Sox5通过上皮间质转化促进前列腺癌转移

发布时间：2018-03-21 07:07

  本文选题：前列腺癌　切入点：Sox5　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：前列腺癌在我国已成为泌尿系统中发病率最高的肿瘤,目前的治疗方法有手术、放射治疗和化疗、免疫治疗及激素治疗等,药物治疗主要是针对雄激素剥夺治疗(Androgen deprivation therapy,ADT),主要有比卡鲁胺、MDV3100、醋酸阿比特龙等抑制雄激素产生或者抑制其功能,即便有着越来越多的药物可供选择,绝大多数患者在经过ADT治疗后的一到两年内疾病继续恶化,发展为去势抵抗性前列腺癌(castration resistance prostate cancer,CRPC),而且前列腺癌转移发生率高,许多患者诊断时已经伴随骨转移,骨转移不仅影响患者预后,其带来的并发症如骨痛、病理性骨折、神经压迫症状等还严重降低患者生活质量。深入研究CRPC的形成以及肿瘤转移机制,对于患者的治疗至关重要。在肝癌、垂体瘤等肿瘤的研究中发现Sox5与增殖转移密切相关,对于前列腺癌患者肿瘤的原位组织标本行免疫组化检测发现Sox5表达增高患者的往往伴随着转移的发生,获取淋巴结转移灶的标本Sox5的表达也较高,而且Sox5表达升高的患者间质标志物表达也高,Sox5与N-cad的表达呈现显著正相关。对前列腺癌细胞系22Rv1、PC3敲减Sox5,划痕实验及迁移实验均提示细胞的迁移能力明显下降,蛋白电泳检测上皮标志物增加,间质标志物减少,提示Sox5与上皮间质转化(epithelial mesenchymal transition,EMT)的发生相关,而且敲减Sox5后细胞的增殖能力也减低。利用22Rv1细胞系敲减Sox5进行小鼠前列腺原位种瘤,发现敲减Sox5后的肿瘤发生淋巴结等转移相对较少,而且皮下种瘤的瘤体体积也较小,提示Sox5与前列腺癌进展相关。对LNCa P细胞应用TGF-β进行EMT诱导时发现Sox5的表达也增加,在应用TGF-β的同时敲减Sox5可以逆转EMT的发生,在TGF-β信号通路中Smad2/3发挥着重要作用,利用JASPAR在线预测软件发现Smad3可以结合Sox5的promotoer区域,利用Smad3行CHIP以及Luciferase实验验证了这一直接调节作用。根据文献报道及JASPAR预测Sox5可以调节TWIST1的表达,敲减Sox5后检测TWIST1的表达下降,提示Sox5调节Twist1的表达进而调控EMT,利用Sox5行CHIP以及Luciferase实验验证了这一直接调节作用。为了更好研究CRPC的形成,我们利用实验室已有的去雄激素血清长期培养LNCa P细胞系建立去雄激素抵抗的细胞系CR-LN,在长期去雄培养以后,细胞的Sox5的表达增加,细胞的增殖能力回升,而且细胞发生EMT的转变,对CR-LN敲减Sox5后,细胞的间质标志物表达下降,但是对CR-LN进行TGF-β诱导时未发生明显EMT,提示CR-LN的TGF-β信号通路的自身活化。对CR-LN敲减Sox5后行迁移实验发现细胞的迁移能力下降,MTT实验发现细胞的活力也下降,球形成实验表明细胞的自我更新能力下降,提示Sox5对于CRPC的形成也发挥着一定作用。上述结果提示,在持续性雄激素剥夺治疗前列腺癌过程中,由于AR对于Sox5的抑制作用解除,导致Sox5的表达升高,肿瘤细胞的增殖能力也逐渐恢复,而且细胞可能受肿瘤微环境中TGF-β等细胞因子作用发生EMT,促进肿瘤的转移。对于CRPC的治疗,阻断Sox5及其下游可能是新的治疗策略。
[Abstract]:Prostate cancer in China has become the highest incidence of cancer in the urinary system, the current treatment methods include surgery, radiotherapy and chemotherapy, immune therapy and hormone therapy, drug treatment is mainly for androgen deprivation therapy (Androgen deprivation, therapy, ADT), the main bicalutamide, MDV3100, etc. abbitt acetic acid inhibits androgen Liuzhou to produce or inhibit its function, even if there are more and more drugs are available, most of the patients after ADT treatment after one to two years the disease continues to deteriorate, the development of adenocarcinoma before castration resistant (castration resistance prostate cancer column, CRPC), and the high rate of metastasis of prostate cancer, many patients have been diagnosed with bone metastasis, bone metastasis not only affects the prognosis of the patients, the complications such as bone ache, pathological fracture, nerve compression symptoms also seriously reduce the quality of life of patients . to research the mechanism of the CRPC formation and tumor metastasis, is essential for the treatment of patients. In the liver of pituitary tumor and tumor was found in Sox5 and the proliferation of tumor metastasis for patients with prostate cancer in situ tissue specimens for immunohistochemical detection showed that the expression of Sox5 was increased in patients with often accompanied with metastasis, access the expression of metastatic lymph node specimens of Sox5 were higher, and the expression of Sox5 increased in patients with interstitial