本文選題：miRNA + microarray　； 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：腎細(xì)胞癌(renal cell carcinoma,RCC,簡(jiǎn)稱“腎癌”)是泌尿系統(tǒng)中致死性最強(qiáng)的惡性腫瘤。盡管近幾年靶向藥物顯著改善了晚期腎癌患者的治療結(jié)局,但其有效性非常有限。微小RNA (microRNA, miRNA)是一類經(jīng)典的非編碼RNA,具有長(zhǎng)度短、作用廣、穩(wěn)定性強(qiáng)的特點(diǎn),被認(rèn)為是腫瘤治療領(lǐng)域的“新靶標(biāo)”。我們前期通過高通量microarray分析腎透明細(xì)胞癌(clear cell RCC, ccRCC)的癌組織及其癌旁組織的miRNA表達(dá)譜,發(fā)現(xiàn)miRNA在癌組織中以下調(diào)為主,其中miR-141下調(diào)最為明顯。迄今,miR-141在腎癌中的作用及其機(jī)制缺乏全面而深入的研究。本研究旨在鑒定miR-141在腎癌中的臨床價(jià)值和生物學(xué)作用及其機(jī)制,為將來運(yùn)用于臨床診治提供理論依據(jù)。我們將通過檢測(cè)多種腎腫瘤標(biāo)本的miR-141表達(dá)水平,明確miR-141在腎癌診斷及預(yù)后判斷中的價(jià)值；通過體內(nèi)體外全面的功能研究,揭示miR-141在腎癌發(fā)生發(fā)展中的作用及其具體機(jī)制；通過ccRCC細(xì)胞培養(yǎng)液的分析,了解胞外miR-141的特性。本研究分為三部分： 第一部分腎癌組織miRNA表達(dá)譜分析及miR-141臨床價(jià)值鑒定 目的：探索ccRCC癌組織和癌旁組織中miRNA的表達(dá)差異,篩選并鑒定可能在腎癌發(fā)生發(fā)展中起關(guān)鍵作用的miRNA(s)的臨床價(jià)值。 方法：用microarray生物芯片高通量分析ccRCC患者手術(shù)切除的癌組織及其癌旁組織中miRNA的表達(dá)情況,篩選出表達(dá)差異最明顯的niRNA,運(yùn)用實(shí)時(shí)定量PCR(qRT-PCR)在較大腎腫瘤樣本組織中分析miRNA的臨床意義。 結(jié)果：按照倍數(shù)2和P0.05標(biāo)準(zhǔn),我們發(fā)現(xiàn)相對(duì)于癌旁組織,44個(gè)miRNAs在ccRCC癌組織中下調(diào),30個(gè)miRNAs上調(diào)。其中,miR-141在癌組織中下調(diào)最明顯。qRT-PCR結(jié)果顯示miR-141在92.6%(63/68)的ccRCC中明顯下調(diào)(p0.0001)。受試者工作特征曲線(ROC)分析顯示miR-141鑒別ccRCC與正常組織的AUC(曲線下面積,即準(zhǔn)確性)為0.93(95%CI,0.881to0.981)。癌組織中miR-141的表達(dá)水平與腫瘤分期、分級(jí)、大小均無(wú)統(tǒng)計(jì)學(xué)相關(guān)性。miR-141在腎癌細(xì)胞系和其他亞型腎腫瘤中也顯著低表達(dá),包括腎嫌色細(xì)胞癌(chRCC)、肉瘤樣腎細(xì)胞癌和腎血管平滑肌脂肪瘤(AML)。 結(jié)論：在腎癌發(fā)病中,miRNA異常表達(dá)以下調(diào)為主。miR-141可能作為鑒別ccRCC及正常組織有力的診斷標(biāo)志物。miR-141的下調(diào)可能促進(jìn)腎腫瘤的發(fā)生發(fā)展。 第二部分胞內(nèi)和胞外miR-141對(duì)腎癌細(xì)胞生物學(xué)特性的影響 目的：通過體內(nèi)體外研究,明確內(nèi)源性miR-141在調(diào)控腎癌惡性活性中的關(guān)鍵作用,探索腎癌細(xì)胞胞外miR-141的生物學(xué)特性。 方法：用慢病毒構(gòu)建穩(wěn)定高表達(dá)miR-141及其對(duì)照的腎癌細(xì)胞系,體外分析miR-141在腎癌細(xì)胞增殖(MTT)、周期(PI染色)、遷移侵襲(Transwell)、凋亡和藥物敏感性(MTT和FACS)方面的調(diào)控作用；裸鼠原位腎腫瘤模型分析miR-141對(duì)腫瘤成瘤率、腫瘤大小、重量、局部侵襲率和遠(yuǎn)處轉(zhuǎn)移率的影響；收集細(xì)胞培養(yǎng)液,提取RNA,qRT-PCR分析胞外miR-141的表達(dá)水平,并將含有miR-141的培養(yǎng)液培養(yǎng)對(duì)照細(xì)胞,觀察其對(duì)受體腎癌細(xì)胞遷移侵襲能力的影響。 