过表达TFE3基因在Xp11.2易位性肾癌中的作用及其分子机制研究

发布时间：2018-02-14 18:12

本文关键词： TFE3 慢病毒过表达 Xp11.2易位性肾癌 ACHN细胞 mTOR pS6　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的:Xp11.2易位性肾癌是WHO新分类的一种肾癌,发病年龄轻、预后较差,主要特征为TFE3基因发生易位,导致其过表达。因目前尚无针对过表达TFE3建立的肾癌细胞学模型,故作用机制尚不明确,涉及的信号通路有待研究,其诊断及治疗缺乏理论基础。目的:1.筛选出适合构建过表达TFE3慢病毒感染的肾癌细胞系,构建并鉴定特异性慢病毒载体颗粒。2.建立慢病毒过表达TFE3稳定感染的肾癌细胞系,研究过表达TFE3对其生物学影响。3.明确mTOR及pS6在Xp11.2易位性肾癌及过表达TFE3细胞系中的表达,探讨PI3K/AKT/mTOR通路在该类肿瘤中的作用。4.Affymetrix基因芯片检测Xp11.2易位性肾癌相关的差异基因,筛选出更多精确的靶基因。方法:1.Real time PCR和Western blot方法检测8种肾源性的细胞系中TFE3的表达水平,筛选一株进行后续实验,构建特异性慢病毒过表达载体颗粒并鉴定其感染效率。2.构建稳定感染过表达TFE3的肾癌细胞系,分为过表达组(OE),阴性对照组(NC)和空载对照组(CON),并用Western blot进行验证。3.MTT、平板克隆法检测不同实验组的细胞增殖和克隆形成;利用流式细胞仪检测其细胞周期。4.免疫组化方法检测12例Xp11.2易位性肾癌和27例非Xp11.2易位性肾癌中mTOR和pS6的表达情况,同时用Western blot方法检测不同细胞实验组中两者的差异。5.Affymetrix基因芯片分析2例Xp11.2易位性肾癌患者癌组织与癌旁组织之间基因表达谱的差异。结果:1.Real-time PCR结果显示TFE3基因在ACHN中表达丰度为中,Western blot结果显示在ACHN中无目的条带,因此选取ACHN细胞系作为靶细胞进行慢病毒感染。成功构建人TFE3特异性慢病毒过表达载体并获得相应高效率慢病毒。2.在实验组中,OE组病毒滴度=2×108TU/ml,MOI=10,Western blot结果显示OE组中检测到95KD的目的条带,表明过表达TFE3稳定感染ACHN细胞成功。OE组的细胞增殖水平和克隆数较NC组显著升高(P0.05),说明过表达TFE3能够促进ACHN细胞的增殖和克隆形成。细胞周期结果显示,OE组较NC组的S期延长(P0.05),说明过表达TFE3能够调控ACHN细胞的周期从而促进细胞增殖。3.免疫组化法检测石蜡标本中的mTOR和pS6在Xp 11.2易位性肾癌较非Xp 11.2易位性肾癌中阳性率显著升高(P0.05),两种蛋白在OE组中表达显著增高(P0.05),说明该肿瘤在组织学和细胞学层面都存在PI3K/AKT/mTOR通路的过度激活。4.Affymetrix基因芯片检测Xp11.2易位性肾癌患者相关差异表达的基因。共筛选出差异表达基因1479个,癌组织中上调基因790个,下调基因689个。其中泛素化相关的RNF180基因下调5倍以上,提示该种肿瘤可能与泛素化有关。结论:1.过表达TFE3能够促进ACHN细胞的增殖,调控其细胞周期。2.在Xp 11.2易位性肾癌中存在PI3K/AKT/mTOR通路的过度激活。3.Xp11.2易位性肾癌可能与泛素化途径相关。
[Abstract]:Background and objective: Xp11.2 translocated renal cell carcinoma (RCC) is a new type of renal cell carcinoma (RCC) classified by WHO. Its age is young and its prognosis is poor. The main feature is translocation of TFE3 gene, which results in its overexpression. There is no cytological model of RCC established for overexpression of TFE3 at present. Therefore, the mechanism of action is unclear, the signal pathway involved remains to be studied, and its diagnosis and treatment lack theoretical basis. Objective: 1. To screen and construct a cell line of renal cell carcinoma with overexpression of TFE3 lentivirus infection. To construct and identify the specific lentivirus vector particle. 2. To establish a cell line with stable infection of lentivirus overexpression TFE3, and to study the biological effects of overexpression of TFE3 on the cell line. 3. To clarify the expression of mTOR and pS6 in Xp11.2 translocated renal cell carcinoma and TFE3 cell line. To investigate the role of PI3K/AKT/mTOR pathway in this type of tumor. 4. Affymetrix gene chip was used to detect differentially expressed genes related to Xp11.2 translocation of renal cell carcinoma, and more accurate target genes were screened. Methods: 1. Real time PCR and Western blot were used to detect the expression of TFE3 in eight renal cell lines. One cell strain was screened for subsequent experiments to construct specific lentivirus overexpression vector particles and to identify its infection efficiency. 2. To construct a renal carcinoma cell line with stable overexpression of TFE3. Western blot was used to test the cell proliferation and clone formation in different experimental groups. The expression of mTOR and pS6 in 12 cases of Xp11.2 translocated renal cell carcinoma and 27 cases of non-#en1# translocated renal cell carcinoma were detected by flow cytometry. At the same time, Western blot method was used to detect the difference between the two cell groups. 5.Affymetrix gene chip was used to analyze the difference of gene expression profiles between cancer tissues and adjacent tissues of two patients with Xp11.2 translocated renal cell carcinoma. The expression abundance of ACHN was determined by Western blot. The results showed that there was no objective band in ACHN. Therefore, ACHN cell line was selected as the target cell for lentivirus infection. The human TFE3 specific lentivirus overexpression vector was successfully constructed and the corresponding high efficiency lentivirus was obtained. In the experimental group, the titer of OE group virus was 2 脳 10 8 TUU / ml moi 10 blot and the results showed that the human TFE3 specific lentivirus overexpression vector was found in the OE group. The target band of 95KD was detected, The results showed that the proliferation level and clone number of ACHN cells infected by overexpression of TFE3 were significantly higher than those of NC group, which indicated that overexpression of TFE3 could promote the proliferation and clone formation of ACHN cells. The cell cycle results showed that the cell cycle of OE group was higher than that of NC group. The S-phase prolongation (P0.05) of group S indicates that overexpression of TFE3 can regulate the cell cycle of ACHN cells and thus promote cell proliferation .3.Immunohistochemical method is used to detect the positive rate of mTOR and pS6 in Xp11.2 translocated renal cell carcinoma compared with non-Xp11.2 translocated renal cell carcinoma. The expression of two proteins in OE group was significantly higher than that in OE group, indicating that there was overexpression of PI3K/AKT/mTOR pathway in the tumor. 4. Affymetrix gene chip was used to detect the differentially expressed genes in patients with Xp11.2 translocation of renal cell carcinoma. A total of 1479 differentially expressed genes were screened. There were 790 up-regulated genes and 689 down-regulated genes in cancer tissues. The RNF180 gene associated with ubiquitization was down-regulated by more than 5 times, suggesting that the tumor might be related to ubiquitization. Conclusion: 1. Overexpression of TFE3 can promote the proliferation of ACHN cells. Regulation of cell cycle. 2. Overexpression of PI3K/AKT/mTOR pathway in Xp11.2 translocation RCC. 3.Xp11.2 translocation RCC may be associated with Ubiquitin pathway.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.11

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