RHBDD1在结直肠癌中的功能研究

发布时间：2018-08-03 10:18

【摘要】：结直肠癌是世界上最常见的癌症之一,也是死亡率最高的癌症之一。当患者确诊为结直肠癌时,约1/4的患者已经发生了体内癌组织的转移,并且大部分的结直肠癌患者最终会发展为转移癌。转移性结直肠癌的5年生存率较低,死亡率高。目前,对于结直肠癌的发生,尤其是转移的分子机制的相关研究还不是很详尽,结直肠癌的治疗也未能取得良好的效果。因此,发现和鉴定与结直肠癌发生和转移相关的基因,对于研究结直肠癌的发生和转移的分子机制具有非常重要的意义,不仅有助于我们了解和认识结直肠癌,而且可以为结直肠癌的治疗提供新的思路。Rhomboid domain containing protein 1(RHBDD1)是我们实验室前期在筛选睾丸cDNA文库时发现的一个高表达的新基因,最初我们将该基因命名为HSD50并提交至GenBank。因其含有rhomboid结构域所以又称为RHBDD1。RHBDD1是跨膜蛋白,在人中表达全长315aa,含有6个跨膜结构,其中包含保守的Rhomboid domain,具有蛋白切割活性,是Rhomboid家族成员之一。根据我们实验室前期研究表明,RHBDD1可以切割tumour suppressor activated pathway-6 (TSAP6)、凋亡前提蛋白Bik和转化生长因子α(transforming growth factor alpha, TGF-a)。我们实验室近期发现RHBDD1在结直肠癌中有较高的蛋白表达水平,其可以通过切割TGF-a从而激活表皮生长因子受体(epidermal growth factor receptor, EGFR)下游信号通路,从而对肿瘤的生长发挥重要的作用。而RHBDD1和EGFR之间其他可能的关系还尚无相关研究,本文围绕RHBDD1和EGFR的相互作用关系开展了深入的研究。我们首先研究了RHBDD1对肿瘤生长的影响。通过体外实验,利用结直肠癌细胞系研究发现,当RHBDD1敲除后细胞的生长增殖能力明显降低。接着在裸鼠体内皮下成瘤实验中观察到,在RHBDD1敲除的实验组中,肿瘤的体积明显减小,并且当在RHBDD1敲除的细胞系中恢复RHBDD1后,小鼠皮下肿瘤明显增大。接着观察了RHBDD1对肿瘤转移的影响。首先我们在临床结直肠癌术后复发的患者中发现,RHBDD1表达阳性和远端器官转移显著相关。接着应用细胞迁移和侵袭这两个经典的观察癌转移特性的实验发现,RHBDD1敲低后,结直肠癌细胞系的迁移和侵袭能力明显降低。我们采用免疫缺陷鼠尾静脉注射结直肠癌细胞系实验观察癌细胞的转移时发现,RHBDD1敲除或敲低时,形成转移灶的能力显著降低并且转移灶的体积显著减小。以上实验说明RHBDD1可以影响结直肠癌细胞的生长和转移,其可能在结直肠癌的发生和发展中发挥重要的作用。为了研究RHBDD1在结直肠癌发生发展过程中具体分子机制,我们通过免疫共沉淀(Co-immunoprecipitation, CO-IP)实验验证RHBDD1和EGFR的相互作用关系,发现RHBDD1可以与EGFR发生相互作用。通过进一步的实验发现,RHBDD1可以和EGFR家族的四个成员(EGFR、HER2、HER3和HER4)都发生相互作用。又通过免疫荧光实验(Immunofluorescence, IF)发现,RHBDD1和EGFR在细胞中存在共定位情况。我们检测了RHBDD1敲除后EGFR的蛋白表达,发现EGFR的蛋白表达量明显降低,并且HER2和HER4的表达量也明显降低。在敲低RHBDD1后也可以观察到EGFR蛋白表达减少。接着通过放线菌酮(cycloheximide,CHX)实验观察RHBDD1对EGFR蛋白稳定性的影响,发现当RHBDD1敲除后,EGFR蛋白质稳定性降低,并且RHBDD1恢复表达后,EGFR蛋白质的稳定性升高。我们对细胞给予EGF刺激,发现RHBDD1敲除的细胞EGFR降解加剧。同样在RHBDD1敲低的细胞中也观察到相同的现象。在观察RHBDD1敲低后EGFR下游信号通路变化时发现,p38蛋白质磷酸化水平升高。我们进一步观察了RHBDD1对EGFR mRNA的影响。我们发现当敲低RHBDD1后EGFR mRNA的表达显著降低,并且在RHBDD1敲除的细胞中重复实验时,观察到一致的现象。接着我们发现RHBDD1敲低和敲除后EGFR蛋白质和c-Jun蛋白质表达都明显减少。在RHBDD1敲除的细胞中外源表达c-Jun后发现EGFR表达升高,并且呈现剂量依赖性,并且检测到EGFR mRNA的表达显著升高。我们也在小鼠皮下的肿瘤组织中检测到RHBDD1敲低和敲除时,EGFR和c-Jun的蛋白质表达量均显著降低。接着在结直肠癌患者组织标本中,通过蛋白免疫印迹实验检测到RHBDD1、EGFR和c-Jun的表达水平趋势一致。综上所述,RHBDD1与EGFR存在相互作用的关系,发挥稳定EGFR蛋白质的作用。并且RHBDD1可以通过调节c-Jun来调节EGFR mRNA表达,从而影响EGFR在结直肠癌发生发展中的作用。
[Abstract]:Colorectal cancer is one of the most common cancers in the world and one of the most fatal cancers. When patients are diagnosed with colorectal cancer, about 1/4 of the patients have undergone metastasis in the body, and most of the colorectal cancer patients eventually develop to metastatic cancer. The 5 year survival rate of metastatic colorectal cancer is low and the mortality rate is high. Previous studies on the occurrence of colorectal cancer, especially the molecular mechanisms of metastasis, are not very detailed, and the treatment of colorectal cancer has failed to achieve good results. Therefore, the discovery and identification of genes associated with the occurrence and metastasis of colorectal cancer is very important for the study of the molecular mechanisms of the occurrence and metastasis of colorectal cancer. It not only helps us understand and understand colorectal cancer, but also provides a new idea for the treatment of colorectal cancer.Rhomboid domain containing protein 1 (RHBDD1) is a highly expressed new gene found in our early laboratory screening of testicular cDNA library, and first we named the gene HSD50 and submitted to GenBank.. Because it contains the rhomboid domain so that RHBDD1.RHBDD1 is a transmembrane protein, it expresses a full length 315aa and contains 6 transmembrane structures, including the conservative Rhomboid domain, which is one of the members of the Rhomboid family. According to our previous research in the laboratory, RHBDD1 can cut tumour suppressor activate. D pathway-6 (TSAP6), apoptosis precondition protein Bik and transforming growth factor alpha (transforming growth factor alpha, TGF-a). Our laboratory recently found that RHBDD1 has a high protein expression level in colorectal cancer, and it can activate the downstream letter of