【摘要】：研究背景：結(jié)直腸癌,又稱大腸癌,是世界上第三大常見的惡性腫瘤。在我國,結(jié)直腸癌的發(fā)病率和死亡率一直呈上升趨勢。直腸癌是大腸癌中最常見的一類腫瘤,約占60%左右。惡性腫瘤的一個最主要生物學特征就是轉(zhuǎn)移,就是癌細胞從原發(fā)病灶遷移到其它部位形成轉(zhuǎn)移灶,并維持復發(fā)腫瘤的生長。腫瘤一旦發(fā)生轉(zhuǎn)移,其預后將極差。大多數(shù)癌癥患者并非死于原發(fā)性癌而是死于轉(zhuǎn)移性癌。因此,如何預防直腸癌轉(zhuǎn)移是直腸癌治療成敗的關(guān)鍵。盡管外科手術(shù)一直以來都是治療直腸癌的主要手段,但術(shù)后復發(fā)率高。目前,術(shù)前放療已經(jīng)成為Ⅱ/Ⅲ期直腸癌的標準療法。放射治療可以最大限度地將放射線的劑量集中照射到病灶內(nèi),殺滅腫瘤細胞,同時使周圍的正常組織和關(guān)鍵器官免受或盡可能少受不必要的照射。術(shù)前放療可以使腫瘤降期、降體積,甚至達到病理學上的完全消失,從而提高手術(shù)的局部根治率,還可以減少術(shù)中腫瘤種植機會,減少術(shù)后腫瘤的復發(fā)率,進而提高患者的長期生存率。但是術(shù)前放療使直腸癌細胞發(fā)生了哪些分子變化尚不清楚�；蛐酒夹g(shù)是一種高通量、快速、全基因組分析技術(shù),是研究基因功能強有力的工具之一,現(xiàn)在已普遍應用于醫(yī)學研究的各個領(lǐng)域�；虮磉_譜是一種生物體或一種細胞對環(huán)境、遺傳或者生化信號發(fā)生反應時,基因表達被激活或抑制數(shù)倍甚至數(shù)千倍,形成的特征性的基因表達圖譜。表達譜基因芯片可用來檢測基因的表達水平,通過比較不同條件下基因表達的差異情況,可分析疾病形成的基因水平原理、研究基因功能以及與疾病相關(guān)的通路,具有準確和簡便兩大優(yōu)點�；蛐酒臄�(shù)據(jù)分析通常都需要一定的流程處理,也就是生物信息學分析。生物信息學是一門整合了統(tǒng)計學、信息學和計算機學等多種技術(shù)的交叉學科。該技術(shù)利用現(xiàn)有的分析工具和公共數(shù)據(jù)庫,先對生物芯片的海量數(shù)據(jù)進行篩選,再采用序列比對、統(tǒng)計分析、生物聚類、功能或通路分析、可視化作圖等方式,挖掘關(guān)鍵生物分子及其潛在機制,從而在分子水平上對疾病進行分析,豐富人們對疾病發(fā)生、治療和預后等方面的認識。隨著各種模式生物基因組測序工作的完成,生物信息學已經(jīng)進入了功能基因組學時代。功能基因組學利用結(jié)構(gòu)基因組所提供的信息和產(chǎn)物,發(fā)展和應用新的實驗手段,通過在基因組或系統(tǒng)水平上全面分析基因的功能,使得生物學研究從對單一基因或蛋白質(zhì)的研究轉(zhuǎn)向多個基因或蛋白質(zhì)同時進行系統(tǒng)的研究。目前,利用基因表達譜芯片來分析術(shù)前放療對直腸癌分子學方面的影響還很少。研究目的和意義：(1)通過放療前后直腸癌基因表達譜數(shù)據(jù)的分析,篩選放療后直腸癌中的差異表達基因(DEG),并分析它們參與的功能和通路以及相互作用關(guān)系,預測調(diào)控這些DEG的microRNA和轉(zhuǎn)錄因子(TF),構(gòu)建基因調(diào)控網(wǎng)絡(luò),找出放療后直腸癌中發(fā)生顯著變化的基因和關(guān)鍵功能通路,從分子層面揭示術(shù)前放療治療直腸癌的分子機制。(2)驗證上述預測結(jié)果的準確性,并檢測在相關(guān)基因沉默前和沉默后,直腸癌細胞增殖能力和轉(zhuǎn)移能力的變化,以及不同X射線放射劑量對直腸癌細胞增殖能力和轉(zhuǎn)移能力的影響,為臨床放射治療直腸癌提供實驗依據(jù)。研究方法：(1)從GEO公共數(shù)據(jù)庫里下載直腸癌放療前后的mRNA表達譜數(shù)據(jù),利用Bioconductor中的limma軟件包對該數(shù)據(jù)集中所有樣本進行差異表達分析,篩選出放療后直腸癌細胞中的DEG(差異倍數(shù)大于1,并且p值小于0.05),并利用DAVID在線軟件對這些DEG進行GO (Gene Ontology)富集分析和KEGG (Kyoto Encyclopedia of Genes and Genomes)通路富集分析(p值小于0.05)；利用STRING在線數(shù)據(jù)庫分析DEG對應的蛋白質(zhì)相互作用關(guān)系,并用Cytoscape作圖軟件構(gòu)建蛋白互作網(wǎng)絡(luò)；利用UCSC數(shù)據(jù)庫找出DEG中的TF,確定它們調(diào)控的差異表達基因,然后利用Cytoscape作圖軟件來構(gòu)建它們的調(diào)控網(wǎng)絡(luò)；最后利用多個miRNA數(shù)據(jù)庫找出DEG與miRNA的調(diào)控關(guān)系,利用Cytoscape作圖軟件來構(gòu)建它們的調(diào)控網(wǎng)絡(luò)。(2)選取放療后直腸癌細胞中差異表達倍數(shù)較大,并且與細胞增殖和轉(zhuǎn)移功能相關(guān)的基因作為實驗驗證的對象,選用高轉(zhuǎn)移性、惡性程度高的結(jié)直腸癌細胞系SW620為研究材料,利用MTT法和Transwell法檢測不同X射線放射劑量對SW620細胞的增殖能力和轉(zhuǎn)移能力的影響,用RT-PCR實驗驗證不同劑量的X射線輻照對SW620細胞相關(guān)基因表達的影響。采用siRNA法沉默相關(guān)基因,并用RT-PCR和western blot檢測沉默效果。同樣地,利用MTT法和Transwell法檢測相關(guān)基因沉默后SW620細胞的增殖能力和轉(zhuǎn)移能力。研究結(jié)果：(1)通過基因表達譜分析,共篩選出606個在放療后直腸癌中差異表達的基因(表達差異1倍以上,并且p值小于0.05),其中上調(diào)基因271個,下調(diào)基因335個,其中MMP1的差異表達倍數(shù)最大,且p值最小。(2)GO功能富集分析結(jié)果顯示,表達上調(diào)的差異基因主要顯著富集在鐵轉(zhuǎn)運、對無機物和金屬離子的應答等功能上,如SLC6A3、SLC30A4、RYR2和NEDD4L等。表達下調(diào)的差異基因主要和細胞信號傳遞、細胞增殖以及膠原代謝有關(guān),其中SLC6A4和PDX1等參與了細胞間的信號轉(zhuǎn)導功能,PTGS2和CDH5等基因參與了細胞的增殖調(diào)控,而MMP10、COL1A1、MMP3和MMP1主要和膠原蛋白的代謝過程有關(guān)。