of PHlpp1 and PHLpp2 was significantly correlated with the clinical stage of HSCC ( P0.05 ) , pathological differentiation ( P0.05 ) , and cervical lymph node metastasis ( P0.05 ) . The Kaplan - Meier analysis showed that the overall survival ( OS ) rate of patients with low expression of PHLpp1 and PHLpp2 was lower than that in PHLpp1 and PHLpp2 high expression patients ( P = 0.004 and 0.008 , respectively ) . The three - year survival rate was 46.2 % and 82.2 % in PHLpp1 patients with low expression .
The three - year survival rate of patients with low expression of PHLpp2 was 46.7 % , and the three - year survival rate was 78.8 % in patients with high expression of PHLpp1 . The results of Cox regression analysis showed that the expression of PHPLC in HSCC was significantly associated with the prognosis of patients with HSCC .
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R739.63

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