下咽鳞状细胞癌患者IncRNA和mRNA表达谱的基因芯片分析以及PHLPPs的表达水平及其临床意义

发布时间：2018-05-20 23:00

本文选题：下咽鳞状细胞癌 + lncRNA　；参考：《山东大学》2016年博士论文

【摘要】：第一部分：下咽鳞状细胞癌患者lncRNA和mRNA表达谱的基因芯片分析以及生物信息学研究研究背景：长链非编码RNA (Long non-coding RNA, lncRNA)是一种序列长度超过200个核苷酸(Nucleotide, nt)的非编码RNA (non-coding RNA, ncRNA)的总称。大量的研究表明,lncRNA数量庞大,调控方式多样,它们可以通过与DNA、RNA或者蛋白质的相互作用,以RNA的形式在生命活动调控网络的多个层面(表观遗传调控、转录调控以及转录后调控等)中调控基因的表达,从而参与X染色体沉默、基因组印记、染色质修饰、转录激活、转录干扰,以及核内运输等多种重要生命活动的调控过程。有研究表明,lncRNA参与了多种肿瘤的发生发展、侵袭转移、放化疗抵抗等。然而,lncRNA在下咽鳞状细胞癌(Hypopharyngeal Squamous Cell Carcinoma, HSCC)发生、发展中所起的作用,以及其作用形式和机制等方面目前仍尚不清楚。实验方法：我们选取23例初次接受手术治疗的HSCC患者作为研究对象,于术中收集HSCC组织样本和对应的癌旁非肿瘤粘膜组织样本。其中3对组织利用Arraystar lncRNA芯片检测了HSCC组织和癌旁非肿瘤粘膜组织中lncRNA和mRNA的表达谱,并分析了其差异表达；其余20对组织利用实时定量PCR (Quantitative Real-time Polymerase Chain Reaction, qRT-PCR)方法对芯片结果进行验证；进一步利用生物信息学手段如：京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)分析、基因本体(Gene Ontology, GO)分析、类增强子功能lncRNA谱(Enhancer lncRNA profiling)、基因间lncRNA谱(Intergenic lncRNA profi ling)、同源盒聚类谱(HOXcluster profiling)等分析方法,分析差异表达lncRNAs和mRNAs可能的生物学功能：筛选出一批针对HSCC有进一步研究价值的候选lncRNAs和mRNAs。实验结果：1.与癌旁非肿瘤粘膜组织相比,669条lncRNAs在HSCC组织中表达显著增高,630条lncRNAs在HSCC组织中表达显著降低；684条mRNAs在HSCC组织中表达显著增高,748条mRNAs在HSCC组织中表达显著降低。2.随机选取2条差异表达的lncRNAs (ab209630, ab019562)和2条差异表达的mRNAs (SPP1,TJP2),应用qRT-PCR方法在20例HSCC患者的HSCC组织和癌旁非肿瘤粘膜组织中对基因芯片结果进行了验证。qRT-PCR验证表明,基因芯片结果准确、可靠。3.差异表达的lncRNAs和mRNAs分布在包括X和Y染色体的所有染色体中。KEGG路径分析表明,差异表达mRNAs的生物学功能与48条细胞通路相关,而这些细胞通路可能与HSCC的发生发展相关。GO分析表明,593条与生物进程相关的mRNAs,50条与细胞组成相关的uRNAs,以及46条与分子功能相关的mRNAs在HSCC中高表达；280条与生物进程相关的mRNAs,58条与细胞组成相关的mRNAs,以及71条与分子功能相关的mRNAs在HSCC中低表达。4.此外,我们发现有8条增强型lncRNAs (Enhancer-like lncRNAs)和21条基因间lncRNAs (Intergenic lncRNAs)与其相邻的mRNA间有协同调节转录关系。实验结论：这些发现将有助于我们进一步阐明HSCC的发生机制,并为HSCC新的治疗靶点的提出和肿瘤生物标志物的发现提供参考。第二部分：PHLPP1和PHLPP2在下咽鳞状细胞癌患者中的表达水平及其临床意义研究背景：PHLPP (Pleckstrin Homology [PH] Domain Leucine-rich Repeat [LRR] Protein Phosphatase)家族是新发现的一类丝氨酸/苏氨酸蛋白磷酸酶(Ser/Thr Protein Phosphatases),该家族包含了PHLPP 1和PHLPP2两个成员。PHLPPs在多种肿瘤的形成和转移中发挥着重要的抑制作用,然而,PHLPPs在下咽鳞状细胞癌(Hypopharyngeal Squamous Cell Carcinoma, HSCC)患者中的表达水平及其临床意义目前未有研究报道。实验方法：选取138例初次接受手术治疗的HSCC患者作为研究对象,术中收集138例HSCC组织样本和64例癌旁非肿瘤粘膜组织样本。