(3) KEGG通路富集分析結(jié)果顯示,表達上調(diào)的差異基因主要顯著富集在甾類激素的生物合成、鈣信號通路、雄性激素和雌性激素的新陳代謝、刺激神經(jīng)組織的配體和受體相互作用以及年輕人的成年型糖尿病這5條通路上,其中HSD3B2、UGT2A3、SULT1E1和UGT2B15這4個基因參與了甾類激素的生物合成通路以及雄性激素和雌性激素的新陳代謝通路,CYSLTR2、CHRM1和HTR6這3個基因主要參與了鈣信號和刺激神經(jīng)組織的配體和受體相互作用這兩條通路。表達下調(diào)的差異基因顯著富集在細胞外基質(zhì)與受體的相互作用以及補體和凝血級聯(lián)這兩條通路上,其中COL4A2、COL4A1和COL6A3等7個編碼膠原蛋白的基因參與細胞外基質(zhì)與受體的相互作用這條通路上,SERPINE1、SERPIND1、F7、PLAU和F2R這5個基因主要參與補體和凝血級聯(lián)通路。(4)DEG的蛋白互作網(wǎng)絡(luò)分析結(jié)果顯示,該網(wǎng)絡(luò)共包含241個蛋白的410個相互作用關(guān)系對,連接度大于等于10的節(jié)點共有20個。其中COL1A2和COL1A1的連接度都是18,MMP1的連接度是11。(5)通過UCSC數(shù)據(jù)庫分析,共找出5個是TF的差異表達基因,包含PAX6、 PLAU、FOXL1、NKX2-2和FOSL1。在DEG與TF的調(diào)控網(wǎng)絡(luò)中,共有77個調(diào)控關(guān)系對,其中,PLAU除了調(diào)控MMP1、COL1A1等DEG,還調(diào)控NKX2-2和PAX6這兩個轉(zhuǎn)錄因子。(6)通過7個miRNA數(shù)據(jù)庫的綜合分析,共篩選出177對miRNA和差異基因的調(diào)控關(guān)系,包含145個miRNA和40個差異表達基因,如hsa-miR-29c調(diào)控COL1A1、COL1A2、COL4A1和COL4A2這4個基因,MMP1被hsa-miR-222調(diào)控,MMP3被hsa-miR-204調(diào)控。(7)選取MMP1進行實驗驗證。MMP1沉默前,通過RT-PCR檢測發(fā)現(xiàn),SW620細胞中MMP1的1mRNA水平在0.1 GY、0.5 GY、1 GY、3GY口6 GY這5種輻射劑量下,相對于空白對照組(0 GY)都是降低的,并且輻射劑量在0.5 GY內(nèi),MMP1的表達量隨著輻射劑量的增加大幅度降低。(8)通過MTT實驗和Transwell實驗發(fā)現(xiàn),MMP1沉默前,SW620細胞的增殖能力和轉(zhuǎn)移能力明顯高于沉默后的(p值小于0.5),并且在輻射劑量6 GY內(nèi),SW620細胞的增殖能力和轉(zhuǎn)移能力隨著輻射劑量的增加逐漸下降。結(jié)論：(1)放射治療使直腸癌中一些參與金屬離子應答的基因(如SLC6A3、 SLC30A4、RYR2和NEDD4L)、參與鈣離子信號通路與刺激神經(jīng)組織的配體和受體相互作用通路的基因(如CYSLTR2和CHRM1)、與細胞增殖調(diào)控和補體凝血級聯(lián)有關(guān)的基因(如PLAU、FOSL1和SERPINE1)、參與膠原代謝的基因(如MMP1和MMP3)以及一些參與細胞外基質(zhì)與受體的相互作用通路的基因(如COL1A2、COL1A1和COL4A1等)發(fā)生了明顯的表達量變化,這些基因?qū)Ψ暖煯a(chǎn)生了顯著的應答反應。(2)對差異表達基因具有調(diào)控作用的一些miRNAs(如hsa-miR-29c、 hsa-miR-224、hsa-miR-204和hsa-miR-222),以及一些轉(zhuǎn)錄因子(如PLAU和FOSL1等),可能在直腸癌的放療過程中具有重要的調(diào)控作用。(3)不同劑量的X射線輻射能使SW620細胞中的MMP1下調(diào)表達,并且輻射劑量在0.5 GY內(nèi),MMP1的表達量隨著輻射劑量的增加大幅度降低。(4)MMP1對SW620細胞的增殖和轉(zhuǎn)移具有關(guān)鍵的促進作用。本研究有助于揭示術(shù)前放療治療直腸癌的分子機制,并首次驗證了不同放射劑量對直腸癌細胞中MMP1表達水平的影響,實驗證明了MMP1在直腸癌細胞的增殖和轉(zhuǎn)移過程中發(fā)揮的重要作用。這些結(jié)果為直腸癌的臨床放射治療敏感性檢測提供了很好的理論和實驗依據(jù)。
[Abstract]:Background: Colorectal cancer, also known as colorectal cancer, is the third most common malignant tumor in the world. In China, the incidence and mortality of colorectal cancer has been on the rise. Rectal cancer is the most common type of colorectal cancer, accounting for about 60%. Once metastasis occurs, the prognosis will be extremely poor. Most cancer patients die not from primary cancer but metastatic cancer. Therefore, how to prevent metastasis of rectal cancer is the key to the success or failure of rectal cancer treatment. Currently, preoperative radiotherapy has become the standard therapy for stage II/III rectal cancer. Radiotherapy can maximize the dose of radiation to the focus, kill tumor cells, and protect the surrounding normal tissues and key organs from or from unnecessary radiation. Preoperative radiation therapy can reduce the tumor stage, volume, and even