TIP30低表达与膀胱癌患者临床预后相关性、促进膀胱癌细胞增殖、转移及相关机制研究

发布时间：2018-04-02 14:31

本文选题：TIP30　切入点：抑癌基因　出处：《南方医科大学》2017年博士论文

【摘要】：研究背景和目的膀胱肿瘤(bladdercancer,BC)是世界上最常见的恶性肿瘤之一,74%的膀胱肿瘤初诊时都是浅表性膀胱肿瘤[1]。目前对于浅表性膀胱癌的治疗主要包括手术加术后膀胱内化疗,但是大部分患者仍然出现复发及进展,直至进入肿瘤晚期,而对于晚期膀胱爱,目前缺乏有效的治疗手段。因此寻找新的有用的膀胱肿瘤治疗手段以提高治疗效果具有非常重要的临床意义。TIP30基因是一种抑癌基因[2]。文献证实TIP30在多种恶性肿瘤中表达水平下降,如肺癌、乳腺癌、结肠癌和肝细胞癌等[2-12]。上调TIP30可以抑制多种肿瘤细胞生长,促进肿瘤细胞凋亡,抑制肿瘤血管形成及转移[3,4,13,14]。目前国内有研究发现TIP30蛋白在膀胱肿瘤中低表达,但具体机制未有研究,且国内外均无相关的文献报道。因此探索TIP30在膀胱肿瘤的发生发展中的作用及机制,有望为膀胱肿瘤的基因治疗提供实验依据。方法:1.用免疫组化检测TIP30在膀胱肿瘤和正常膀胱黏膜中的表达,分析TIP30表达强度与患者多项临床病理参数的关系,分析TIP30的表达和无进展生存期和总生存期的关系。2.构建载体过表达T24细胞中TIP30基因,检测过表达后肿瘤细胞生长能力、细胞周期及膀胱肿瘤细胞迁移能力和侵袭能力。3.运用WB检测过表达TIP30后EGFR/AKT信号传导通路相关蛋白表达。统计学处理采用SPSS17.0行统计学分析,不同组间TIP30表达MOD值比较采用两独立样本的t检验和单因素方差分析(One-Way ANOVA),多个样本均数两两比较采用Dunnett T3法。生存分析采用Kaplan-Meier法,差异性检验采用log-rank法。采用多因素Cox回归模型对影响患者预后的相关因素进行分析。检验水准α=0.05,P0.05认为差异有统计学意义。结果1.免疫组化结果发现,和正常组织相比,TIP30在膀胱肿瘤组织中的表达明显下降。低级别(G1)组膀胱肿瘤组织中TIP30表达较高级别(G2+G3)组明显下降。非肌层浸润组TIP30的表达明显高于肌层浸润组。2.进展组患者TIP30的表达明显低于非进展组。TIP30高表达组的总生存时间明显高于低表达组,但两组患者的无瘤生存时间无明显差异。3.TIP30过表达后T24细胞中TIP30mRNA及TIP30蛋白表达均明显上升,进而抑制细胞生长、诱导细胞周期G1期阻滞、诱导凋亡。Transwell侵袭及迁移能力检测提示过表达TIP30基因能够抑制T24细胞的侵袭和迁移能力。4.TIP30过表达后抑制EGFR/AKT的表达,促进Bax和P21表达上调及抑制Bcl2,cyclinD1和cyclinE的表达抑制T24细胞增殖周期,通过激活caspase3引起T24细胞凋亡。通过下调MMP2,6,9表达抑制T24细胞转移。结论TIP30可能参与了膀胱肿瘤发生、发展。TIP30作为抑癌基因,可作为膀胱肿瘤治疗的一个靶基因。TIP30在T24细胞中可能通过EGFR/AKT信号通路发挥抑癌作用。
[Abstract]:Background and objective bladder cancer BCis one of the most common malignant tumors in the world.At present, the treatment of superficial bladder cancer mainly includes surgery and postoperative intravesical chemotherapy, but most of the patients still have recurrence and progress until the advanced stage of the tumor, but for the late bladder love, there is a lack of effective treatment.Therefore, it is very important to find a new and useful therapy for bladder cancer in order to improve the therapeutic effect. TIP30 gene is a tumor suppressor gene [2].The literature confirmed that the expression of TIP30 decreased in many malignant tumors, such as lung cancer, breast cancer, colon cancer and hepatocellular carcinoma [2-12].Upregulation of TIP30 can inhibit the growth of many kinds of tumor cells, promote the apoptosis of tumor cells, and inhibit the angiogenesis and metastasis of tumor cells.At present, low expression of TIP30 protein in bladder tumors has been found in China, but the specific mechanism has not been studied, and there is no related literature report at home and abroad.Therefore, to explore the role and mechanism of TIP30 in the occurrence and development of bladder tumors, it is expected to provide experimental evidence for gene therapy of bladder tumors.Method 1: 1.The expression of TIP30 in bladder tumor and normal bladder mucosa was detected by immunohistochemistry. The relationship between the expression of TIP30 and several clinicopathological parameters was analyzed, and the relationship between TIP30 expression and progressive survival and total survival was analyzed.The TIP30 gene was overexpressed in T24 cells. The growth ability, cell cycle, migration ability and invasion ability of bladder tumor cells were detected.WB was used to detect the expression of EGFR/AKT signal transduction pathway associated protein after overexpression of TIP30.The MOD of TIP30 expression in different groups was compared with that of two independent samples by t-test and one-way ANOVAN analysis of variance, and the Dunnett T3 method was used to compare the mean of multiple samples.Kaplan-Meier method was used for survival analysis and log-rank method was used for difference test.Multivariate Cox regression model was used to analyze the related factors affecting the prognosis of patients.The test level was 0.05%, and the difference was statistically significant.Result 1.Immunohistochemical results showed that the expression of TIP30 in bladder neoplasms was significantly lower than that in normal tissues.The expression of TIP30 in low grade G 1 group was significantly lower than that in high grade G 2 G 3 group.The expression of TIP30 in non-myometrial infiltrating group was significantly higher than that in myometrial infiltration group.The expression of TIP30 in the advanced group was significantly lower than that in the non-progressive group. The total survival time of the high expression group of TIP30 was significantly higher than that of the low expression group. However, there was no significant difference in the tumor-free survival time between the two groups. 3. After overexpression of TIP30, the expression of TIP30mRNA and TIP30 protein in T24 cells increased significantly.The results showed that overexpression of TIP30 gene could inhibit the invasion and migration of T24 cells. 4. Overexpression of TIP30 could inhibit the expression of EGFR/AKT.Promote the up-regulation of Bax and P21 expression and inhibit the expression of Bcl-2 cyclin D1 and cyclinE to inhibit the proliferation of T24 cells, and induce the apoptosis of T24 cells by activating caspase3.By down-regulating the expression of MMP2 + 6 + 9, the metastasis of T 24 cells was inhibited.Conclusion TIP30 may be involved in the development of bladder tumor. TIP30, as a tumor suppressor gene, can be used as a target gene for the treatment of bladder cancer. TIP30 may play an inhibitory role in T24 cells through EGFR/AKT signaling 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】：R737.14

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