|YB-1 regulates angiogenesis in bladder cancer via miR-29b-3p-VEGFA pathway
|Place of Conferral
|目的：膀胱癌是危害人类生命和健康的常见恶性肿瘤，目前膀胱癌的治疗主要是依据病理分期，以手术为主，辅以化学治疗，存在着高费用、高复发的缺点。为此，有关膀胱癌的研究越来越集中于靶向药物的研发，其中抗肿瘤血管生成是靶向药物研发的重要领域。通过文献调研以及生信分析，我们发现YB-1与膀胱癌患者的预后相关;YB-1可以抑制miR-29b-3p的成熟；miR-29b-3p的靶基因包括VEGFA。提出如下假说“YB-1通过抑制miR-29b-3p的成熟促进VEGFA的表达从而介导膀胱癌的血管生成”。本实验拟通过阐释YB-1与膀胱癌血管生成的内在机制，为膀胱癌的治疗提供新的靶点和研究方向。方法：Western Blot检测EJ、UMUC3、RT4、SW780 4种膀胱癌细胞系中YB-1的表达，在高表达YB-1的EJ、UMUC3细胞系中用RNA干扰技术敲减YB-1，并用EJ细胞建立对照稳转株和YB-1敲减稳转株。通过qRT-PCR、Western blot检测其下游基因VEGFA、miR-29b-3p的表达；用RNA干扰技术在YB-1敲减稳转株中敲减miR-29b-3p，qRT-PCR、Western blot检测VEGFA表达的变化，探讨miR-29b-3p在YB-1调节VEGFA 表达通路中的作用。CCK8法、克隆形成实验、血管形成实验检测并分析YB-1基因表达下调对膀胱癌EJ细胞增殖活力、克隆形成能力、诱导血管生成的能力的影响。在体内水平探讨YB-1对膀胱癌发生、发展的影响，用对照稳转株和YB-1敲减稳转株在裸鼠皮下建立肿瘤模型，观察肿瘤的生长状态，绘制生长曲线，免疫组化检测肿瘤模型中的微血管密度，揭示YB-1与肿瘤血管生成的关系。回顾性，免疫组化检测膀胱癌临床标本中YB-1、VEGFA的表达，分析YB-1与VEGFA表达的关系。结果：EJ、UMUC3、RT、SW780 4种膀胱癌细胞系中YB-1的表达情况为EJ＞UMUC3＞SW780＞RT4,成功地在EJ细胞中建立了对照稳转株和YB-1敲减稳转株。在EJ、UMUC3细胞系中敲减YB-1后，VEGFA表达下降，miR-29b-3p表达升高，在YB-1敲减稳转株中敲减miR-29b-3p后VEGFA表达升高。在EJ细胞中，YB-1基因表达下调后，其诱导血管生成的能力下降，细胞增殖活力、克隆形成能力无明显变化。动物实验中，对照稳转株肿瘤模型生长速度明显快于YB-1敲减稳转株肿瘤模型，而且前者的微血管密度明显高于后者。对56例膀胱癌标本进行了免疫组化检测分析，膀胱癌标本中YB-1高表达样本总体较YB-1低表达样本的VEGFA表达高。结论：YB-1通过抑制miR-29b-3p的成熟促进VEGFA的表达从而介导膀胱癌中血管的生成，其有望成为治疗膀胱癌的有效靶点。
|Objective：Bladder cancer is a common malignant tumor that endangers human life and health. At present, the treatment of bladder cancer is mainly based on surgery, supplemented by chemotherapy according to pathological staging. This treatment has the disadvantages of high cost and high risk of recurrence. To this end, research on bladder cancer is increasingly focused on the development of targeted drugs, where anti-tumor angiogenesis has an important position. Through literature research and bioinformatics analysis, we found that YB-1 is associated with the prognosis of patients with bladder cancer. YB-1 can inhibit the maturation of miR-29b-3p, and the target genes of miR-29b-3p include VEGFA. So we proposed the hypothesis that " YB-1 mediates angiogenesis by promoting VEGFA expression by inhibiting the maturation of miR-29b-3p in bladder cancer." This experiment intended to provide a new target and research direction for the treatment of bladder cancer by interpreting the intrinsic mechanism between YB-1 and bladder cancer angiogenesis.Methods: We detected the expressions of YB-1 in bladder cancer cell lines of EJ, UMUC3, RT4 and SW780 by Western blot. YB-1 was knocked down with RNA interference technology in the EJ and UMUC3 cell lines with high expressions of YB-1, and EJ cells were used to establish control steady cell line and YB-1 knocked-down steady cell line. The expressions of VEGFA and miR-29b-3p were detected by qRT-PCR and Western blot. The miR-29b-3p was knocked down with RNA interference technique and changes of VEGFA expressions were detected by qRT-PCR and Western blot in YB-1 knocked-down steady cell lines, for exploring the role of miR-29b-3p in the pathway of YB-1 regulating VEGFA expression. We analyzed influence of down-regulation of YB-1 expressions by detecting the ability of proliferation, colony formation and induction of angiogenesis of EJ cell. To investigate the effect of YB-1 on the occurrence and development of bladder cancer in vivo, we established tumor models under the skin of nude mice with control steady cell line and YB-1 knocked-down steady cell line. We observed the growth state of the tumor models, and draw growth curves of them. Immunohistochemistry was used to detect microvessel density in tumor models to reveal the relationship between YB-1 and tumor angiogenesis. Retrospectively, the expressions of YB-1 and VEGFA in clinical specimens of bladder cancer were detected by immunohistochemistry，to analysis the relationship between YB-1 and VEGFA expression. Results: The expressions of YB-1 in bladder cancer cell lines of EJ, UMUC3, RT and SW780 was EJ>UMUC3>SW780>RT4. Control steady cell line and YB-1 knocked-down steady cell lines were successfully established in EJ cells. After down-regulation of YB-1 expression in EJ and UMUC3 cell lines, VEGFA expression decreased, while miR-29b-3p expression increased. And VEGFA expression increased after down-regulation of miR-29b-3p in YB-1 knocked-down steady cell lines. In EJ cell, after down-regulation of YB-1 expression, its ability to induce angiogenesis decreased, but cell proliferation activity and clone formation ability did not change significantly. In animal experiments, tumor models of control steady cell line grew significantly faster than those of YB-1 knocked-down steady cell line, and the microvessel density of the former was significantly higher than that of the latter. We performed immunohistochemical analysis of 56 specimens of bladder cancer. On the whole, the YB-1 high expression samples had higher VEGFA expression than those of YB-1 low expression in bladder cancer specimens.Conclusion: YB-1 mediates angiogenesis by promoting VEGFA expression by inhibiting the maturation of miR-29b-3p in bladder cancer.
|First Author Affilication
|Second Clinical School
郜栋阳. YB-1通过miR-29b-3p-VEGFA途径调控膀胱癌的血管生成[D]. 兰州. 兰州大学,2019.
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