MiR-429负反馈调控CRKL抑制肾透明细胞癌发生及其作用机制研究

发布时间：2018-04-26 09:20

本文选题：肾透明细胞癌 + CRKL　；参考：《大连医科大学》2017年硕士论文

【摘要】：肾细胞癌(Renal cell carcinoma,RCC)是常见的恶性肿瘤,约占人类恶性肿瘤的3%,肾细胞癌分四类,肾透明细胞癌(clear cell renal carcinoma,ccRCC),乳头状肾癌(papillary carcinoma)和嫌色肾细胞癌(chromophobe renal carcinoma)。肾透明细胞癌是最常见的,也极具侵略性的肾细胞癌,手术治疗仍是目前最有效的治疗方案,但术后复发的患者约占20%~40%,因此研究肾癌发生发展中的分子水平变化及调控机制,并提供肾癌诊治标志物,意义重大。CRK最初作为一种癌基因产物发现于禽类肉瘤病毒CT10(chicken tumor 10)中,主要由SH2(src homology 2)和SH3(src homology 3)结构域组成。CRK家族包括三个成员:CRKI、CRKII和CRKL,在各个组织中广泛表达。近年来的研究表明:CRKL异常表达与肿瘤生物学行为密切相关,主要通过参与调控细胞骨架重组,信号传导等。然而对于CRKL与肾透明细胞癌(ccRCC)发病机制及可能参与的信号通路的研究仍是一片空白,这值得我们进行探究。MicroRNAs(miRNAs)是一类包含18-24个碱基的高度保守的单链非编码RNA。miRNAs通过与AGO2蛋白结合形成RNA诱导沉默复合体(RNA-induced silencing complex,RISC),进而结合靶基因mRNA的3’-UTR区引发靶基因mRNA降解或抑制其翻译过程。MiR-200家族包括miR-141,200c,200a,200b,和429,这类mi RNA最早被发现参与犬肾细胞的上皮细胞间质化的过程(Epithelial to mesenchymal transition,EMT),后续研究同时发现其在多种癌症中作为肿瘤抑制因子参与调控肿瘤转移。MiR-429作为miR-200家族一员在肾癌中的研究仍然较少,因此针对mir-429进一步探求其可能潜在的其他靶点,深入研究其参与肾透明细胞癌转移的潜在机制。在targetscan,mirdb等生物信息学软件上均发现mir-429与crkl存在靶向关系,因此mir-429与crkl的二者的调控关系在肾透明细胞癌发生发展中的相互作用值得深入探究。目的:1.crkl水平与肾透明细胞癌和786-o细胞的关系探究;2.mir-429水平与肾透明细胞癌和786-o细胞的关系探究;3.明确二者之间的靶向结合以及相互调控关系。方法:1.qrt-pcr和wb检测肾透明细胞癌临床样本中mir-429与crkl的相对表达量;2.利用lipofectaminetm2000介导法转染mir-429mimic/mir-429inhibitor以及阴性对照ncmimic/ncinhibitor,并用qrt-pcr检测转染效率;3.transwell小室测定mir-429表达变化对786-o细胞迁移和侵袭能力的影响;4.利用lipofectaminetm2000介导法转染sir-crkl-pool/pcdh-crkl以及阴性对照sir-nc-pool/pcdh-nc,并用wb检测转染效率;5.transwell小室测定crkl表达变化对786-o细胞迁移和侵袭能力的影响;6.双荧光素酶载体psicheck-mutcrkl-site1、psicheck-mutcrkl-site2、psicheck-wtcrkl-site1、psicheck-wtcrkl-site2及psicheck-wtcrkl-site1+site2构建,并检测mir-429对crkl的靶向作用;7.共转染crklcds区与mir-429检测mir-429是否还能具有调控作用;8.tgf-β诱导实验检测mir-429对内源性crkl调控作用;9.ip和wb检测crkl结合调控蛋白分子;10、鬼笔环肽染色检测细胞骨架构象改变。结果:1.28例肾透明细胞癌临床样本检测中,25例样本的mir-429呈现低表达,23例样本的crkl呈现高表达,二者之间具有负相关的关系;2.qrt-pcr与transwell小室法表明mir-429水平与786-o细胞迁移侵袭能力负相关;3.wb与transwell小室法证明crkl水平与细胞迁移侵袭能力正相关;4.成功构建双荧光素酶载体psicheck-mutcrkl-site1、psicheck-mutcrkl-site2、psicheck-wtcrkl-site1、psicheck-wtcrkl-site2及psicheck-wtcrkl-site1+site2,并测序正确;5.双荧光素酶报告试验检测发现mir-429可以靶向调控crkl3’-utr区;6.共转染crklcds区与mir-429证明mir-429仅调控crkl3’-utr区;7.tgf-β诱导实验发现mir-429能阻断tgf-β对786-o细胞内源性的crkl诱导作用;8.ip实验发现CRKL可以通过和CRKⅡ相互结合作用;9.鬼笔环肽染色发现miR-429增加会减弱细胞骨架的形成。结论:1.miR-429与CRKL的表达关系在肾透明细胞癌样本中呈现负相关;2.miR-429水平与786-O细胞的迁移侵袭能力负相关,CRKL水平与786-O细胞的迁移侵袭能力正相关;3.miR-429能靶向调控CRKL 3’-UTR,并且两个靶向位点中CRKL 3’-UTR 3728-3735区是主要调控位点;4.miR-429能抑制786-O细胞中TGF-β对CRKL的激活以及迁移侵袭能力的促进作用;5.miR-429通过抑制CRKL表达,影响CRKL和CRKⅡ相互结合作用,阻碍细胞的运动能力。
[Abstract]:Renal cell carcinoma (RCC) is a common malignant tumor, accounting for about 3% of human malignant tumors, four types of renal cell carcinoma, clear cell carcinoma of the kidney (clear cell renal carcinoma, ccRCC), papillary renal cell carcinoma (papillary carcinoma) and chromophobe renal cell carcinoma (chromophobe). Invasive renal cell carcinoma (RCC) is still the most effective treatment, but the postoperative recurrence is about 20%~40%. Therefore, the changes in molecular level and regulation mechanism in the development of renal carcinoma are studied, and the diagnosis and treatment marker of renal cancer is provided. The significance of.CRK was found at first as a kind of oncogene product in avian sarcoma virus CT10 (chick EN tumor 10) consists mainly of the SH2 (SRC homology 2) and SH3 (SRC homology 3) domains that comprise the.CRK family including three members: CRKI, CRKII and CRKL, which are widely expressed in various tissues. Recent studies have shown that abnormal expression is closely related to the biological behavior of tumor, mainly by regulating cytoskeleton recombination and signal transduction. The study of the pathogenesis of CRKL and renal clear cell carcinoma (ccRCC) and the possible signaling pathway is still a blank. It is worth exploring that.MicroRNAs (miRNAs) is a highly conservative, single chain non coding RNA.miRNAs containing 18-24 bases, which combines with AGO2 egg white to form a RNA induced silence complex (RNA-induced sil). Encing complex, RISC), and then combined with the 3 '-UTR region of the target gene mRNA, triggering the degradation of the target gene mRNA or the inhibition of the