新型小分子化合物WJ460通过靶向Myoferlin抑制乳腺癌转移的性质与机理研究

发布时间：2018-03-18 07:31

  本文选题：乳腺癌　切入点：乳腺癌转移　出处：《华东师范大学》2015年博士论文　论文类型：学位论文

【摘要】：乳腺癌是女性最常见肿瘤。在过去的几十年间,随着早诊断的意识和治疗手段的提高,乳腺癌患者的死亡率有了一定程度的降低。但是,每年仍有数以万计的患者死于乳腺癌。对于乳腺癌患者来说,最差的预后是发生了肿瘤转移,因为绝大部分的乳腺癌患者直接或间接地死于乳腺癌转移。传统的乳腺癌疗法包括手术治疗,放射疗法,以及系统疗法。手术疗法联合放疗能清除绝大部分的原位乳腺肿瘤。手术治疗前或后对患者施以系统疗法(包括化疗,激素疗法和分子靶向疗法)在清除一些残余的原位肿瘤细胞的同时也能缩小转移灶的大小。然而,这些治疗方法其目的均是针对肿瘤细胞的生存。因此,研发靶向肿瘤转移的新型药物能为临床治疗乳腺癌提供新的治疗策略并且能降低乳腺癌转移的风险。1999年,Davis及其同事发现了一个调控肌肉萎缩症的ferlin家族的新成员。他们将这个蛋白命名为Myoferlin (MYOF)。近年来,人们也逐渐意识到MYOF在乳腺癌中的重要作用。已有研究人员证明MYOF在临床的浸润性乳腺癌患者的样本中有异常高的表达;此外,还有证据表明MYOF在乳腺癌的浸润和上皮-间质转化(EMT)过程中起重要作用,提示MYOF是一个乳腺癌转移的相关蛋白且可以作为治疗乳腺癌转移的一个潜在靶标。基于上述背景,我们对本实验室的小分子化合物库进行了筛选。首先,我们利用体外乳腺癌转移相关实验筛选出11个具有较强体外抗乳腺癌转移活性的小分子。利用Matrigel侵袭实验我们对这11个小分子进行了进一步的筛选。我们从中得到了一个新型的具最强抗乳腺癌转移活性的小分子WJ460。随后,我们利用小鼠乳腺癌自发性转移模型检测了WJ460是否也具有较强的体内抗乳腺癌转移的能力。实验结果表明,WJ460能显著抑制乳腺癌细胞向小鼠各远端器官的转移。为了探究WJ460抑制乳腺癌转移的具体分子机制,我们借助了生物素分子探针。通过将生物素偶联到小分子WJ460上,我们成功“钓取”了WJ460的直接作用靶蛋白。通过质谱分析,我们发现并鉴定该蛋白是MYOF。此外,我们通过亲和pull-down实验对这一结果进行了验证。于此同时,免疫荧光的结果也显示WJ460和MYOF确实存在共定位。为了寻找MYOF和WJ460结合的具体部位,我们构建了不同的MYOF缺失体。通过亲和pull-down实验我们发现只有含有C2D结构域的MYOF缺失体能与WJ460相结合。接下来,我们探究了WJ460是否影响MYOF所调节的与乳腺癌转移相关的生物学过程。实验结果表明,WJ460能明显抑制MYOF的靶基因基质金属蛋白酶1(MMP1)的基因及蛋白表达。通过3D培养,免疫荧光,实时定量PCR及蛋白免疫印迹实验,我们发现WJ460能逆转乳腺癌的上皮-间质转化(EMT)过程从而降低乳腺癌转移能力。此外,我们利用人源的受体酪氨酸激酶芯片检测了WJ460对受体酪氨酸激酶活性(磷酸化形式)的影响。实验结果表明,多个受体酪氨酸激酶活性均能被WJ460所抑制。最后,通过小鼠实验性肺转移模型,我们发现knock-down MYOF和WJ460处理有类似的抑制乳腺癌转移的效果。同时,我们的结果还显示,knock-down MYOF和WJ460处理均能抑制乳腺癌肺转移的外渗与恶性增殖。综上所述,我们的研究表明WJ460可作为第一个靶向MYOF的先导化合物,同时为WJ460治疗乳腺癌转移提供了一定的理论依据。
[Abstract]:Breast cancer is the most common female cancer. In the past few decades, with the awareness and treatment of early diagnosis of breast cancer mortality has decreased to a certain degree. However, there are still tens of thousands of patients die of breast cancer each year. For patients with breast cancer, the worst prognosis is the occurrence of the tumor metastasis that is because most of the breast cancer patients directly or indirectly died of breast cancer metastasis. Breast cancer therapy including traditional surgery, radiotherapy, and systemic therapy. Surgical therapy combined with radiotherapy can remove most of the in situ mammary gland tumor. Surgical treatment before or after therapy to patients of system (including chemotherapy, hormone therapy and molecular targeted therapy) can also reduce the size of the metastases in the removal of some in situ residual tumor cells at the same time. However, these treatments the aim is for students of tumor cells Deposit. Therefore, development of new drugs targeting tumor metastasis can provide new therapeutic strategies for the treatment of breast cancer and can reduce the risk of breast cancer metastasis in.1999, Davis and colleagues found a new member of the ferlin family regulate muscle atrophy. They will be the protein named Myoferlin (MYOF) in recent years. Also, people gradually realized the important role of MYOF in breast cancer. Researchers have demonstrated that MYOF in the clinical expression of abnormally high of breast cancer patients in the sample; in addition, there is evidence that MYOF in breast cancer invasion and epithelial mesenchymal transition (EMT) plays an important role in the process, prompt MYOF is a potential target for a breast cancer metastasis related protein and can be used as a treatment for breast cancer metastasis. Based on the above background, we in the laboratory of small molecular libraries were screened. First of all, we benefit The selected 11 has strong anti breast cancer activity in vitro transfer of small molecules in vitro and metastasis of breast cancer. The use of Matrigel invasion experiment we consider these 11 small molecules were further screened. We get a new type of small molecules with the strongest activity against breast cancer metastasis WJ460. then, we use the mice breast cancer model with spontaneous metastasis detected whether WJ460 also has anti breast cancer metastasis ability in vivo. The experimental results show that WJ460 can significantly inhibit the metastasis of breast cancer cells into mice of the remote organs. In order to explore the molecular mechanism of WJ460 inhibiting the metastasis of breast cancer, we used the biotin molecules through biological probe. By coupling the small molecule WJ460, we successfully obtained the direct effect of WJ460 target protein. By mass spectrum analysis, we found and identified the protein is MYOF. addition ,鎴戜滑閫氳繃浜插拰pull-down瀹為獙瀵硅繖涓，

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