CASC2在肾细胞癌中的功能和调控机制研究

发布时间：2018-06-03 09:17

本文选题：CASC2 + 肾细胞癌　；参考：《苏州大学》2016年博士论文

【摘要】：肾细胞癌(renal cell carcinoma,RCC)是泌尿系统最常见的恶性肿瘤之一,发病率占全身恶性肿瘤的3%,透明细胞肾细胞癌(clear cell renal cell carcinoma,cc RCC)和乳头状肾细胞癌是最常见的RCC类型,在肾癌中的比例分别为60%~85%和7%~14%。RCC其起源于肾小管上皮细胞。局限性肾癌患者中约有30%会在术后出现局部复发或远处转移,是导致患者死亡的主要原因,而肾癌发生复发、转移的机制尚不清楚。癌易感性候选基因2(cancer susceptibility candidate 2,CASC2)是属于长链非编码RNA(long non?coding RNA,lnc RNA),定位于染色体的10q26,最初发现CASC2在子宫内膜癌中表达下调,起到抑癌基因的作用,外源性上调CASC2的表达可以显著抑制未分化的子宫内膜癌细胞生长,CASC2还可以抑制胶质瘤细胞的转移,但是,CASC2在RCC中的表达情况及其功能尚不清楚。微小RNA(micro RNA,miRNA)也是非编码RNA分子,其在细胞的增殖、转移、侵袭、凋亡和细胞周期等生物进程中发挥重要的调节作用,有研究表明lnc RNA是新的miRNA作用靶点,本研究检测了CASC2在RCC组织、癌旁正常肾组织及肾癌细胞株中的表达水平,发现CASC2在RCC的表达明显下降,起到抑癌基因作用;miRNA-21(miR-21)以顺序特异性的方式下调CASC2的表达;通过miR-21调节CASC2表达可以部分消除CASC2介导的肿瘤抑制作用。因此,CASC2可能是RCC新的预后标志物和有效的治疗靶点。第一部分CASC2在肾细胞癌中的表达及意义目的:研究RCC癌组织及癌旁组织中CASC2的表达,不同RCC细胞株中CASC2的表达,探讨CASC2在RCC的可能作用。方法:采用实时定量逆转录-聚合酶链反应(Real-time Quantitative PCR,q PCR)检测32例癌组织和癌旁组织中CASC2的表达水平。根据病理分期,患者预后,分析其相关性。检测RCC细胞株786?O、A498和人胚胎肾细胞(HEK 293)中CASC2基因表达情况,并对其进行比较和相关分析。结果:q PCR结果显示:RCC组织与相应癌旁正常肾组织中的CASC2表达有差异,在RCC组织中CASC2的表达明显均低于癌旁组织,差异具有统计学意义;分析了CASC2表达水平与病理分期的关系:发现CASC2低表达的比例在低分期组(p T1+p T2)中较低,高分期组(p T3+p T4)较高,分别是44.8%(13/29)和66.7%(2/3);Kaplan-Meir生存曲线分析发现CASC2高表达组患者的预后明显优于CASC2低表达组,具有统计学意义。CASC2在RCC细胞株786?O、A498中的表达明显低于HEK293细胞株,差异具有统计学意义。结论:RCC组织和RCC细胞株中CASC2的表达明显降低,表明CASC在肾癌中起到抑癌基因的作用。随着肿瘤进展分期升高,CASC2低表达的比例越高,CASC2低表达组患者预后不佳,CASC2低表达可能是患者预后不佳的标志物。第二部分:调控CASC2表达水平对肾细胞癌增殖和迁移的影响目的:通过检测上调CASC2表达对RCC细胞增殖、迁移能力的影响,验证CASC2在RCC中的作用。方法:构建高表达质粒pc DNA3.1(+)-CASC2;通过高表达质粒pc DNA3.1(+)-CASC2转染上调CASC2表达后,使用MTT法和细胞划痕实验,观察786-O和A498细胞株的增殖、迁移是否受影响。结果:pc DNA3.1(+)-CASC2过表达质粒转染后,786-O和A498细胞的OD值分析表明细胞增殖明显减少;同样,过表达CASC2可以明显抑制786-O和A498细胞的迁移能力。结论:上调CASC2的表达水平可以抑制RCC细胞的增殖和迁移,CASC2有肿瘤抑制作用。第三部分:miR-21可特异性结合CASC2并调控其功能目的:本研究通过生物信息学分析寻找CASC2的关联miRNA,确认其与CASC2特异性结合能力和靶向调控作用。方法:根据miRanda(http://www.microrna.org)和miRcode(http://www.mircode.org)数据库,通过计算机辅助得出CASC2具有潜在的miR-21结合位点。用miR-21 mimic模拟miR-21高表达,双荧光素酶报告分析786-O和A498细胞株中miR-21高表达对CASC2表达水平的影响,进而检测CASC2对786-O和A498的肿瘤抑制作用是否受miR-21干扰影响。结果:通过生物信息学分析CASC2具有miR-21的结合位点。双荧光素酶报告基因分析确认在CASC2-WT质粒转染组,miR-21高表达可以明显抑制相应的荧光活性,而在突变组(CASC2-MUT)miR-21的未有这种抑制作用,因此CASC2与miR-21结合是特异性的。miR-21高表达后,786-O和A498细胞株的CASC2表达水平下降,CASC2对786-O和A498细胞株的增殖与迁移的抑制作用也被部分消除。结论:CASC2具有miR-21的结合位点,miR-21可特异性结合CASC2,部分消除CASC2对RCC细胞株增殖与迁移的抑制作用。CASC2可能还有其他途径实现肿瘤抑制功能。
[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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.11

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