本文選題：胰腺癌 + miRNAs　； 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：一、研究背景胰腺導(dǎo)管腺癌是全球最為致命的惡性腫瘤之一,在惡性腫瘤致死率中排名第4位,其5年生存率僅6%左右。由于胰腺癌具有明顯的侵襲性,80%的胰腺癌患者在疾病確診時就發(fā)現(xiàn)了早期的胰腺外轉(zhuǎn)移,使得手術(shù)及藥物治療效果很差,導(dǎo)致很高的疾病致死率和極差的預(yù)后。究其原因,其一是胰腺導(dǎo)管腺癌的早期診斷存在短板,早期預(yù)警缺乏特異性及敏感性較高的分子標(biāo)記,大多數(shù)患者在出現(xiàn)癥狀時,往往已出現(xiàn)局部侵犯和遠(yuǎn)處轉(zhuǎn)移,已經(jīng)失去了最佳的手術(shù)治療時機。其二,胰腺導(dǎo)管腺癌是“高度異質(zhì)性”疾病,致病基因多變而復(fù)雜。因此,更為敏感、特異的早期胰腺癌分子標(biāo)記亟待發(fā)掘,臨床需求呼吁更為深入地研究胰腺癌發(fā)生、發(fā)展的分子機制和調(diào)控通路,并可能為開發(fā)新的治療胰腺癌的分子靶點提供思路。MicroRNA是近年來目前研究最為廣泛的一種ncRNA,它在編碼蛋白基因的轉(zhuǎn)錄后水平進(jìn)行調(diào)控。它被證實在腫瘤細(xì)胞的發(fā)生、發(fā)展、增殖、分化、凋亡和侵襲等過程中有重要的調(diào)節(jié)作用。這些miRNAs具有穩(wěn)定性、組織特異性、易獲得性和高度保守性,可以作為很好的腫瘤疾病診斷標(biāo)志物,同時這些功能性miRNAs的作用機制還遠(yuǎn)沒有被人類所認(rèn)識。二、研究目的本研究旨在研究miR-377在胰腺癌中的表達(dá)特征及其在胰腺癌細(xì)胞增殖和凋亡中的調(diào)控作用及其分子機制。三、材料和方法1.本研究組先運用生物信息學(xué)方法在GEO胰腺癌miRNAs芯片數(shù)據(jù)庫和TCGA胰腺癌miRNAs表達(dá)譜數(shù)據(jù)庫中篩選出差異表達(dá)的miRNAs。其中,miR-377在胰腺癌中表達(dá)特異性下調(diào),并與臨床預(yù)后相關(guān)。2.擴大臨床樣本量,通過QT-PCR技術(shù)分別檢測30例胰腺導(dǎo)管腺癌癌組織/癌旁組織,以及胰腺癌細(xì)胞/正常胰腺導(dǎo)管上皮細(xì)胞中miR-377的表達(dá)水平,明確其表達(dá)差異。同時利用統(tǒng)計學(xué)方法分析miR-377的表達(dá)水平與患者臨床參數(shù)之間的相關(guān)性。3.為了研究miR-377在胰腺癌中的功能,我們構(gòu)建了異位miR-377過表達(dá)和沉默慢病毒載體,建立穩(wěn)轉(zhuǎn)胰腺癌細(xì)胞系以進(jìn)行更為深入的功能研究。通過CCK-8實驗、細(xì)胞平板克隆形成實驗、Transwell遷移實驗、流式細(xì)胞技術(shù)檢測細(xì)胞周期、流式細(xì)胞技術(shù)檢測細(xì)胞凋亡、TUNEL染色檢測細(xì)胞凋亡研究miR-377對胰腺癌細(xì)胞的增殖、生長活性、遷移能力、細(xì)胞周期分布、細(xì)胞凋亡的影響。4.通過生物信息學(xué)預(yù)測工具分析miR-377潛在的靶基因,結(jié)果提示PIM-3可能是miR-377的靶控基因。通過QT_PCR、WB檢測異位表達(dá)miR-377的胰腺癌細(xì)胞中PIM-3mRNA水平及蛋白水平。QT_PCR檢測胰腺癌組織中miR-377與PIM-3表達(dá)量之間的相關(guān)性。WB檢測胰腺癌組織與胰腺癌細(xì)胞中PIM-3蛋白水平的差異。雙熒光素酶報告基因系統(tǒng)鑒定miR-377與PIM-3的直接結(jié)合活性。5.通過WB技術(shù)檢測異位表達(dá)miR-377后,其靶蛋白PIM-3及下游通路蛋白的改變。6.通過siRNA沉默PIM-3的表達(dá)后,檢測胰腺癌細(xì)胞生物學(xué)功能的改變。四、統(tǒng)計學(xué)方法本實驗數(shù)據(jù)均用SPSS 23.0軟件(Windows)進(jìn)行統(tǒng)計,并運用Graph Pad Prism 5.0.軟件進(jìn)行統(tǒng)計圖的繪制。組間計量資料的比較采用重復(fù)測量的單因素方差分析(兩兩比較時采用配對t檢驗)。運用Person相關(guān)系數(shù)分析miR-377與PIM-3 mRNA之間的相關(guān)性。Kaplan-Meier生存分析來檢測miR-377與臨床生存期的關(guān)系。PIM-3 mRNA的表達(dá)水平與臨床參數(shù)之間的關(guān)系采用χ2檢驗。計量資料采用均數(shù)±標(biāo)準(zhǔn)差來表示,P0.05作為顯著統(tǒng)計學(xué)差異的標(biāo)準(zhǔn)。五、結(jié)果1.公共數(shù)據(jù)分析結(jié)果支持miR-377在胰腺導(dǎo)管上皮腺癌組織中下調(diào)表達(dá)。并與臨床生存預(yù)后顯著相關(guān)。2.miR-377在胰腺導(dǎo)管腺癌癌組織和細(xì)胞株中特異性低表達(dá),相對于正常胰腺組織和正常胰腺導(dǎo)管上皮細(xì)胞;且miR-377在胰腺癌中的表達(dá)水平與腫瘤大小、局部淋巴結(jié)有無轉(zhuǎn)移顯著相關(guān)。3.在胰腺癌細(xì)胞中過表達(dá)miR-377后可抑制細(xì)胞增殖、生長、遷移能力,阻滯胰腺癌細(xì)胞周期于G1/S轉(zhuǎn)換,并促進(jìn)胰腺癌細(xì)胞凋亡,減少胰腺癌細(xì)胞對于化療藥物的耐藥性。4.雙熒光素酶報告基因證實了miR-377的靶基因是PIM-3,并通過直接結(jié)合于它的3’-UTR區(qū)對其起到負(fù)向調(diào)控作用。QT-PCR的結(jié)果顯示:miR-377的表達(dá)水平與PIM-3mRNA的表達(dá)量呈負(fù)向相關(guān)。WB結(jié)果也顯示:miR-377是通過負(fù)向調(diào)控PIM-3蛋白水平,而PIM-3則通過磷酸化Ser112 Bad蛋白,抑制Bad-Bcl-XL抗凋亡通路參與胰腺癌細(xì)胞增殖、凋亡的調(diào)控。5.應(yīng)用siRNA沉默PIM-3基因后,胰腺癌細(xì)胞的增殖、生長能力、克隆形成能力、遷移能力均得到抑制,并導(dǎo)致細(xì)胞周期阻滯、凋亡增強,而使穩(wěn)定沉默miR-377表達(dá)的作用被抵消。六、結(jié)論綜合實驗數(shù)據(jù)可顯示:miR-377在胰腺癌中特異性下調(diào)表達(dá),并與臨床不良預(yù)后相關(guān),其表達(dá)水平與腫瘤大小、淋巴結(jié)有無轉(zhuǎn)移密切相關(guān);miR-377可抑制胰腺癌細(xì)胞增殖、生長及遷移,并引起細(xì)胞周期阻滯、凋亡增強、對化療藥物敏感性上升;miR-377通過負(fù)向調(diào)控靶基因PIM-3,抑制Bad-Bcl-XL抗凋亡通路。因此miR-377顯示出一定的抑制腫瘤的特征,miR-377-Pim-3-p BAD112調(diào)控通路可能為研究胰腺癌潛在的新治療靶點提供實驗依據(jù)。