結(jié)果：含有miR-141的慢病毒能顯著提高786-0和SN12-PM6細(xì)胞中的miR-141表達(dá)水平,分別為400倍和2400倍。全面的功能研究顯示,miR-141極大地抑制了腎癌細(xì)胞的遷移侵襲能力(p0.0001),部分抑制細(xì)胞的生長(zhǎng)(p0.05),誘使細(xì)胞周期阻滯在Go/G1期,降低S期細(xì)胞數(shù)。然而,miR-141未能誘導(dǎo)腎癌細(xì)胞形態(tài)發(fā)生變化,對(duì)凋亡無(wú)直接促進(jìn)作用,也未提高腎癌細(xì)胞對(duì)藥物順鉑(DDP)、5-氟尿嘧啶(5-FU)和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TRAIL)的敏感性。相對(duì)于對(duì)照組,過表達(dá)miR-141組的裸鼠腎腫瘤大小、重量明顯降低。植入腫瘤細(xì)胞后6周和8-9周,miR-141組腎腫瘤未出現(xiàn)局部侵襲和遠(yuǎn)處轉(zhuǎn)移,而對(duì)照組腫瘤明顯向腎外侵襲性生長(zhǎng),多個(gè)臟器出現(xiàn)轉(zhuǎn)移病灶。此外,帶有miR-141的慢病毒感染腎癌細(xì)胞,可增加培養(yǎng)液中胞外miR-141的表達(dá)水平；胞外miR-141可進(jìn)入受體腎癌細(xì)胞內(nèi),抑制受體細(xì)胞的遷移侵襲能力而對(duì)細(xì)胞增殖凋亡無(wú)明顯影響。786-0和SN12-PM6細(xì)胞的胞外/胞內(nèi)miR-141比值分別低于2.2%和0.01%。 結(jié)論：通過抑制腎癌細(xì)胞的增殖和轉(zhuǎn)移,miR-141可作為腎癌重要的抑癌基因。miR-141可分泌入細(xì)胞外,發(fā)揮細(xì)胞間的信息傳遞作用,但這種作用有限。 第三部分在腎癌組織中高表達(dá)的EphA2是miR-141的一個(gè)新靶點(diǎn) 目的：探索miR-141在腎癌中發(fā)揮抑癌作用的具體機(jī)制。 方法：用Targetscan.miRanda、PicTar等多個(gè)靶點(diǎn)預(yù)測(cè)軟件篩選miR-141的靶點(diǎn),構(gòu)建EphA23'UTR野生型和突變型熒光素酶報(bào)告基因,qRT-PCR、Western blot、IHC等方法在體外細(xì)胞水平、體內(nèi)臨床標(biāo)本、裸鼠腎腫瘤標(biāo)本分析EphA2和miR-141的表達(dá)水平。將EphA2siRNA轉(zhuǎn)染腎癌細(xì)胞,分析敲除EphA2與過表達(dá)miR-141對(duì)腎癌細(xì)胞生物學(xué)功能影響的相似性。將EphA2siRNA和miR-141抑制劑共轉(zhuǎn)染入腎癌細(xì)胞,設(shè)計(jì)“拯救”實(shí)驗(yàn)。查閱文獻(xiàn)尋找與EphA2結(jié)構(gòu)和功能相關(guān)的基因,Vestern blot分析過表達(dá)miR-141和敲除EphA2對(duì)腎癌細(xì)胞中FAK、p-FAK、AKT、p-AKT、MMP-2、MMP-9蛋白表達(dá)的影響。 結(jié)果：多個(gè)靶點(diǎn)軟件均預(yù)測(cè)到miR-141種子序列與EphA23'UTR相結(jié)合。雙熒光素酶報(bào)告基因結(jié)果顯示,過表達(dá)miR-141明顯降低EphA23'UTR野生型的熒光素酶活性,而對(duì)突變型無(wú)明顯影響。過表達(dá)miR-141的細(xì)胞和裸鼠腎腫瘤標(biāo)本中的EphA2mRNA和蛋白水平明顯下調(diào)。盡管ccRCC癌組織與癌旁正常組織中EphA2mRNA的表達(dá)水平無(wú)明顯差異,但癌組織中EphA2mRNA與：miR-141的表達(dá)水平呈明顯負(fù)相關(guān)(Pearson相關(guān)分析,R2=0.3661,p=0.0047)。更值得關(guān)注的是,EphA2蛋白在85%(17/20)的ccRCC中高表達(dá)。而且,敲除EphA2顯著削弱腎癌細(xì)胞的增殖、遷移侵襲能力,阻滯細(xì)胞周期在Go/G1期,其作用與過表達(dá)miR-141的類似。miR-141抑制劑可以通過增加EphA2的表達(dá)增強(qiáng)腎癌細(xì)胞的遷移侵襲能力,該增強(qiáng)作用可被EphA2siRNA所中和。以往研究表明FAK和AKT是兩個(gè)與EphA2結(jié)構(gòu)和功能密切相關(guān)的基因,MMP-2和MMP-9是FAK和AKT的下游信號(hào)基因。