分别应用实时定量PCR (Quantitative Real-time Polymerase Chain Reaction, qRT-PCR)和免疫组织化学染色(Immunohistochemistry Staining, IHC)检测HSCC组织和癌旁组织中PHLPP1和PHLPP2的mRNA和蛋白相对表达水平。HSCC组织与配对癌旁组织PHLPP 1和PHLPP2的mRNA相对表达量分析采用配对Wilcoxon秩和检验；采用独立样本Mann-Whitney双尾检验比较两组样本间的蛋白表达情况；PHLPP 1与PHLPP2两者的相关性采用Spearman检验；采用Pearson卡方检验或Fisher精确检验分析PHLPP1和PHLPP2的表达情况与患者临床病理特征之间的关系；生存曲线的绘制采用Kaplan-Meier分析法,应用log-rank检验分析曲线间的差异；应用Cox单因素和多因素回归模型分析影响HSCC患者生存时间的危险因素。实验结果：1.在配对的64例HSCC组织和癌旁组织中,PHLPP 1-mRNA在HSCC组织中相对表达量为0.0037±0.0024,显著低于配对的癌旁组织的相对表达量(0.0060±0.0035),差异有统计学意义(P0.0001)。PHLPP2-mRNA在HSCC组织中相对表达量为0.0030±0.0023,而在配对的癌旁组织中的相对表达量为0.0054±0.0036,两者差异有统计学意义(P0.0001)。Spearman相关分析结果表明：PHLPP 1-mRNA和PHLPP2-mRNA无论在HSCC组织中(相关系数r=0.678,P0.001)还是在癌旁组织中(相关系数r=0.460,P0.001)均存在着正相关。2. PHLPP 1和PHLPP2蛋白在肿瘤细胞和癌旁非肿瘤组织上皮细胞中均主要表达于细胞膜和细胞质。PHLPP1在87.5%(56/64)癌旁组织中,PHLPP2在85.9%(55/64)癌旁组织中呈现出高表达。与之形成鲜明对比的是,PHLPP1在83.3%(115/138)的HSCC组织中,PHLPP2在82.6%(114/138)的HSCC组织中呈现出低表达。两种PHLPP亚型在HSCC组织中表达均明显降低(P0.0001)。两种PHLPP亚型的染色强度在HSCC组织中呈明显的正相关(相关系数r=0.873,P0.001),说明在同一患者的HSCC组织中,两种PHLPP亚型表达常同时降低。3. Pearson卡方检验或Fisher精确检验分析PHLPP1和PHLPP2的蛋白表达情况与患者临床病理特征之间的关系表明：两种PHLPP亚型蛋白表达水平与HSCC临床分期(P0.05)、病理分化(P0.05)以及颈部淋巴结转移(P0.05)明显相关。PHLPP1与肿瘤T分期(P=0.047)明显相关。4. Kaplan-Meier分析表明,PHLPP1和PHLPP2低表达患者的总体生存(overall survival, OS)率低于PHLPP1和PHLPP2高表达患者(两者的P值分别为0.004和0.008)。低表达PHLPP 1患者的三年生存率为46.2%,高表达PHLPP1患者的三年生存率为82.2%；低表达PHLPP2患者的三年生存率为46.7%,高表达PHLPP1患者的三年生存率为78.8%。单因素Cox回归分析结果表明颈部淋巴结转移、T分期、病理分化、临床分期以及PHLPPK PHLPP2蛋白表达均和患者的预后有关。进一步的多因素Cox回归分析表明,颈部淋巴结转移(P=0.042,危险比[Hazard Ratio, HR]=1.617,95%可信区间[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蛋白表达(P=0.018,HR=0.402,95%CI:0.189-0.854)是HSCC患者预后的独立危险因素,而病理分化、临床分期和PHLPP2均不能作为独立预后因素。实验结论：PHLPPs在HSCC中的表达较癌旁非肿瘤粘膜明显降低,其异常表达与肿瘤分化、临床分期和颈部淋巴结转移明显相关。PHLPPs的表达异常与HSCC患者的总体生存率显著降低相关。此外,PHLPP 1是HSCC患者术后生存率的独立预后因素。这些结果表明,PHLPPs可能为未来HSCC的治疗提供新选择。
[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 卤 0.0024 , which was significantly lower than that of paired adjacent tissues ( 0.0054 卤 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 .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R739.63

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