VTCN1基因在膀胱癌发生发展中作用及机制研究

发布时间：2018-05-24 12:28

本文选题：膀胱肿瘤 + VTCN1基因　；参考：《武汉大学》2014年博士论文

【摘要】：研究目的与背景：一些肿瘤细胞系组成性表达VTCN1基因。在肺癌、卵巢癌、肾细胞癌均有VTCN1蛋白的表达；乳腺癌和子宫内膜癌几乎普遍存在VTCN1基因的表达,且不依赖肿瘤分级和分期；VTCN1基因高表达是明显增加胃癌病例死亡风险的独立因素。但VTCN1基因在膀胱癌肿瘤组织表达的确切临床意义还有待进一步验证,它是否可以作为膀胱癌早期诊断和预后评价的指标也值得进一步深入研究。VTCN1基因家族负性分子在肿瘤中的异常表达提示其在肿瘤发生发展中发挥了重要作用,这其中涉及的机制将为干预肿瘤的发生发展提供有效途径,具有较大的潜在应用价值。但其在膀胱癌发生发展中的作用及机制还有待进一步研究。因此本研究拟探讨VTCN1基因在膀胱癌组织中的表达及其与临床病理及预后的关系；通过RNA干扰技术下调VTCN1基因在人膀胱癌HT-1376细胞株中的表达,探讨VTCN1基因表达下调对膀胱癌细胞生物学行为的影响及分子机制。研究方法：第一部分：以酶联免疫反应检测膀胱癌患者外周血中可溶性VTCN1蛋白(sVTCN1)的含量,荧光定量逆转录聚合酶链反应(real time RT-PCR)技术及免疫组织化学染色检测膀胱癌患者手术切除组织标本中VTCN1基因的mRNA及蛋白表达水平。第二部分：将针对VTCN1基因的siRNA序列重组入真核表达载体中构建pU-VTCN1-siRNA,转染至膀胱癌HT-1376细胞株中,采用逆转录聚合酶链反应(RT-PCR)、免疫印迹法检测转染pU-VTCN1-siRNA后的HT-1376细胞中VTCN1基因在基因表达水平和蛋白质表达水平的变化。采用流式细胞仪分析转染pU-VTCN1-siRNA后的HT-1376细胞的细胞周期变化,应用Transwell侵袭小室模型观察转染pU-VTCN1-siRNA后HT-1376细胞侵袭力的变化。研究结果：第一部分：膀胱癌患者外周血中sVTCN1的含量(45.25士8.93μg/L)明显高于膀胱良性肿瘤患者(34.41士6.91μg/L)和正常人群(31.20士6.28μg/L)(P0.01)。VTCN1基因mRNA在30例膀胱癌组织和癌旁组织中均有表达,膀胱癌组织中表达明显高于癌旁组织(P0.01),且癌旁组织中的表达明显高于膀胱良性肿瘤组织(P0.01)。检测70例膀胱癌组织中VTCN1蛋白的表达,其阳性表达率68.57%,其中77.08%为高表达,明显高于膀胱良性肿瘤组织(P0.01)。VTCN1蛋白的表达与膀胱癌TNM分期和盆腔淋巴转移密切相关(P0.05),而与患者性别、年龄及肿瘤组织病理分级无关(P0.05)。VTCN1高表达患者者生存率明显低于低表达患者(P0.05)。第二部分：转染pU-VTCN1-siRNA后的HT-1376细胞中,VTCN1基因的基因表达水平和蛋白质表达水平显著下降(P0.05),细胞周期被阻滞在G1期,细胞生长缓慢,体外侵袭能力明显下降(P0.05)。研究结论： (1) VTCN1基因在膀胱癌组织表达状态与膀胱癌的转移及预后关系密切,可以作为反映膀胱癌生物学行为和判断预后的有效指标。(2)通过下调VTCN1基因的表达,能有效抑制HT-1376细胞的生长、增殖和侵袭能力,提示VTCN1基因可能在膀胱癌的发生发展过程中起促进作用。
[Abstract]:Study Purpose and Background :

The expression of VTCN1 gene was expressed in some tumor cell lines . The expression of VTCN1 protein was found in lung cancer , ovarian cancer and renal cell carcinoma .
The expression of VTCN1 gene is almost universal in breast cancer and endometrial carcinoma , and does not depend on tumor grading and staging ;
The high expression of VTCN1 gene is an independent factor that significantly increases the risk of death in gastric cancer . However , the exact clinical significance of VTCN1 gene in the expression of tumor tissue of bladder cancer is to be further studied . The mechanism of VTCN1 gene family negative molecule plays an important role in the development of tumor .
The expression of VTCN1 gene in human bladder cancer HT - 1376 cell line was downregulated by RNA interference technique .

Study method :

The contents of soluble VTCN1 protein ( sVTCN1 ) , real time RT - PCR and immunohistochemical staining were used to detect the mRNA and protein expression level of VTCN1 gene in bladder cancer patients .

The expression level of VTCN1 in HT - 1376 cells transfected with pU - VTCN1 - siRNA was detected by reverse transcription polymerase chain reaction ( RT - PCR ) , and the cell cycle of HT - 1376 cells transfected with pU - VTCN1 - siRNA was detected by flow cytometry .

Results of the study :

The expression of VTCN1 protein in bladder cancer tissues was significantly higher than that in bladder benign tumor tissues ( P0.01 ) . The expression of VTCN1 protein was significantly higher than that in benign tumor tissues ( P0.01 ) . The expression of VTCN1 protein was significantly higher than that in benign tumor tissues ( P0.05 ) .

In the second part , in HT - 1376 cells transfected with pU - VTCN1 - siRNA , the expression level of VTCN1 gene and the level of protein expression decreased significantly ( P0.05 ) . Cell cycle was blocked in G1 phase , cell growth was slow and invasion ability in vitro was significantly decreased ( P0.05 ) .

Conclusions of the study :

( 1 ) The expression of VTCN1 gene is closely related to the metastasis and prognosis of bladder cancer . It can be used as an effective index to reflect the biological behavior and prognosis of bladder cancer . ( 2 ) By downregulating the expression of VTCN1 gene , the growth , proliferation and invasion ability of HT - 1376 cells can be effectively inhibited , suggesting that the VTCN1 gene may play a role in the development of bladder cancer .
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.14

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