據(jù)此,我們推測(cè)p-FAK/p-AKT/MMPs是miR-141-EphA2發(fā)揮抗癌作用的“效應(yīng)器”。Western blot顯示,過表達(dá)miR-141和敲除EphA2均能充分抑制腎癌細(xì)胞的p-FAK、p-AKT、MMP-2和MMP-9的表達(dá),且miR-141抑制劑可增加FAK和AKT的磷酸化水平,該增加作用可被EphA2siRNA中和。 結(jié)論：EphA2是miR-141的一個(gè)具有功能的直接靶點(diǎn)。EphA2在腎癌組織中高表達(dá)可能是由miR-141的缺失所致。miR-141-EphA2抑制腫瘤活性可能是通過p-FAK/p-AKT/MMPs通路實(shí)現(xiàn)的。
[Abstract]:Renal cell carcinoma (RCC) is the most fatal malignant tumor in the urinary system. Although targeted drugs have significantly improved the treatment outcome of patients with advanced renal cancer in recent years, its effectiveness is very limited. Small RNA (microRNA, miRNA) is a class of classic non coded RNA with short length, wide action and stability. The qualitative characteristics are considered to be the "new target" in the field of cancer treatment. We have analyzed the miRNA expression profiles of the cancerous tissues and adjacent tissues of clear cell RCC (ccRCC) by high throughput microarray. It was found that miRNA was the dominant factor in the cancer tissue, and the most obvious down regulation was miR-141. So far, miR-141 is in renal cancer. The purpose of this study is to identify the clinical value and biological role of miR-141 in renal carcinoma and to provide a theoretical basis for clinical diagnosis and treatment in the future. We will examine the miR-141 expression of a variety of renal tumor specimens and determine miR-141 in the diagnosis and prognosis of renal cancer. The role of miR-141 in the development of renal cell carcinoma and its specific mechanism through comprehensive functional study in vitro and in vivo, and through the analysis of ccRCC cell culture fluid to understand the characteristics of extracellular miR-141. This study is divided into three parts:
Part one miRNA expression profiling of renal cell carcinoma and evaluation of miR-141 clinical value
Objective: To explore the difference in the expression of miRNA in ccRCC and para cancerous tissues, and to screen and identify the clinical value of miRNA (s), which may play a key role in the development of renal carcinoma.
