【摘要】：目的:結(jié)直腸癌是世界范圍內(nèi)常見(jiàn)的惡性腫瘤之一。我國(guó)的結(jié)直腸癌發(fā)病率和死亡率在不斷上升。由于結(jié)直腸癌通常起病隱匿,主要就診癥狀如便血、大便習(xí)慣改變等多與某些良性疾病相似,早期診斷較為困難,因此大部分患者確診時(shí)已處于中晚期。越來(lái)越多的證據(jù)顯示,微小RNA(miRNA)參與著細(xì)胞增殖、分化、凋亡等重要生命過(guò)程,與多種疾病密切相關(guān),在腫瘤發(fā)生發(fā)展中類(lèi)似抑癌基因或癌基因的功能。MiRNA在相關(guān)癌癥患者的腫瘤組織或血液循環(huán)中存在著特異性的表達(dá),可以用許多常規(guī)方法進(jìn)行差異表達(dá)分析及定量檢測(cè),具有良好的應(yīng)用前景。因此我們研究直腸癌組織中miRNA的特異性表達(dá),并探索其是否可以作為直腸癌診斷和評(píng)估預(yù)后的預(yù)測(cè)因子。方法:收集2015年12月至2016年6月期間就診于安徽省立醫(yī)院并經(jīng)病理科醫(yī)師確診后的直腸癌患者手術(shù)切除的組織,包括腫瘤組織和距離腫瘤2cm以外的周?chē)ｐつそM織,所有患者均未進(jìn)行過(guò)任何術(shù)前放療或化療。取同一病例的癌組織和癌旁正常組織為1對(duì),組織送檢后立刻行福爾馬林固定。所有患者的臨床分期經(jīng)過(guò)專(zhuān)科醫(yī)師評(píng)判。隨機(jī)選取5對(duì)組織進(jìn)行基因芯片檢測(cè),初步篩選出具有差異表達(dá)的miRNA,再利用qRT-PCR的方法驗(yàn)證兩種方法檢測(cè)結(jié)果的一致性,隨后篩選出1~2種最優(yōu)的miRNA擴(kuò)大樣本量進(jìn)行進(jìn)一步驗(yàn)證。證實(shí)差異顯著性后,分析miRNA表達(dá)量與臨床病理參數(shù)之間的相關(guān)性,探討其可能的臨床意義。結(jié)果:經(jīng)過(guò)基因芯片分析,從3100種miRNA中檢測(cè)出上調(diào)的共有138種,下調(diào)的有116種。結(jié)合差異倍數(shù)和探針信號(hào)值初步篩選出5種,分別是上調(diào)的hsa-miR-93-5p,hsa-miR-20a-5p,hsa-miR-106a-5p,和下調(diào)的hsa-miR-27a-3p,hsa-miR-4707-5p。在對(duì)這5對(duì)樣本進(jìn)行qRT-PCR檢測(cè)之后我們發(fā)現(xiàn),這5種miRNA在PCR中檢測(cè)出的趨勢(shì)與芯片檢測(cè)均一致,但hsa-miR-20a-5p(P=0.3221)與hsa-miR-4707-5p(P=0.9369)的表達(dá)量在腫瘤組織與正常組織之間差異無(wú)統(tǒng)計(jì)學(xué)意義。比較結(jié)果并查找相關(guān)文獻(xiàn)后,我們最終選取進(jìn)行后續(xù)實(shí)驗(yàn)的上調(diào)miRNA為hsa-miR-93-5p(腫瘤組織表達(dá)量是正常組織的3.165倍,P=0.0058),下調(diào)miRNA為hsa-mi R-27a-3p(腫瘤組織表達(dá)量是正常組織的0.392倍,p=0.0251)。隨后的擴(kuò)大樣本實(shí)驗(yàn)中,miR-93-5p在腫瘤組織中過(guò)表達(dá)的病例數(shù)占64.79%(46/71),其在腫瘤組織與正常組織中的表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05,n=71)。而mi R-27a-3p在腫瘤組織中呈現(xiàn)過(guò)表達(dá)的病例數(shù)占54.9%(39/71),腫瘤組織中的表達(dá)量高于正常組織,且差異具有統(tǒng)計(jì)學(xué)意義,與前期實(shí)驗(yàn)結(jié)果不一致,另外mi R-27a-3p的數(shù)據(jù)標(biāo)準(zhǔn)差較大,因此最終選擇miR-93-5p進(jìn)行臨床病理參數(shù)分析,發(fā)現(xiàn)腫瘤組mi R-93-5p的表達(dá)量與腫瘤體積大小(P=0.004)、治療前CEA水平(P=0.001)和淋巴結(jié)轉(zhuǎn)移陽(yáng)性患者其受侵淋巴結(jié)數(shù)目相關(guān)(rs=0.534,P=0.005)。結(jié)論:在直腸癌組織中存在特異性表達(dá)的miRNA。miR-93-5p在直腸癌中呈現(xiàn)出高表達(dá),并與腫瘤體積,治療前CEA水平和淋巴結(jié)轉(zhuǎn)移陽(yáng)性患者受侵淋巴結(jié)數(shù)目存在相關(guān)性,也許能在今后作為新型生物標(biāo)記物在臨床醫(yī)師的診療過(guò)程中提供一定的指導(dǎo)意義。
[Abstract]:Objective:Colorectal cancer is one of the most common malignant tumors in the world.The morbidity and mortality of colorectal cancer is increasing in China.Because colorectal cancer usually has a hidden onset and its main symptoms such as blood in stool and changes in stool habits are similar to some benign diseases,early diagnosis is difficult,so most patients have been diagnosed at the time of diagnosis. More and more evidences show that microRNAs (microRNAs) are involved in cell proliferation, differentiation, apoptosis and other important life processes, closely related to a variety of diseases, similar to tumor suppressor genes or oncogene function in the