miR-141抑制肾癌细胞增殖和转移的新机制

发布时间：2018-05-29 13:29

本文选题：miRNA + microarray　；参考：《华中科技大学》2014年博士论文

【摘要】：肾细胞癌(renal cell carcinoma,RCC,简称“肾癌”)是泌尿系统中致死性最强的恶性肿瘤。尽管近几年靶向药物显著改善了晚期肾癌患者的治疗结局,但其有效性非常有限。微小RNA (microRNA, miRNA)是一类经典的非编码RNA,具有长度短、作用广、稳定性强的特点,被认为是肿瘤治疗领域的“新靶标”。我们前期通过高通量microarray分析肾透明细胞癌(clear cell RCC, ccRCC)的癌组织及其癌旁组织的miRNA表达谱,发现miRNA在癌组织中以下调为主,其中miR-141下调最为明显。迄今,miR-141在肾癌中的作用及其机制缺乏全面而深入的研究。本研究旨在鉴定miR-141在肾癌中的临床价值和生物学作用及其机制,为将来运用于临床诊治提供理论依据。我们将通过检测多种肾肿瘤标本的miR-141表达水平,明确miR-141在肾癌诊断及预后判断中的价值；通过体内体外全面的功能研究,揭示miR-141在肾癌发生发展中的作用及其具体机制；通过ccRCC细胞培养液的分析,了解胞外miR-141的特性。本研究分为三部分：第一部分肾癌组织miRNA表达谱分析及miR-141临床价值鉴定目的：探索ccRCC癌组织和癌旁组织中miRNA的表达差异,筛选并鉴定可能在肾癌发生发展中起关键作用的miRNA(s)的临床价值。方法：用microarray生物芯片高通量分析ccRCC患者手术切除的癌组织及其癌旁组织中miRNA的表达情况,筛选出表达差异最明显的niRNA,运用实时定量PCR(qRT-PCR)在较大肾肿瘤样本组织中分析miRNA的临床意义。结果：按照倍数2和P0.05标准,我们发现相对于癌旁组织,44个miRNAs在ccRCC癌组织中下调,30个miRNAs上调。其中,miR-141在癌组织中下调最明显。qRT-PCR结果显示miR-141在92.6%(63/68)的ccRCC中明显下调(p0.0001)。受试者工作特征曲线(ROC)分析显示miR-141鉴别ccRCC与正常组织的AUC(曲线下面积,即准确性)为0.93(95%CI,0.881to0.981)。癌组织中miR-141的表达水平与肿瘤分期、分级、大小均无统计学相关性。miR-141在肾癌细胞系和其他亚型肾肿瘤中也显著低表达,包括肾嫌色细胞癌(chRCC)、肉瘤样肾细胞癌和肾血管平滑肌脂肪瘤(AML)。结论：在肾癌发病中,miRNA异常表达以下调为主。miR-141可能作为鉴别ccRCC及正常组织有力的诊断标志物。miR-141的下调可能促进肾肿瘤的发生发展。第二部分胞内和胞外miR-141对肾癌细胞生物学特性的影响目的：通过体内体外研究,明确内源性miR-141在调控肾癌恶性活性中的关键作用,探索肾癌细胞胞外miR-141的生物学特性。方法：用慢病毒构建稳定高表达miR-141及其对照的肾癌细胞系,体外分析miR-141在肾癌细胞增殖(MTT)、周期(PI染色)、迁移侵袭(Transwell)、凋亡和药物敏感性(MTT和FACS)方面的调控作用；裸鼠原位肾肿瘤模型分析miR-141对肿瘤成瘤率、肿瘤大小、重量、局部侵袭率和远处转移率的影响；收集细胞培养液,提取RNA,qRT-PCR分析胞外miR-141的表达水平,并将含有miR-141的培养液培养对照细胞,观察其对受体肾癌细胞迁移侵袭能力的影响。结果：含有miR-141的慢病毒能显著提高786-0和SN12-PM6细胞中的miR-141表达水平,分别为400倍和2400倍。全面的功能研究显示,miR-141极大地抑制了肾癌细胞的迁移侵袭能力(p0.0001),部分抑制细胞的生长(p0.05),诱使细胞周期阻滞在Go/G1期,降低S期细胞数。