translation process of the.MiR-200 family including miR-141200c, 200A, 200B, and 429. This class mi RNA was found to be involved in the process of epithelial cell homogenization of the canine renal cells. It is found that it is involved in the regulation of tumor metastasis as a tumor suppressor in various cancers as a member of the.MiR-429 family as a member of the miR-200 family in renal cancer. Therefore, the potential potential other targets for mir-429 are further explored and the potential mechanism of its involvement in the transfer of renal clear cell carcinoma. In targetscan, mirdb and other biological letters It is found that the relationship between mir-429 and CRKL has a target relationship, so the interaction between the two of mir-429 and CRKL in the development of renal clear cell carcinoma should be deeply explored. Objective: the relationship between the level of 1.crkl and the renal clear cell carcinoma and 786-o cells; the relationship between the level of 2.mir-429 and the renal clear cell carcinoma and 786-o cells. 1.qrt-pcr and WB were used to detect the relative expression of mir-429 and CRKL in the clinical samples of renal clear cell carcinoma; 2. by lipofectaminetm2000 mediated transfection of mir-429mimic/mir-429inhibitor and negative ncmimic/ncinhibitor, and qRT-PCR was used to detect transfection. Efficiency; the effect of mir-429 expression on the migration and invasion ability of 786-o cells in 3.transwell cells; 4. transfection of sir-crkl-pool/pcdh-crkl and negative control sir-nc-pool/pcdh-nc by lipofectaminetm2000 mediated method, and WB detection efficiency; 5.transwell chamber was used to determine the migration and invasion ability of CRKL expression to 786-o cells. The 6. double luciferase vector psicheck-mutcrkl-site1, psicheck-mutcrkl-site2, psicheck-wtcrkl-site1, psicheck-wtcrkl-site2 and psicheck-wtcrkl-site1+site2 were constructed, and the targeting effect of mir-429 on CRKL was detected. 7. whether the co transfection of crklcds region and mir-429 detected mir-429 could be regulated by mir-429; 8.tgf- beta induction test The regulatory role of mir-429 on endogenous CRKL; 9.ip and WB detection of CRKL binding protein molecules; 10, cytoskeleton conformation changes. Results: in the detection of 1.28 cases of renal clear cell carcinoma, 25 samples of mir-429 showed low expression, CRKL in 23 samples showed high expression, and the relationship between the two was negative correlation; 2.qrt -pcr and Transwell chamber method showed that mir-429 level was negatively related to migration and invasion of 786-o cells; 3.wb and Transwell chamber method showed that CRKL level was positively related to cell migration and invasion; 4. the successful construction of dual luciferase carrier psicheck-mutcrkl-site1, psicheck-mutcrkl-site2, psicheck-wtcrkl-site1, psicheck-wtcrkl-site2 and psicheck. -wtcrkl-site1+site2, and sequenced correctly; 5. double Luciferase Report test found that mir-429 could target crkl3 '-utr region; 6. co transfection of crklcds region and mir-429 showed that mir-429 only regulated crkl3' -utr region; 7.tgf- beta induced experimental discovery that mir-429 could block the inducement of tgf- beta. It can be combined with CRK II; 9. phallo cyclic peptide staining found that the increase of miR-429 would weaken the formation of cytoskeleton. Conclusion: the relationship between the expression of 1.miR-429 and CRKL is negative in the samples of renal clear cell carcinoma; the level of 2.miR-429 is negatively related to the migration and invasion ability of 786-O cells, and the CRKL level and the migration and invasion ability of 786-O cells Positive correlation; 3.miR-429 can target CRKL 3 '-UTR, and CRKL 3' 3728-3735 region is the main regulatory site in the two target loci; 4.miR-429 can inhibit the activation of TGF- beta in 786-O cells and the promoting effect of migration and invasion. 5.miR-429 inhibits the interaction between CRKL and mineral II and hinders the cell by inhibiting the CRKL table. The ability to exercise.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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