development of tumors. MiRNA in the tumor tissues or blood circulation of cancer patients with specific expression, can be. Therefore, we studied the specific expression of microRNAs in rectal cancer tissues and explored whether microRNAs could be used as predictors for the diagnosis and prognosis of rectal cancer. All patients had not received any preoperative radiotherapy or chemotherapy. One pair of cancerous tissues and normal tissues adjacent to the cancer were taken from the same patient and fixed with formalin immediately after examination. Five pairs of tissues were randomly selected for microarray detection, and the results of differentially expressed microRNAs were preliminarily screened out. The consistency of the two methods was verified by qRT-PCR, and then 1-2 optimal microRNAs were selected for further validation. Results: A total of 138 kinds of up-regulated and 116 kinds of down-regulated microRNAs were detected from 3100 kinds of microRNAs by microarray analysis. Five kinds of up-regulated hsa-microRNA-93-5p, hsa-microRNA-20a-5 were preliminarily screened by combining differential multiples and probe signal values. P, hsa-microRNA-106a-5p, and down-regulated hsa-microRNA-27a-3p, hsa-microRNA-4707-5p. After qRT-PCR detection of these five pairs of samples, we found that the five microRNAs detected in PCR had the same trend as chip detection, but the expression of hsa-microRNA-20a-5p (P = 0.3221) and hsa-microRNA-4707-5p (P = 0.9369) had no significant difference between tumor tissue and normal tissue. Comparing the results and looking up the relevant literature, we finally selected the up-regulated microRNA for follow-up experiments as hsa-microRNA-93-5p (tumor tissue expression was 3.165 times of normal tissue, P = 0.0058), down-regulated microRNA as hsa-mi R-27a-3p (tumor tissue expression was 0.392 times of normal tissue, P = 0.0251). Subsequently, in the expanded sample experiment, microRNA-9 The overexpression of 3-5p in tumor tissues accounted for 64.79% (46/71), and the expression of 3-5p in tumor tissues was significantly different from that in normal tissues (P 0.05, n = 71). In addition, the standard deviation of MIR-27a-3p was large. Therefore, the expression of MIR-93-5p in tumor group was correlated with tumor size (P = 0.004), CEA level before treatment (P = 0.001) and the number of invasive lymph nodes in patients with positive lymph node metastasis (rs = 0.534, P = 0.004). CONCLUSION: There is a high expression of microRNAs. MicroRNAs-93-5p in rectal cancer tissues, which is correlated with tumor volume, CEA level before treatment and the number of invaded lymph nodes in patients with positive lymph node metastasis. It may provide a new biomarker for clinicians in the future. Definite guiding significance.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.37
，

本文編號(hào)：2241320