completely disappear pathologically, thus increasing the local radical rate of surgery, reducing the chance of intraoperative tumor implantation, reducing the recurrence rate of postoperative tumor, and thus improving the long-term survival rate of patients. However, what molecular changes have taken place in rectal cancer cells after preoperative radiation therapy? Gene chip technology is a high-throughput, rapid, genome-wide analysis technology, is one of the powerful tools for studying gene function, and is now widely used in various fields of medical research. Gene microarray can be used to detect the level of gene expression. By comparing the differences of gene expression under different conditions, the principle of gene level in disease formation can be analyzed, and the function of gene and the pathway related to disease can be studied. Bioinformatics is an interdisciplinary subject that integrates many technologies such as statistics, informatics and computer science. The technology uses existing analytical tools and public databases to perform mass data on biochips first. Screening, sequence alignment, statistical analysis, biological clustering, functional or pathway analysis, visual mapping and other methods are used to explore key biological molecules and their underlying mechanisms, so as to analyze diseases at the molecular level and enrich people's understanding of disease occurrence, treatment and prognosis. With the completion of bioinformatics, functional genomics has entered the era of functional genomics. Using the information and products provided by structural genomics, functional genomics has developed and applied new experimental methods to comprehensively analyze the functions of genes at the genome or system level, thus making biological research shift from single gene or protein to multiple ones. The purpose and significance of this study are: (1) Screening differentially expressed genes (DEG) in rectal cancer before and after radiotherapy by analyzing the gene expression profiles of rectal cancer before and after radiotherapy. They participate in the function and pathway and interaction, predict the microRNA and transcription factor (TF) that regulate these DEGs, construct gene regulatory network, identify significant changes in the gene and key functional pathways in rectal cancer after radiotherapy, reveal the molecular mechanism of preoperative radiotherapy for rectal cancer. (2) Verify the predicted results. Methods: (1) To download the data from GEO public database, and to analyze the effects of different X-ray doses on the proliferation and metastasis of rectal cancer cells. Using limma software package in Bioconductor, the differential expression profiles of all samples in the data set were analyzed before and after radiotherapy for colorectal cancer. The DEGs in the rectal cancer cells after radiotherapy were