本文選題：CASC2 + 腎細(xì)胞癌　； 參考：《蘇州大學(xué)》2016年博士論文

【摘要】：腎細(xì)胞癌(renal cell carcinoma,RCC)是泌尿系統(tǒng)最常見(jiàn)的惡性腫瘤之一,發(fā)病率占全身惡性腫瘤的3%,透明細(xì)胞腎細(xì)胞癌(clear cell renal cell carcinoma,cc RCC)和乳頭狀腎細(xì)胞癌是最常見(jiàn)的RCC類型,在腎癌中的比例分別為60%~85%和7%~14%。RCC其起源于腎小管上皮細(xì)胞。局限性腎癌患者中約有30%會(huì)在術(shù)后出現(xiàn)局部復(fù)發(fā)或遠(yuǎn)處轉(zhuǎn)移,是導(dǎo)致患者死亡的主要原因,而腎癌發(fā)生復(fù)發(fā)、轉(zhuǎn)移的機(jī)制尚不清楚。癌易感性候選基因2(cancer susceptibility candidate 2,CASC2)是屬于長(zhǎng)鏈非編碼RNA(long non?coding RNA,lnc RNA),定位于染色體的10q26,最初發(fā)現(xiàn)CASC2在子宮內(nèi)膜癌中表達(dá)下調(diào),起到抑癌基因的作用,外源性上調(diào)CASC2的表達(dá)可以顯著抑制未分化的子宮內(nèi)膜癌細(xì)胞生長(zhǎng),CASC2還可以抑制膠質(zhì)瘤細(xì)胞的轉(zhuǎn)移,但是,CASC2在RCC中的表達(dá)情況及其功能尚不清楚。微小RNA(micro RNA,miRNA)也是非編碼RNA分子,其在細(xì)胞的增殖、轉(zhuǎn)移、侵襲、凋亡和細(xì)胞周期等生物進(jìn)程中發(fā)揮重要的調(diào)節(jié)作用,有研究表明lnc RNA是新的miRNA作用靶點(diǎn),本研究檢測(cè)了CASC2在RCC組織、癌旁正常腎組織及腎癌細(xì)胞株中的表達(dá)水平,發(fā)現(xiàn)CASC2在RCC的表達(dá)明顯下降,起到抑癌基因作用;miRNA-21(miR-21)以順序特異性的方式下調(diào)CASC2的表達(dá);通過(guò)miR-21調(diào)節(jié)CASC2表達(dá)可以部分消除CASC2介導(dǎo)的腫瘤抑制作用。因此,CASC2可能是RCC新的預(yù)后標(biāo)志物和有效的治療靶點(diǎn)。第一部分CASC2在腎細(xì)胞癌中的表達(dá)及意義目的:研究RCC癌組織及癌旁組織中CASC2的表達(dá),不同RCC細(xì)胞株中CASC2的表達(dá),探討CASC2在RCC的可能作用。方法:采用實(shí)時(shí)定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(Real-time Quantitative PCR,q PCR)檢測(cè)32例癌組織和癌旁組織中CASC2的表達(dá)水平。根據(jù)病理分期,患者預(yù)后,分析其相關(guān)性。檢測(cè)RCC細(xì)胞株786?O、A498和人胚胎腎細(xì)胞(HEK 293)中CASC2基因表達(dá)情況,并對(duì)其進(jìn)行比較和相關(guān)分析。結(jié)果:q PCR結(jié)果顯示:RCC組織與相應(yīng)癌旁正常腎組織中的CASC2表達(dá)有差異,在RCC組織中CASC2的表達(dá)明顯均低于癌旁組織,差異具有統(tǒng)計(jì)學(xué)意義;分析了CASC2表達(dá)水平與病理分期的關(guān)系:發(fā)現(xiàn)CASC2低表達(dá)的比例在低分期組(p T1+p T2)中較低,高分期組(p T3+p T4)較高,分別是44.8%(13/29)和66.7%(2/3);Kaplan-Meir生存曲線分析發(fā)現(xiàn)CASC2高表達(dá)組患者的預(yù)后明顯優(yōu)于CASC2低表達(dá)組,具有統(tǒng)計(jì)學(xué)意義。CASC2在RCC細(xì)胞株786?O、A498中的表達(dá)明顯低于HEK293細(xì)胞株,差異具有統(tǒng)計(jì)學(xué)意義。結(jié)論:RCC組織和RCC細(xì)胞株中CASC2的表達(dá)明顯降低,表明CASC在腎癌中起到抑癌基因的作用。隨著腫瘤進(jìn)展分期升高,CASC2低表達(dá)的比例越高,CASC2低表達(dá)組患者預(yù)后不佳,CASC2低表達(dá)可能是患者預(yù)后不佳的標(biāo)志物。第二部分:調(diào)控CASC2表達(dá)水平對(duì)腎細(xì)胞癌增殖和遷移的影響目的:通過(guò)檢測(cè)上調(diào)CASC2表達(dá)對(duì)RCC細(xì)胞增殖、遷移能力的影響,驗(yàn)證CASC2在RCC中的作用。方法:構(gòu)建高表達(dá)質(zhì)粒pc DNA3.1(+)-CASC2;通過(guò)高表達(dá)質(zhì)粒pc DNA3.1(+)-CASC2轉(zhuǎn)染上調(diào)CASC2表達(dá)后,使用MTT法和細(xì)胞劃痕實(shí)驗(yàn),觀察786-O和A498細(xì)胞株的增殖、遷移是否受影響。結(jié)果:pc DNA3.1(+)-CASC2過(guò)表達(dá)質(zhì)粒轉(zhuǎn)染后,786-O和A498細(xì)胞的OD值分析表明細(xì)胞增殖明顯減少;同樣,過(guò)表達(dá)CASC2可以明顯抑制786-O和A498細(xì)胞的遷移能力。結(jié)論:上調(diào)CASC2的表達(dá)水平可以抑制RCC細(xì)胞的增殖和遷移,CASC2有腫瘤抑制作用。第三部分:miR-21可特異性結(jié)合CASC2并調(diào)控其功能目的:本研究通過(guò)生物信息學(xué)分析尋找CASC2的關(guān)聯(lián)miRNA,確認(rèn)其與CASC2特異性結(jié)合能力和靶向調(diào)控作用。方法:根據(jù)miRanda(http://www.microrna.org)和miRcode(http://www.mircode.org)數(shù)據(jù)庫(kù),通過(guò)計(jì)算機(jī)輔助得出CASC2具有潛在的miR-21結(jié)合位點(diǎn)。用miR-21 mimic模擬miR-21高表達(dá),雙熒光素酶報(bào)告分析786-O和A498細(xì)胞株中miR-21高表達(dá)對(duì)CASC2表達(dá)水平的影響,進(jìn)而檢測(cè)CASC2對(duì)786-O和A498的腫瘤抑制作用是否受miR-21干擾影響。結(jié)果:通過(guò)生物信息學(xué)分析CASC2具有miR-21的結(jié)合位點(diǎn)。雙熒光素酶報(bào)告基因分析確認(rèn)在CASC2-WT質(zhì)粒轉(zhuǎn)染組,miR-21高表達(dá)可以明顯抑制相應(yīng)的熒光活性,而在突變組(CASC2-MUT)miR-21的未有這種抑制作用,因此CASC2與miR-21結(jié)合是特異性的。miR-21高表達(dá)后,786-O和A498細(xì)胞株的CASC2表達(dá)水平下降,CASC2對(duì)786-O和A498細(xì)胞株的增殖與遷移的抑制作用也被部分消除。結(jié)論:CASC2具有miR-21的結(jié)合位點(diǎn),miR-21可特異性結(jié)合CASC2,部分消除CASC2對(duì)RCC細(xì)胞株增殖與遷移的抑制作用。CASC2可能還有其他途徑實(shí)現(xiàn)腫瘤抑制功能。