[Abstract]:First, pancreatic duct adenocarcinoma is one of the most deadly malignant tumors in the world. It ranks fourth in the mortality rate of malignant tumor, and its 5 year survival rate is only about 6%. Because of the obvious invasiveness of pancreatic cancer, 80% of the patients with pancreatic cancer found early extrapancreatic metastases at the time of diagnosis, making the surgical and drug treatment effect very effective. The reason is that the early diagnosis of pancreatic duct adenocarcinoma is short plate, early warning is short of specific and sensitive molecular markers. Most patients often have local invasion and distant metastasis when they have symptoms, and they have lost the best surgical treatment. Secondly, pancreatic ductal adenocarcinoma is a "highly heterogeneous" disease, and the pathogenic gene is changeable and complex. Therefore, it is more sensitive, specific early molecular markers of pancreatic cancer need to be unearthed urgently. Clinical requirements call for more in-depth study of the pathogenesis of pancreatic cancer, the molecular mechanisms and regulatory pathways of development, and the possibility of developing new molecules for the treatment of pancreatic cancer. .MicroRNA is the most widely studied ncRNA in recent years, which regulates the post transcriptional level of the encoded protein gene. It has been proved to play an important role in the pathogenesis, development, proliferation, differentiation, apoptosis and invasion of the tumor cells. These miRNAs are stable, tissue specific and easy to obtain. And highly conserved, it can be used as a good marker for diagnosis of tumor disease, and the functional mechanisms of these functional miRNAs are still far from being recognized by humans. Two. The purpose of this study was to study the expression of miR-377 in pancreatic cancer and its regulatory role in the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. Three Materials and methods 1. study group first used bioinformatics methods to select the differentially expressed miRNAs. in the miRNAs chip database of GEO pancreatic cancer and the miRNAs expression database of TCGA pancreatic cancer. The specific down-regulation of miR-377 in pancreatic cancer was expressed, and the clinical samples were enlarged by.2. in the clinical prognosis, and 30 were detected by QT-PCR technology respectively. The expression level of miR-377 in pancreatic ductal carcinoma tissue / paracancerous tissue and pancreatic carcinoma cell / normal pancreatic duct epithelial cells, and the correlation between the expression level of miR-377 and the clinical parameters of the patients was analyzed by statistical methods.3. in order to study the function of miR-377 in pancreatic cancer, we construct it. The ectopic miR-377 overexpression and the silencing of the lentivirus vector to establish a stable pancreatic cancer cell line for further functional study. Through the CCK-8 experiment, the cell plate clone formation experiment, the Transwell migration experiment, the flow cytometry test cell cycle, the flow cytometry to detect the cell apoptosis, and the TUNEL staining to detect the apoptosis of the cells, MI The effects of R-377 on the proliferation, growth activity, migration, cell cycle distribution and apoptosis of pancreatic cancer cells.4. can be used to analyze the potential target genes of miR-377 through bioinformatics prediction tools. The results suggest that PIM-3 may be the target gene of miR-377. WB detection of PIM-3mRNA levels and eggs in pancreatic cancer cells with ectopic expression of miR-377 through QT_PCR Correlation between miR-377 and PIM-3 expression in pancreatic cancer tissues by white level.QT_PCR.WB detection of PIM-3 protein levels in pancreatic cancer tissues and pancreatic cancer cells. The double luciferase reporter gene system identified the direct binding activity of