screened out (the difference multiple was greater than 1, and the p value was less than 0.05). The DAVID online software was used to enrich the DEGs by GO (Gene Ontology) and KEG (KEG). G (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis (p value less than 0.05); STRING online database was used to analyze the protein-protein interaction relationship corresponding to DEG, and Cytoscape mapping software was used to construct protein-protein interaction network; UCSC database was used to identify TF in DEG, determine the differentially expressed genes regulated by them, and Cyt was used to analyze the protein-protein interaction relationship. Osape mapping software was used to construct their regulatory networks. Finally, multiple microRNA databases were used to find the regulatory relationship between DEG and microRNAs, and Cytoscape mapping software was used to construct their regulatory networks. (2) Genes related to cell proliferation and metastasis were selected as the experiment. The proliferation and metastasis of colorectal cancer SW620 cells were examined by MTT and Transwell methods. The effects of different doses of X-ray irradiation on the expression of related genes in SW620 cells were examined by RT-PCR. Similarly, MTT and Transwell methods were used to detect the proliferation and metastasis of SW620 cells. Results: (1) A total of 606 differentially expressed genes (tables) were screened out by gene expression profile analysis. Among them, 271 genes were up-regulated and 335 genes were down-regulated. MMP1 had the largest differential expression multiple and the smallest P value. (2) GO enrichment analysis showed that the up-regulated genes were mainly concentrated in iron transport, inorganic substances and metal ions, such as SLC6A3, SLC30A4, and so on. The down-regulated genes of RYR2 and NEDD4L are mainly related to cell signal transduction, cell proliferation and collagen metabolism. SLC6A4 and PDX1 are involved in cell-to-cell signal transduction, PTGS2 and CDH5 are involved in cell proliferation regulation, while MMP10, COL1A1, MMP3 and MMP1 are mainly related to collagen metabolism. The results of GG pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in the biosynthesis of steroids, calcium signaling pathway, metabolism of androgens and estrogens, interaction of ligands and receptors in nerve tissue, and adult diabetes mellitus in young adults, including HSD3B2, UGT2A3, SULT1E1 and SULT1E1. UGT2B15 genes are involved in the biosynthetic pathways of steroids and the metabolic pathways of androgens and estrogens. CYSLTR2, CHRM1 and HTR6 genes are involved in calcium signaling and ligand-receptor interactions that stimulate nerve tissue. The down-regulated genes are significantly enriched in extracellular matrix. Seven genes encoding collagen, COL4A2, COL4A1 and COL6A3, are involved in the interaction between ECM