然而,miR-141未能诱导肾癌细胞形态发生变化,对凋亡无直接促进作用,也未提高肾癌细胞对药物顺铂(DDP)、5-氟尿嘧啶(5-FU)和肿瘤坏死因子相关凋亡诱导配体(TRAIL)的敏感性。相对于对照组,过表达miR-141组的裸鼠肾肿瘤大小、重量明显降低。植入肿瘤细胞后6周和8-9周,miR-141组肾肿瘤未出现局部侵袭和远处转移,而对照组肿瘤明显向肾外侵袭性生长,多个脏器出现转移病灶。此外,带有miR-141的慢病毒感染肾癌细胞,可增加培养液中胞外miR-141的表达水平；胞外miR-141可进入受体肾癌细胞内,抑制受体细胞的迁移侵袭能力而对细胞增殖凋亡无明显影响。786-0和SN12-PM6细胞的胞外/胞内miR-141比值分别低于2.2%和0.01%。结论：通过抑制肾癌细胞的增殖和转移,miR-141可作为肾癌重要的抑癌基因。miR-141可分泌入细胞外,发挥细胞间的信息传递作用,但这种作用有限。第三部分在肾癌组织中高表达的EphA2是miR-141的一个新靶点目的：探索miR-141在肾癌中发挥抑癌作用的具体机制。方法：用Targetscan.miRanda、PicTar等多个靶点预测软件筛选miR-141的靶点,构建EphA23'UTR野生型和突变型荧光素酶报告基因,qRT-PCR、Western blot、IHC等方法在体外细胞水平、体内临床标本、裸鼠肾肿瘤标本分析EphA2和miR-141的表达水平。将EphA2siRNA转染肾癌细胞,分析敲除EphA2与过表达miR-141对肾癌细胞生物学功能影响的相似性。将EphA2siRNA和miR-141抑制剂共转染入肾癌细胞,设计“拯救”实验。查阅文献寻找与EphA2结构和功能相关的基因,Vestern blot分析过表达miR-141和敲除EphA2对肾癌细胞中FAK、p-FAK、AKT、p-AKT、MMP-2、MMP-9蛋白表达的影响。结果：多个靶点软件均预测到miR-141种子序列与EphA23'UTR相结合。双荧光素酶报告基因结果显示,过表达miR-141明显降低EphA23'UTR野生型的荧光素酶活性,而对突变型无明显影响。过表达miR-141的细胞和裸鼠肾肿瘤标本中的EphA2mRNA和蛋白水平明显下调。尽管ccRCC癌组织与癌旁正常组织中EphA2mRNA的表达水平无明显差异,但癌组织中EphA2mRNA与：miR-141的表达水平呈明显负相关(Pearson相关分析,R2=0.3661,p=0.0047)。更值得关注的是,EphA2蛋白在85%(17/20)的ccRCC中高表达。而且,敲除EphA2显著削弱肾癌细胞的增殖、迁移侵袭能力,阻滞细胞周期在Go/G1期,其作用与过表达miR-141的类似。miR-141抑制剂可以通过增加EphA2的表达增强肾癌细胞的迁移侵袭能力,该增强作用可被EphA2siRNA所中和。以往研究表明FAK和AKT是两个与EphA2结构和功能密切相关的基因,MMP-2和MMP-9是FAK和AKT的下游信号基因。据此,我们推测p-FAK/p-AKT/MMPs是miR-141-EphA2发挥抗癌作用的“效应器”。Western blot显示,过表达miR-141和敲除EphA2均能充分抑制肾癌细胞的p-FAK、p-AKT、MMP-2和MMP-9的表达,且miR-141抑制剂可增加FAK和AKT的磷酸化水平,该增加作用可被EphA2siRNA中和。结论：EphA2是miR-141的一个具有功能的直接靶点。EphA2在肾癌组织中高表达可能是由miR-141的缺失所致。miR-141-EphA2抑制肿瘤活性可能是通过p-FAK/p-AKT/MMPs通路实现的。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

【共引文献】