miR-377 and PIM-3 by the WB technique to detect the expression of miR-377 by WB technique, and the target protein was PIM-3 and lower. The changes in the pathway protein.6. were used to detect the changes in the biological function of pancreatic cancer cells after siRNA silencing the expression of PIM-3. Four, the statistical methods were statistically used by the SPSS 23 software (Windows), and the Graph Pad Prism 5.0. software was used to draw the statistical map. The comparison of the data between groups was compared with the single cause of the repeated measurement. Analysis of prime variance (22 compared with paired t test). Person correlation coefficient was used to analyze the correlation between miR-377 and PIM-3 mRNA to determine the relationship between miR-377 and the clinical survival time. The relationship between the expression level of.PIM-3 mRNA and the clinical parameters was tested by the chi 2 test. The results indicated that P0.05 was a significant statistical difference. Five, results 1. the results of public data analysis supported the downregulation of miR-377 in pancreatic ductal epithelial adenocarcinoma. It was significantly related to the survival prognosis of.2.miR-377 in the specific low expression of.2.miR-377 in pancreatic ductal adenocarcinoma tissue and cell lines, relative to normal pancreatic tissue and normal pancreas. The expression level of miR-377 in pancreatic cancer is significantly related to the size of the tumor and the size of the tumor and the metastasis of the local lymph nodes..3. can inhibit the proliferation, growth and migration of miR-377 in pancreatic cancer cells, block the cell cycle of pancreatic cancer in G1/S conversion, and promote the apoptosis of pancreatic cancer cells and reduce the pancreatic cancer cells. The drug resistance.4. double luciferase reporter gene confirmed that the target gene of miR-377 is PIM-3, and the result of the negative regulation of.QT-PCR through its 3 '-UTR region shows that the expression level of miR-377 and the expression of PIM-3mRNA are negatively related.WB results also show that miR-377 is through the negative regulation of PIM-3. Protein level, and PIM-3 by phosphorylated Ser112 Bad protein, inhibit Bad-Bcl-XL anti apoptosis pathway to participate in the proliferation of pancreatic cancer cells. Apoptosis regulation,.5. application siRNA silent PIM-3 gene, pancreatic cancer cell proliferation, growth, clone formation ability, migration ability to inhibit, and lead to cell cycle block, apoptosis enhancement, and cause The effect of stable silence miR-377 expression was offset. Six, conclusion the comprehensive experimental data can show that miR-377 is specific down expression in pancreatic cancer and is related to poor clinical prognosis. The expression level is closely related to the size of the tumor and the metastasis of lymph nodes. MiR-377 can inhibit the proliferation, growth and migration of pancreatic adenocarcinoma cells and cause cell cycle. Blocking, increasing apoptosis and increasing sensitivity to chemotherapeutic drugs; miR-377 through the negative regulation of target gene PIM-3, inhibiting the anti apoptosis pathway of Bad-Bcl-XL. Therefore, miR-377 shows certain characteristics of tumor inhibition, and miR-377-Pim-3-p BAD112 regulation pathway may provide experimental basis for the study of potential new therapeutic targets for pancreatic cancer.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R735.9

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本文編號：1940939