Methods: microarray biochip was used to analyze the expression of miRNA in the cancerous tissues and adjacent tissues of ccRCC patients with high throughput. The most distinct niRNA was screened out, and the significance of miRNA in the large renal tumor samples was analyzed by real-time quantitative PCR (qRT-PCR).
Results: according to the multiplier 2 and P0.05 standards, we found that 44 miRNAs were downregulated and 30 miRNAs up-regulated in ccRCC cancer tissues relative to the paracancerous tissue. Among them, the most obvious down-regulation of miR-141 in the cancer tissue showed that miR-141 was down significantly down in ccRCC (63/68) in 92.6% (P0.0001). The subject work characteristic curve (ROC) analysis showed miR-141 The identification of ccRCC and normal tissue AUC (the area under the curve, that is, accuracy) was 0.93 (95%CI, 0.881to0.981). The expression level of miR-141 in cancer tissues was not statistically correlated with tumor stages, classification, size and.MiR-141 in renal cell carcinoma cell lines and other subtype renal tumors, including renal cell carcinoma (chRCC) and sarcomatoid renal cells Carcinoma and renal angiomyolipoma (AML).
Conclusion: in the pathogenesis of renal carcinoma, the abnormal expression of miRNA as the main.MiR-141 may be a powerful diagnostic marker for ccRCC and normal tissue, the downregulation of.MiR-141 may promote the development of renal tumor.
The second part is the effect of intracellular and extracellular miR-141 on the biological characteristics of renal cell carcinoma.
Objective: To investigate the key role of endogenous miR-141 in regulating the malignant activity of renal carcinoma in vitro and in vitro, and to explore the biological characteristics of extracellular miR-141 in renal cell carcinoma cells.
Methods: the renal carcinoma cell lines with high expression of miR-141 and its control were constructed with lentivirus. The regulation of miR-141 in renal cell carcinoma cell proliferation (MTT), cycle (PI staining), migration invasion (Transwell), apoptosis and drug sensitivity (MTT and FACS) was analyzed in vitro. The tumor formation rate and tumor size of miR-141 for tumor were analyzed by miR-141 in nude mice. The effects of weight, local invasion rate and distant metastasis rate were collected, cell culture fluid was collected, RNA, qRT-PCR was extracted to analyze the expression level of extracellular miR-141, and the culture medium containing miR-141 was used to culture control cells to observe the influence of the cell migration and invasion ability of RCC cells.
Results: the lentivirus containing miR-141 could significantly increase the level of miR-141 expression in 786-0 and SN12-PM6 cells, 400 times and 2400 times respectively. Comprehensive functional study showed that miR-141 greatly inhibited the migration and invasion ability of renal cell carcinoma cells (P0.0001), partially inhibited the growth of cells (P0.05), induced cell cycle arrest in Go/G1 phase and reduced S. However, miR-141 failed to induce renal cell carcinoma cell morphogenesis, had no direct promoting effect on apoptosis, and did not increase the sensitivity of renal cancer cells to cisplatin (DDP), 5- fluorouracil (5-FU) and tumor necrosis factor related apoptosis inducing ligand (TRAIL). In the 6 and 8-9 weeks after the implantation of tumor cells, there was no local invasion and distant metastasis in miR-141 group, while the tumor in the control group was obviously invasive out of the kidney, and the metastasis of multiple organs appeared. In addition, the expression level of extracellular miR-141, extracellular miR, was added to the renal cancer cells with miR-141. -141 can enter receptor renal cell carcinoma cells, inhibit the migration and invasion of receptor cells and have no significant effect on cell proliferation and apoptosis, and the extracellular / intracellular miR-141 ratio of.786-0 and SN12-PM6 cells is lower than 2.2% and 0.01%., respectively.