and receptor. SERPINE1, SERPIND1, F7, PLAU and F2R are involved in the complement and coagulation cascade pathways. The results of network analysis showed that the network contained 410 interaction pairs of 241 proteins and 20 nodes with connectivity greater than or equal to 10. The connectivity of COL1A2 and COL1A 1 was 18 and that of MMP1 was 11. (5) Five differentially expressed TF genes including PAX6, PLAU, FOXL1, NKX2-2 and FOSL were identified by UCSC database analysis. 1. In the regulatory network of DEG and TF, there are 77 regulatory relationship pairs. PLAU regulates NKX2-2 and PAX6 in addition to MMP1 and COL1A1. MMP1 was regulated by hsa-microarray-222, and MMP3 was regulated by hsa-microarray-204. (7) MMP1 was selected for experimental verification. MMP1 mRNA levels in SW620 cells were detected by RT-PCR before silencing. MMP1 mRNA levels in SW620 cells were decreased at 0.1 GY, 0.5 GY, 1 GY, and 6 GY of 3GY compared with the blank control group (0 GY). (8) MTT and Transwell experiments showed that the proliferation and metastasis abilities of SW620 cells before MMP1 silencing were significantly higher than those after MMP1 silencing (p < 0.5), and the proliferation and metastasis abilities of SW620 cells within 6 GY of radiation dose were significantly higher than those after MMP1 silencing (p < 0.5). CONCLUSIONS: (1) Radiotherapy causes some genes (such as SLC6A3, SLC30A4, RYR2 and NEDDD4L) involved in metal ion response in rectal cancer, genes (such as CYSLTR2 and CHRM1) involved in calcium signaling pathways and ligand-receptor interaction pathways (such as CYSLTR2 and CHRM1) that stimulate nerve tissue, to regulate and complement cell proliferation. Genes related to coagulation cascade (such as PLAU, FOSL1 and SERPINE1), genes involved in collagen metabolism (such as MMP1 and MMMP3), and some genes involved in extracellular matrix-receptor interaction pathways (such as COL1A2, COL1A1 and COL4A1) have undergone significant changes in expression, and these genes respond significantly to radiotherapy. Some microRNAs (such as hsa-microRNAs-29c, hsa-microRNAs-224, hsa-microRNAs-204, hsa-microRNAs-222) and some transcription factors (such as PLAU and FOSL1) may play important roles in the regulation of rectal cancer during radiotherapy. (3) Different doses of X-ray radiation can down-regulate the expression of MMP1 in SW620 cells, and radiation agents can down-regulate the expression of MMP1. The expression of MMP1 decreased significantly with the increase of radiation dose within 0.5 GY. (4) MMP1 plays a key role in promoting proliferation and metastasis of SW620 cells. These results provide a good theoretical and experimental basis for the clinical radiosensitivity detection of rectal cancer.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.37

【共引文獻】

相關(guān)期刊論文 前10條

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