[Abstract]:Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. The incidence of renal cell carcinoma is 3% of the malignant tumor of the whole body. The most common type of RCC is clear cell renal cell carcinoma (clear cell renal cell carcinoma, CC RCC) and papillary renal cell carcinoma. Tubule epithelial cells. About 30% of the patients with localized renal carcinoma may have local recurrence or distant metastasis after operation. It is the main cause of death. The mechanism of the recurrence of renal cancer is not clear. The candidate gene for cancer susceptibility 2 (cancer susceptibility candidate 2, CASC2) is a long chain non coded RNA (long non? Coding RNA,) LNC RNA), located in the chromosome 10q26, it was found that CASC2 was down regulated in endometrial carcinoma and played a role in the tumor suppressor gene. Exogenous up regulation of CASC2 could significantly inhibit the growth of undifferentiated endometrial cancer cells, and CASC2 could inhibit the transfer of glioma cells. However, the expression of CASC2 in RCC and its function still remained. It is not clear that RNA (micro RNA, miRNA) is also a non coding RNA molecule, which plays an important regulatory role in cell proliferation, metastasis, invasion, apoptosis and cell cycle and other biological processes. Studies have shown that LNC RNA is a new target for miRNA action. This study detected CASC2 in RCC tissues, normal renal tissue and renal cell carcinoma cell lines. It was found that the expression of CASC2 decreased significantly in RCC and played a role in the tumor suppressor gene; miRNA-21 (miR-21) downregulated the expression of CASC2 in a sequential specific way; the regulation of CASC2 expression through miR-21 could partially eliminate the tumor inhibitory effect of CASC2 mediated. Therefore, CASC2 may be a new prognostic marker and effective target for the treatment of RCC. The expression of CASC2 in renal cell carcinoma and its purpose: To study the expression of CASC2 in the tissues of RCC and the para cancer tissues, the expression of CASC2 in different RCC cell lines, and to explore the possible role of CASC2 in RCC. Methods: 32 cases of cancer tissues and adjacent tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (Real-time Quantitative PCR, Q PCR). The expression level of C2. According to the pathological stage and the prognosis of the patients, the correlation was analyzed. The expression of CASC2 gene in the RCC cell line 786? O, A498 and human embryonic kidney cells (HEK 293) was detected and compared and analyzed. Results: Q PCR results showed that the RCC tissue was different from the CASC2 expression in the normal renal tissue adjacent to the corresponding cancerous tissues, and the CA was in RCC tissue. The expression of SC2 was significantly lower than that of the paracancerous tissue, and the difference was statistically significant. The relationship between the level of CASC2 expression and pathological staging was analyzed. The low expression of CASC2 was found to be lower in the low stage group (P T1+p T2) and higher in the high staging group (P T3+p T4), which was 44.8% (13/29) and 66.7% (2/3), respectively. Kaplan-Meir survival curve analysis found high