發(fā)布時間：2018-04-26 09:20

  本文選題：腎透明細(xì)胞癌 + CRKL��； 參考：《大連醫(yī)科大學(xué)》2017年碩士論文

【摘要】：腎細(xì)胞癌(Renal cell carcinoma,RCC)是常見的惡性腫瘤,約占人類惡性腫瘤的3%,腎細(xì)胞癌分四類,腎透明細(xì)胞癌(clear cell renal carcinoma,ccRCC),乳頭狀腎癌(papillary carcinoma)和嫌色腎細(xì)胞癌(chromophobe renal carcinoma)。腎透明細(xì)胞癌是最常見的,也極具侵略性的腎細(xì)胞癌,手術(shù)治療仍是目前最有效的治療方案,但術(shù)后復(fù)發(fā)的患者約占20%~40%,因此研究腎癌發(fā)生發(fā)展中的分子水平變化及調(diào)控機(jī)制,并提供腎癌診治標(biāo)志物,意義重大。CRK最初作為一種癌基因產(chǎn)物發(fā)現(xiàn)于禽類肉瘤病毒CT10(chicken tumor 10)中,主要由SH2(src homology 2)和SH3(src homology 3)結(jié)構(gòu)域組成。CRK家族包括三個成員:CRKI、CRKII和CRKL,在各個組織中廣泛表達(dá)。近年來的研究表明:CRKL異常表達(dá)與腫瘤生物學(xué)行為密切相關(guān),主要通過參與調(diào)控細(xì)胞骨架重組,信號傳導(dǎo)等。然而對于CRKL與腎透明細(xì)胞癌(ccRCC)發(fā)病機(jī)制及可能參與的信號通路的研究仍是一片空白,這值得我們進(jìn)行探究。MicroRNAs(miRNAs)是一類包含18-24個堿基的高度保守的單鏈非編碼RNA。miRNAs通過與AGO2蛋白結(jié)合形成RNA誘導(dǎo)沉默復(fù)合體(RNA-induced silencing complex,RISC),進(jìn)而結(jié)合靶基因mRNA的3’-UTR區(qū)引發(fā)靶基因mRNA降解或抑制其翻譯過程。MiR-200家族包括miR-141,200c,200a,200b,和429,這類mi RNA最早被發(fā)現(xiàn)參與犬腎細(xì)胞的上皮細(xì)胞間質(zhì)化的過程(Epithelial to mesenchymal transition,EMT),后續(xù)研究同時發(fā)現(xiàn)其在多種癌癥中作為腫瘤抑制因子參與調(diào)控腫瘤轉(zhuǎn)移。MiR-429作為miR-200家族一員在腎癌中的研究仍然較少,因此針對mir-429進(jìn)一步探求其可能潛在的其他靶點,深入研究其參與腎透明細(xì)胞癌轉(zhuǎn)移的潛在機(jī)制。在targetscan,mirdb等生物信息學(xué)軟件上均發(fā)現(xiàn)mir-429與crkl存在靶向關(guān)系,因此mir-429與crkl的二者的調(diào)控關(guān)系在腎透明細(xì)胞癌發(fā)生發(fā)展中的相互作用值得深入探究。目的:1.crkl水平與腎透明細(xì)胞癌和786-o細(xì)胞的關(guān)系探究;2.mir-429水平與腎透明細(xì)胞癌和786-o細(xì)胞的關(guān)系探究;3.明確二者之間的靶向結(jié)合以及相互調(diào)控關(guān)系。方法:1.qrt-pcr和wb檢測腎透明細(xì)胞癌臨床樣本中mir-429與crkl的相對表達(dá)量;2.利用lipofectaminetm2000介導(dǎo)法轉(zhuǎn)染mir-429mimic/mir-429inhibitor以及陰性對照ncmimic/ncinhibitor,并用qrt-pcr檢測轉(zhuǎn)染效率;3.transwell小室測定mir-429表達(dá)變化對786-o細(xì)胞遷移和侵襲能力的影響;4.利用lipofectaminetm2000介導(dǎo)法轉(zhuǎn)染sir-crkl-pool/pcdh-crkl以及陰性對照sir-nc-pool/pcdh-nc,并用wb檢測轉(zhuǎn)染效率;5.transwell小室測定crkl表達(dá)變化對786-o細(xì)胞遷移和侵襲能力的影響;6.雙熒光素酶載體psicheck-mutcrkl-site1、psicheck-mutcrkl-site2、psicheck-wtcrkl-site1、psicheck-wtcrkl-site2及psicheck-wtcrkl-site1+site2構(gòu)建,并檢測mir-429對crkl的靶向作用;7.共轉(zhuǎn)染crklcds區(qū)與mir-429檢測mir-429是否還能具有調(diào)控作用;8.tgf-β誘導(dǎo)實驗檢測mir-429對內(nèi)源性crkl調(diào)控作用;9.ip和wb檢測crkl結(jié)合調(diào)控蛋白分子;10、鬼筆環(huán)肽染色檢測細(xì)胞骨架構(gòu)象改變。結(jié)果:1.28例腎透明細(xì)胞癌臨床樣本檢測中,25例樣本的mir-429呈現(xiàn)低表達(dá),23例樣本的crkl呈現(xiàn)高表達(dá),二者之間具有負(fù)相關(guān)的關(guān)系;2.qrt-pcr與transwell小室法表明mir-429水平與786-o細(xì)胞遷移侵襲能力負(fù)相關(guān);3.wb與transwell小室法證明crkl水平與細(xì)胞遷移侵襲能力正相關(guān);4.成功構(gòu)建雙熒光素酶載體psicheck-mutcrkl-site1、psicheck-mutcrkl-site2、psicheck-wtcrkl-site1、psicheck-wtcrkl-site2及psicheck-wtcrkl-site1+site2,并測序正確;5.雙熒光素酶報告試驗檢測發(fā)現(xiàn)mir-429可以靶向調(diào)控crkl3’-utr區(qū);6.共轉(zhuǎn)染crklcds區(qū)與mir-429證明mir-429僅調(diào)控crkl3’-utr區(qū);7.tgf-β誘導(dǎo)實驗發(fā)現(xiàn)mir-429能阻斷tgf-β對786-o細(xì)胞內(nèi)源性的crkl誘導(dǎo)作用;8.ip實驗發(fā)現(xiàn)CRKL可以通過和CRKⅡ相互結(jié)合作用;9.鬼筆環(huán)肽染色發(fā)現(xiàn)miR-429增加會減弱細(xì)胞骨架的形成。結(jié)論:1.miR-429與CRKL的表達(dá)關(guān)系在腎透明細(xì)胞癌樣本中呈現(xiàn)負(fù)相關(guān);2.miR-429水平與786-O細(xì)胞的遷移侵襲能力負(fù)相關(guān),CRKL水平與786-O細(xì)胞的遷移侵襲能力正相關(guān);3.miR-429能靶向調(diào)控CRKL 3’-UTR,并且兩個靶向位點中CRKL 3’-UTR 3728-3735區(qū)是主要調(diào)控位點;4.miR-429能抑制786-O細(xì)胞中TGF-β對CRKL的激活以及遷移侵襲能力的促進(jìn)作用;5.miR-429通過抑制CRKL表達(dá),影響CRKL和CRKⅡ相互結(jié)合作用,阻礙細(xì)胞的運動能力。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張秀真,吳澤志;細(xì)胞偽足形成與微絲骨架研究進(jìn)展[J];國外醫(yī)學(xué).臨床生物化學(xué)與檢驗學(xué)分冊;2005年04期

，

本文編號：1805406