Conclusion: by inhibiting the proliferation and metastasis of renal cell carcinoma cells, miR-141 can be used as an important tumor suppressor gene,.MiR-141, to be secreted into cells and play an intercellular information transfer function, but this effect is limited.
The third part is the high expression of EphA2 in renal cell carcinoma, which is a new target for miR-141.
Objective: To explore the specific mechanism of miR-141 in renal cell carcinoma.
Methods: Targetscan.miRanda, PicTar and other target prediction software were used to screen the target of miR-141, and EphA23'UTR wild type and mutant luciferase reporter gene was constructed. QRT-PCR, Western blot, IHC and other methods were used to analyze the expression level of EphA2 and miR-141 in vivo, in vivo and in nude mice kidney tumor markers. The effects of EphA2 and overexpression of miR-141 on the biological function of renal cell carcinoma cells were analyzed. The EphA2siRNA and miR-141 inhibitors were co transfected into the renal cancer cells, and the "rescue" experiment was designed. The literature looked for the genes related to the structure and function of EphA2. Vestern blot analyzed the expression of miR-141 and knockout EphA2 to renal cancer. The expression of FAK, p-FAK, AKT, p-AKT, MMP-2 and MMP-9 proteins in cells.
Results: multiple target software predicted that the miR-141 seed sequence was combined with EphA23'UTR. The results of the double luciferase reporter gene showed that overexpression of miR-141 significantly reduced the luciferase activity of the EphA23'UTR wild type, but had no obvious effect on the mutant type. The EphA2mRNA and protein water in the cells overexpressing miR-141 and the renal tumor specimens of nude mice Although there was no significant difference in the expression level of EphA2mRNA in the ccRCC carcinoma tissue and the normal tissue adjacent to the cancer, the expression level of EphA2mRNA in the cancer tissues was negatively correlated with the expression level of miR-141 (Pearson correlation analysis, R2=0.3661, p=0.0047). It is more important to pay more attention to the high expression of EphA2 egg white in the ccRCC of 85% (17/20). Moreover, the knockout is EphA2. To weaken the proliferation, migration and invasiveness of renal cell carcinoma cells, block cell cycle in Go/G1 phase, its role and.MiR-141 inhibitor, which overexpresses miR-141, can enhance the migration and invasion ability of renal cancer cells by increasing EphA2 expression, which can be neutralized by EphA2siRNA. Previous studies have shown that FAK and AKT are two and EphA2 structures Genes that are closely related to function, MMP-2 and MMP-9 are downstream signal genes of FAK and AKT. Accordingly, we speculate that p-FAK/p-AKT/MMPs is a "effector".Western blot that miR-141-EphA2 plays the role of anti-cancer. The expression of miR-141 and knockout EphA2 can fully inhibit the p-FAK, p-AKT, and inhibition of renal cell carcinoma cells and inhibit the expression of EphA2. Agents can increase the phosphorylation level of FAK and AKT, which can be neutralized by EphA2siRNA.
Conclusion: EphA2 is a functional direct target of miR-141, the high expression of.EphA2 in renal carcinoma tissue may be caused by the deletion of miR-141 caused by.MiR-141-EphA2 inhibition of tumor activity, which may be achieved through p-FAK/p-AKT/MMPs pathway.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.11

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1 徐艷s，

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