【作者】 陈智勇；

【导师】 齐琳；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：研究肾透明细胞癌(ccRCC)患者血循环microRNA的表达,初步建立肾透明细胞癌特征性的循环microRNA表达谱。方法：实验组共收集2008年8月到2009年6月期间肾透明细胞癌20例。其中14例接受根治性肾切除术,5例接受保留肾单位肾部分切除术,1例行肾穿刺活检。所有患者均经病理检查证实为肾透明细胞癌。AJCC肾癌临床分期：Ⅰ期8例,Ⅱ期3例,Ⅲ期5例,Ⅳ期4例。对照组20例为门诊体检健康人群。所有对照组均排除了肾脏疾病和其他部位肿瘤。实验组和对照组均于清晨空腹抽血,在无菌条件下操作,离心吸取血浆,再放入-80℃冰箱保存。挑选实验组和对照组血浆样本各8例,将每组的8例样本等体积混合,取混合后每组血浆2ml,用mirVana PARIS试剂盒分别抽提实验组和对照组的总RNA。测定浓度后使用TaqMan MicroRNA低密度芯片分别检测实验组和对照组的microRNA谱。使用SDS 2.3软件分析TLDA芯片数据；采用SPSS15.0软件进一步统计分析。对比分析两者之间microRNA表达的差异。结果：在肾透明细胞癌组和对照组的血浆中共检测到83种microRNA分子。其中ccRCC组73种表达阳性,对照组55种表达阳性。根据判断标准,我们找到48种在ccRCC组中表达显著上调的microRNA,其中有24种microRNA为肾透明细胞癌组特异性表达,24种表达明显上调。12种microRNA在肾透明细胞癌组中表达下调,其中10种为无表达,2种为表达明显下调。结论：[1]肾透明细胞癌患者血循环microRNA较正常人群有特征性改变。[2]肾透明细胞癌患者血microRNA改变以表达上调为主,这些microRNA改变可以用来区分肾透明细胞癌和正常人群,初步建立了肾透明细胞癌的循环microRNA表达谱。目的：验证miR-21,miR-142-5p和miR-34a在肾透明细胞癌患者血浆中的表达增加,分析其增加的程度与临床分期之间的关系。方法：实验对象同第一部分,包含全部实验组和对照组各20例。我们利用芯片结果结合相关文献筛选出miR-21, miR-142-5p, miR-34a三个microRNA分子。分别抽提实验组和对照组血浆总RNA,通过northern-blot和qRT-PCR对其进行检测。同时分别提取20例不同分期肾癌患者及正常对照血浆的总RNA,检测其miR-21,miR-142-5p和miR-34a在血浆中的表达程度,并分析这三种miRNA表达增加的程度与肾透明细胞癌临床分期之间的关系。结果：Northern-blot结果显示miR-21,miR-142-5p和miR-34a在肾癌患者血浆中表达明显上调。qRT-PCR验证发现,miR-21,miR-142-5p和miR-34a的溶解曲线很好,无非特异性峰。同时结果显示miR-21,miR-142-5p和miR-34a在肾癌患者血浆中表达明显上调,而在正常血浆中表达极低,与芯片结果一致。进一步的对比qRT-PCR检测显示miR-21和miR-142-5p表达的增加与肾透明细胞癌临床分期相关,而miR-34a的表达增加程度与临床分期无明显相关。结论：[1]循环miR-21和miR-142-5p在肾透明细胞癌患者表达增加,其增加的程度与临床分期可能相关。[2]循环miR-34a在肾透明细胞癌患者表达增加,其增加的程度与临床分期可能不相关。[3]循环miR-21,miR-142-5p和miR-34a可用于肾透明细胞癌的血清学诊断标志物。更多还原

【Abstract】 Objective:To investigate the express of microRNA(miRNA) in the blood of clear cell renal cell carcinoma (ccRCC) patients and establish the distinctive circulating miRNA spectrum for the diagnosis and treatment of ccRCC.Methods:The total study population included 20 ccRCC patients and 20 control subjects. Among the ccRCC group there were 14 patients received radical nephrectomy,5 received nephron-spare hemi nephrectomy,1 received renal biopsy. All patients were diagnosed with pathology confirm. AJCC clinical stage included 8 in stageⅠ,3 in stageⅡ,5 in stageⅢand 4 in stageⅣ. The control group was consisted of healthy people. No diabetes in both groups. The blood samples were drawn with empty stomach. The blood samples were centrifugated. Plasma was aspirated and then frozen under the-80℃condition. Eight samples were chosen from ccRCC group and the control group. The plasma of those two groups was mixed with identical volume respectively. 2 ml plasma from each group was used to extract the total RNA by mirVana PARIS kit. After the saturation evaluation and quality control, the total RNA of each group was examed by TaqMan MicroRNA low density assay(TLDA) respectively. The result was analyzed through SDS 2.3 software and SPSS 15.0 software.Results:There were totally 83 miRNAs detected in both groups. There were 73 miRNAs detected in the ccRCC group and 55 detected in control group.28 miRNAs detected only in the ccRCC group and 10 detected only in the control group. According the judgment standard, we found 48 miRNAs up-regulated in ccRCC group and among them,24 were specific expressed. There were 12 miRNAs down-regulated in the ccRCC group contrasted to the control group of which 10 miRNAs were undetectable.Conclusion:[1] The circulating miRNA of ccRCC patients has its specific express spectrum relative to healthy people. [2] The expression changes of circulating miRNA in ccRCC patients mainly are up-regulation, those up-regulated miRNAs can distinct ccRCC patients from healthy people. We preliminarily establish the circulating miRNA express spectrum of ccRCC patients. Objective:To validate the result that miR-21, miR-142-5p and miR-34a were up-regulated in the plasma of ccRCC patients and analyze their relationship to the clinical stages of ccRCC.Methods:All of the samples in ccRCC group and control group were examed in this part. The total RNA of both ccRCC group and control group were extracted and tested respectively. Three of the up-regulated miRNAs, miR-21, miR-142-5p and miR-34a, were validated firstly using northern blot and then qRT-PCR analysis. All the samples of ccRCC group and control group were randomly paired. The miR-21, miR-142-5p and miR-34a of each sample in the ccRCC group were tested using qRT-PCR, the relationship of those miRNAs to the clinical stage of ccRCC were analyzed.Results:Northern blot result revealed that miR-21, miR-142-5p and miR-34a were up-regulated in the plasma of ccRCC patients which confirmed the results of miRNA assay results. qRT-PCR validation showed that the solubility curves of those three miRNA were good and no specific peak was found. The result also showed that miR-21, miR-142-5p and miR-34a were up-regulated in the plasma of ccRCC patients. They had very low expression in the control group. The qRT-PCR test of ccRCC patients showed that the up-regulation degree of miR-21 and miR-142-5p in the plasma had strong relation to the clinical stage. But miR-34a had no statistical significance between the up-regulation degree and the clinical stages of ccRCC.Conclusion:[1] The expression of circulating miR-21 and miR-142-5p in the ccRCC patients were up-regulated and the degree of up-regulation was strongly related to the clinical stages of ccRCC. [2]The expression of circulating miR-34a also was up-regulated in the ccRCC patients but its degree had no significant relation to the clinical stages. [3]Circulating miR-21, miR-142-5p and miR-34a could be used as tumor biomarkers for ccRCC.更多还原