the epidermal growth factor receptor by cutting TGF-a. The pathway of RHBDD1 plays an important role in the growth of the tumor. Other possible relationships between the RHBDD1 and the EGFR are still not related. This article focuses on the interaction of RHBDD1 and EGFR. We first studied the effect of RHBDD1 on the growth of the tumor. Now, the cell growth and proliferation ability of the cells decreased significantly after RHBDD1 knockout. Then, the tumor volume decreased significantly in the RHBDD1 knockout experimental group, and the subcutaneous swelling of the mice increased significantly after the RHBDD1 knockout cell line. Then, the tumor metastasis was observed by RHBDD1. First of all, we found a significant correlation between RHBDD1 expression positive and distal organ metastasis in patients with recurrent rectal cancer after surgery. Then, the two classics of cell migration and invasion were used to observe cancer metastasis. After RHBDD1 knockout, the migration and invasiveness of colorectal cancer cell lines were significantly reduced. When the tumor cell line was injected into the tail vein of the immunodeficient rat, the metastatic lesion was significantly reduced and the volume of the metastases decreased significantly when RHBDD1 was knocked down or knocked down. The above experiment indicated that RHBDD1 could affect the growth and metastasis of colorectal cancer cells, which may be in the occurrence of colorectal cancer and the occurrence of colorectal cancer. In order to study the specific molecular mechanism of RHBDD1 in the development of colorectal cancer, we verified the interaction between RHBDD1 and EGFR through the Co-immunoprecipitation (CO-IP) experiment, and found that RHBDD1 can interact with EGFR. Further experiments found that RHBDD1 can and Four members of the EGFR family (EGFR, HER2, HER3, and HER4) were interacted with each other. By immunofluorescence (Immunofluorescence, IF), it was found that RHBDD1 and EGFR were Co located in the cells. We detected the EGFR protein expression after the RHBDD1 knockout, and found that the expression of the EGFR protein was significantly reduced and the expression of the RHBDD1 was also expressed. EGFR protein expression decreased after knocking down RHBDD1. The effect of RHBDD1 on the stability of EGFR protein was observed by Actinomycate (cycloheximide, CHX), and the stability of EGFR protein was reduced after RHBDD1 knockout, and the stability of EGFR protein was increased after RHBDD1 resumed. Stimulated by EGF, the EGFR degradation of RHBDD1 knockout cells increased. The same phenomenon was also observed in RHBDD1 knockout cells. The phosphorylation level of p38 protein increased when the downstream EGFR signaling pathway of RHBDD1 knocked low. We further observed the effect of RHBDD1 on EGFR mRNA. We found that EGF after low RHBDD1 EGF. The expression of R mRNA was significantly reduced and the same phenomenon was observed during repeated experiments in the RHBDD1 knockout cells. Then we found that the expression of EGFR protein and c-Jun protein decreased significantly after the RHBDD1 knockout and knockout. The expression of EGFR in the RHBDD1 knockout cells showed a rise in the EGFR expression and a dose-dependent manner. The expression of EGFR mRNA was significantly increased. We also detected a significant decrease in the protein expression of EGFR and c-Jun in the tumor tissues of the subcutaneous tissue of the mice. Then in the tissue specimens of the colorectal cancer patients, the expression of RHBDD1, EGFR and c-Jun was consistent with the protein immunoblotting test. To sum up, RHBDD1 and EGFR interact with each other to play a role in stabilizing EGFR proteins. And RHBDD1 can regulate the expression of EGFR mRNA by regulating c-Jun, thereby affecting the role of EGFR in the development of colorectal cancer.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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