核转录因子SATB1表达与肾细胞癌生物学行为的关系及其临床意义

发布时间：2018-04-01 14:24

本文选题：SATB1　切入点：肾细胞癌　出处：《华中科技大学》2014年博士论文

【摘要】：目的：探讨核转录因子SATB1在不同肾癌细胞系中的表达情况,以及SATB1基因表达对肾癌细胞增殖、侵袭能力的影响。方法：分别应用实时定量PCR、Western blotting与免疫荧光染色法检测不同肾癌细胞系中SATB1的表达情况。在构建靶向沉默SATB1的pGenesil2-SATB1-shRNA和SATB1过表达真核表达质粒pcDNA3.1-SATB1的基础上,采用CCK-8比色法评估调控SATB1的表达对RCC细胞增殖能力的影响,应用Transwell侵袭实验分析RCC细胞侵袭性的变化。结果：RCC细胞中SATB1mRNA及其蛋白表达水平显著高于对照组细胞HK-2(P0.001),且SATB1在ACHN、A498及786-0中的表达依次增高。成功构建pGenesil2-SATB1-shRNA和pcDNA3.1-SATB1过表达质粒。通过Western blotting和实时定量PCR法证实：shRNA稳转组786-0细胞中,SATB1mRNA及其蛋白表达均显著降低(P0.001)；而SATB1过表达组ACHN细胞中,SATB1在mRNA和蛋白水平的表达均显著升高(P0.001)。CCK-8研究结果显示：SATB1表达下调可显著抑制786-0细胞的增殖能力,而SATB1过表达则可明显促进ACHN细胞的增殖活性,差异均具有显著的统计学意义(均为P0.05)。Transwell研究表明：shRNA稳转组肾癌细胞786-0的侵袭能力显著降低(P0.001),而pcDNA3.1-SATB1稳转组肾癌细胞ACHN的侵袭能力则显著增强(P0.001)。结论：核转录因子SATB1在RCC细胞中的表达显著上调。SATB1基因过表达在肾癌细胞获得侵袭表型的过程中起关键作用,对促进肾癌侵袭、转移具有重要意义。目的：探讨SATB1和EMT标志基因在ccRCC组织中的表达及其相关性。方法：分别采用免疫组织化学(IHC)染色、Western blotting及实时定量PCR法检测89例ccRCC组织标本中SATB1的表达情况；应用IHC法检测SATB2及EMT标志基因(E-cadherin、ZEB2、vimentin和fibronectin)在ccRCC组织中的表达,并通过Spearman秩次相关分析法评估其与SATB1表达之间的相关性。结果：IHC结果显示89例ccRCC组织中SATB1、SATB2、E-cadherin、ZEB2、vimentin和fibronectin蛋白表达阳性率分别为46.1%、42.7%、44.9%、51.7%、37.1%和49.4%。与对照组正常组织相比,肾癌组织中SATB2和E-cadherin的表达水平均明显下降,而SATB1和ZEB2表达水平则显著升高,且SATB1高表达与患者肿瘤浸润深度(P0.001)、TNM分期(P=0.009)及淋巴结转移状态(P=0.001)之间具有显著关联。此外,Spearman秩次相关分析表明SATB1表达与ZEB2之间呈显著正相关(R=0.262,P=0.013),而与SATB2、E-cadherin之间则呈明显负相关(]R=-0.296, P=0.005; R=-0.427,P0.001)。结论：ccRCC组织中,SATB1表达与EMT标志基因蛋白表达密切相关,将二者联合进行分析,对于进一步明确SATB1与EMT过程在肾癌侵袭转移中所起的作用以及评估肾癌患者预后均具有重要意义。
[Abstract]:Aim: to investigate the expression of nuclear transcription factor SATB1 in different renal cell lines and the effect of SATB1 gene expression on the proliferation and invasion of renal carcinoma cells. Methods: the expression of SATB1 in different RCC cell lines was detected by real-time quantitative PCR Western blotting and immunofluorescence staining. The eukaryotic expression plasmids pcDNA3.1-SATB1 of pGenesil2-SATB1-shRNA and SATB1 targeting silencing SATB1 were constructed. CCK-8 colorimetric assay was used to evaluate the effect of regulating SATB1 expression on the proliferation of RCC cells, and Transwell invasion assay was used to analyze the changes of the invasiveness of RCC cells. Results the expression of SATB1mRNA and its protein was significantly higher in the cell line than that in the control cell line HK-2P 0.001, and the expression of SATB1 was increased in turn in ACHNNAA498 and 786-0. The overexpression plasmids of pGenesil2-SATB1-shRNA and pcDNA3.1-SATB1 were successfully constructed. The stable transformation of pGenesil2-SATB1-shRNA and pcDNA3.1-SATB1 was confirmed by Western blotting and real-time quantitative PCR. In group 786-0, the expression of Tb1 mRNA and its protein decreased significantly, while the expression of mRNA and protein in ACHN cells in SATB1 overexpression group increased significantly. The results of CCK-8 study showed that down-regulation of the expression of TB1 in SATB1 could significantly inhibit the proliferation of 786-0 cells. However, overexpression of SATB1 could significantly promote the proliferation of ACHN cells. The difference was statistically significant (all P0.05).Transwell studies showed that the invasion ability of renal cancer cell line 786-0 was significantly decreased in pcDNA3.1-SATB1 stable group, while the invasion ability of ACHN was significantly enhanced in pcDNA3.1-SATB1 stable group. Conclusion: the expression of nuclear transcription factor SATB1 significantly up-regulates the expression of. SATB1 gene in RCC cells, which plays a key role in the process of invasion phenotype of renal cancer cells, and plays an important role in promoting the invasion and metastasis of renal cell carcinoma. Objective: to investigate the expression and correlation of SATB1 and EMT marker genes in ccRCC tissues. Methods: the expression of SATB1 in 89 cases of ccRCC tissues was detected by immunohistochemical staining blotting and real time quantitative PCR, and the expression of SATB2 and EMT marker genes such as SATB2 and EMT marker ZEB2vimentin and fibronectin in ccRCC tissues were detected by IHC method. The correlation between SATB1 expression and Spearman rank correlation analysis was evaluated. Results the positive rates of SATB2 and E-cadherin in 89 cases of ccRCC were 46.1% and 44.7%, respectively. Compared with the control group, the expression of SATB2 and E-cadherin in RCC were significantly decreased, while the expression of SATB1 and ZEB2 were significantly increased. Moreover, there was a significant correlation between the high expression of SATB1 and the depth of tumor invasion (P0.001, P0.009) and the metastatic status of lymph nodes (P 0.001). In addition, there was a significant positive correlation between the expression of SATB1 and ZEB2, and a significant negative correlation between the expression of SATB1 and ZEB2, but a significant negative correlation with SATB2E-cadherin (] RV -0.296, P0. 005; R- 0. 427 and P0. 0027; P 0. 0013), but negative correlation with SATB2E-cadherin (P 0. 001, P 0. 005, P 0. 005, P 0. 005, P 0. 005, P 0. 427, P 0. 0013). Conclusion the expression of SATB1 is closely related to the expression of EMT marker gene protein in the tissues of WCCRCC, and the expression of SATB1 is associated with the protein expression of the EMT marker gene. It is important to further understand the role of SATB1 and EMT in the invasion and metastasis of RCC and to evaluate the prognosis of RCC.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.11

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