expression. The prognosis of the group was significantly better than that of the CASC2 low expression group, with statistical significance.CASC2 in RCC cell line 786? O, and the expression in A498 was significantly lower than that of HEK293 cell lines. The difference was statistically significant. Conclusion: the expression of CASC2 in RCC tissue and RCC cell lines decreased obviously, indicating that CASC plays the role of tumor suppressor gene in renal carcinoma. The higher the stage, the higher the proportion of CASC2 low expression, the poor prognosis in the CASC2 low expression group, the low expression of CASC2 may be the marker of poor prognosis. Second part: the effect of regulating CASC2 expression level on the proliferation and migration of renal cell carcinoma: the effect of up regulation of CASC2 expression on RCC cell proliferation and migration ability, and to verify CASC2 in CASC2 Method: Construction of high expression plasmid PC DNA3.1 (+) -CASC2; after transfection of high expression plasmid PC DNA3.1 (+) -CASC2 to up regulation of CASC2 expression, MTT method and cell scratch test were used to observe the proliferation of 786-O and A498 cell lines and whether the migration of CASC2 was affected. Analysis showed that cell proliferation decreased obviously; similarly, overexpression of CASC2 could significantly inhibit the migration of 786-O and A498 cells. Conclusion: up regulation of CASC2 expression can inhibit the proliferation and migration of RCC cells, CASC2 has tumor inhibition. The third part: miR-21 can specifically bind CASC2 and regulate its functional purpose: This study through Biology Informatics analysis seeks the associated miRNA of CASC2 to confirm its specific binding ability to CASC2 and target regulation. Methods: according to the database of miRanda (http://www.microrna.org) and miRcode (http://www.mircode.org), the potential miR-21 binding site of CASC2 is obtained by computer assistance. MiR-21 mimic is used to simulate miR-21 high expression, double fluorescent. The effect of miR-21 high expression on the level of CASC2 expression in 786-O and A498 cells was analyzed, and the effect of CASC2 on the tumor inhibition of 786-O and A498 was detected by miR-21 interference. The high expression of miR-21 could obviously inhibit the corresponding fluorescence activity, but there was no inhibition in the mutant group (CASC2-MUT) miR-21, so the combination of CASC2 and miR-21 was the specific.MiR-21 high expression, the CASC2 expression level of 786-O and A498 cells decreased, and the inhibition of CASC2 to the proliferation and migration of 786-O and A498 cell lines was also inhibited. Partial elimination. Conclusion: CASC2 has miR-21 binding site, miR-21 can specifically bind to CASC2, partially eliminate the inhibitory effect of CASC2 on proliferation and migration of RCC cell lines,.CASC2 may have other ways to achieve tumor inhibition.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王淼;黃韜;羅剛;黃超;肖行遠(yuǎn);汪良;蔣國(guó)松;曾甫清;;Long Non-coding RNA MEG3 Induces Renal Cell Carcinoma Cells Apoptosis by Activating the Mitochondrial Pathway[J];Journal of Huazhong University of Science and Technology(medical Sciences);2015年04期

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本文編號(hào)：1972260