marker expression is also high, showing a significant positive correlation with the expression of Sox5 N-cad. 22Rv1 on prostate cancer cell line, PC3 knockdown of Sox5, scratch test and migration experiments indicate that the ability of cell migration was significantly decreased, protein electrophoresis detection of epithelial markers increased, interstitial marker decreased, suggesting that Sox5 and epithelial mesenchymal transition (epithelial mesenchymal, transition, EMT) related to the occurrence, and knock in Sox5 cells by increasing Colonization ability also decreased. Knockdown of Sox5 mouse prostate tumor cell lines by in situ 22Rv1, found that knockdown of Sox5 after tumor lymph node metastasis is relatively small, and the tumor volume of subcutaneous tumor is small, suggesting that progress of Sox5 associated with prostate cancer. The LNCa P cells with TGF- beta induced by EMT to find the expression of Sox5 also increased at the same time, application of TGF- beta knockdown of Sox5 can reverse the occurrence of EMT, in the TGF- beta Smad2/3 pathway plays an important role in predicting software found Smad3 can bind to promotoer region of Sox5 by JASPAR online, using Smad3 CHIP and Luciferase experimental results verify the direct role the expression of JASPAR. According to the literatures and prediction of Sox5 can regulate TWIST1, knockdown of Sox5 expression after detection of TWIST1 decreased, suggesting that Sox5 regulates the expression and regulation of EMT by Twist1, Sox5 CHIP and Lucifera Se experimental results verify the direct role. In order to form a better study of CRPC, we use the serum androgen deprivation long-term cultures of LNCa P cell line to establish androgen resistant cell line CR-LN, after long-term training increased the expression of emasculation, Sox5 rebound, cell proliferation, and cell EMT change of CR-LN knockdown Sox5 cells, interstitial marker expression decreased, but the CR-LN TGF- did not change significantly when induced by EMT, suggesting that TGF- beta CR-LN signaling pathway activation. The knockdown of CR-LN on Sox5 after migration experiments showed decreased cell migration, MTT experiments showed that the cell vitality the ball drops, forming assay showed that cell self-renewal capacity decreased, suggesting that Sox5 for the formation of CRPC also plays a role. The above results suggest that, in the continuous androgen deprivation therapy for prostate In the process of cancer, due to AR for the inhibition of Sox5 release, resulting in increased expression of Sox5, tumor cell proliferation also gradually restored, and TGF- beta cells may be the tumor microenvironment in the role of cytokines EMT promote tumor metastasis. For the treatment of CRPC, blocking Sox5 and its downstream treatment strategies may be new.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

【参考文献】

相关期刊论文 前1条

1 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;Jie He;;Annual report on status of cancer in China, 2011[J];Chinese Journal of Cancer Research;2015年01期

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本文编号：1642787