CD147通过内化循环及贯序水解促进肝细胞肝癌恶性进展

发布时间：2018-04-29 13:27

  本文选题：CD147 + CD98　； 参考：《第四军医大学》2017年博士论文

【摘要】：原发性肝癌俗称“癌中之王”,恶性进展快,治疗难度大,病死率高,是一种非常凶险的疾病,世界范围内肿瘤致死率排名第三位,国内肿瘤致死率的排名第二位,对居民生命健康危害极大。CD147是早期我们科室筛选鉴定的肿瘤药靶分子,CD147在包括肝癌在内多种人类恶性肿瘤中表达,且其高表达是临床不良预后的指标,CD147既可以作为诱导因素,又可以作为效应分子通过不同的机制促进肿瘤细胞的生长代谢,浸润转移,促血管生成,耐药以及死亡抵抗等恶性行为,导致肿瘤进展。生理病理条件下,以跨膜形式定位于细胞膜表面的CD147可以内化进入胞浆而后重新循环到细胞膜上,内化到胞浆的CD147以囊泡形式存在;此外,CD147还可以外泌体的形式或者胞外段被水解后以游离形式(shedding)释放到细胞外基质中。近年来发现,内化循环异常是肿瘤恶性进展过程中获得的表型,这一特点已经逐渐被认可,但是CD147内化循环对于肿瘤进展具有什么意义?同时,CD147水解脱落胞外段之后剩余部分的去向以及有没有生理意义?这些问题的解答有助于我们进一步揭开CD147在肿瘤中发挥重要意义的神秘面纱,并为以CD147为靶点的药物研发提供理论依据。基于此,我们展开下面三部分实验:研究第一部分,我们从CD98与肝癌不良预后密切相关出发,首先证实了细胞膜表达的CD98通过β1-integrin及其下游信号通路在肝癌细胞的铺展和成瘤方面具有重要作用;然后基于CD147正向调节CD98的细胞膜表达水平的流式结果,以及证实CD147与CD98相互作用的共定位、pull-down和SPR等实验结果,推测CD147能够调节CD98的细胞膜定位;进而我们采用K7721细胞过表达荧光蛋白标记的CD98,激光共聚焦结果发现在缺失CD147的情况下,CD98阻滞在内质网以及Rab5阳性的早期内体,而恢复CD147之后,CD98又可以重新定位到细胞膜上;而进一步实验证实,CD147和CD98的内化步骤,也就是囊泡循环的步骤都是由Flotilin-1介导的,而同时也都受Arf6分子的调节。于是,CD147通过与CD98胞外段直接相互作用实现共转运,呈现协同膜表达的现象,而后促进β1-integrin及其下游信号通路的活化,在肝癌细胞铺展和成瘤方面发挥重要作用。因此,CD147能够调节CD98的细胞膜定位和囊泡循环机制解释了CD147和CD98的协同作用,并在肝癌进展中具有重要意义。研究第二部分,我们从CD147分子的内化现象出发,使用胆固醇抑制剂MβCD在阻断CD147内化之后,流式结果却发现CD147在细胞膜上的表达水平反而下降;通过进一步研究,我们发现细胞膜胆固醇水平下降之后,ADAM10对CD147的膜外切割作用(shedding)增强,而使用HAb 18抗体能够阻断这一过程;在分别阻断蛋白酶体降解途径和溶酶体降解途径之后,我们发现CD147胞外切割之后剩余的片段会进一步走向溶酶体处理。于是,CD147的胞外切割以及随后的溶酶体降解可能组成了一种CD147的降解途径。但是,也有可能CD147的胞内段在经历贯续水解之后发挥更为重要的生理意义。研究第三部分,我们首先分析发现CD147胞内段存在一个经典的核定位信号序列,且CD147胞内段能够指引融合的荧光蛋白定位到细胞核;然后,我们通过构建GFP-CD147-mCherry载体并转染入HEK293T细胞,发现由于CD147的贯续水解,CD147胞内段能够指引融合的mCherry定位到细胞核;通过自噬诱导、自噬阻断实验,我们发现CD147胞内段的产生很可能与肝癌细胞自噬过程相关,过表达CD147胞内段之后,肝癌细胞的自噬过程进一步增强;通过荧光报告基因筛选、转录组基因芯片分析以及RT-PCR和Western blot验证,CD147胞内段可能抑制NF-κb活性以及NF-κB-TRAIL-caspase8-ATG3通路使肝癌细胞具有自噬倾向;而具有自噬倾向的肝癌细胞在一定程度上赋予了肝癌细胞化疗药物顺铂抵抗的能力;鉴于HAb18在一定程度上阻断了CD147的贯续水解,我们发现顺铂联合HAb18能够显著抑制肝癌细胞生长。综上所述,我们的研究证实了CD147通过内化循环促进了CD98的膜定位表达,以及其下游的integrin信号通路,在肝癌细胞的粘附以及裸鼠的皮下成瘤方面发挥了重要作用;通过探寻CD147的贯续水解现象,我们发现CD147是在ADAM10和溶酶体的联合作用下,实现了分步切割,这可能是细胞蛋白降解维持稳态的机制;最后我们通过体外过表达CD147胞内段分析发现,具有核定位作用的CD147胞内段通过自噬促进作用增强了肝癌细胞的顺铂耐药能力,HAb18通过抑制CD147的贯续水解,在化疗药物的增敏作用中发挥显著的作用。
[Abstract]:Primary liver cancer, commonly known as "the king of cancer", is fast progresses, difficult to treat, and has a high mortality rate. It is a very dangerous disease. The death rate of tumor in the world ranks third, the death rate in domestic tumor is second, and the damage to life and health of the residents.CD147 is a cancer drug target in the early section of our department, CD1 47 expression in a variety of human malignant tumors, including liver cancer, and its high expression is an indicator of poor clinical prognosis. CD147 can be used as an inducer and can be used as an effector to promote the growth and metabolism of tumor cells, infiltration, angiogenesis, resistance and death resistance. In physiological and pathological conditions, the CD147 that is located on the surface of the cell membrane in the form of transmembrane can be internalized into the cytoplasm and then recirculated to the cell membrane, and the CD147 internalized into the cytoplasm exists in the form of vesicles; in addition, CD147 can also be released into the extracellular matrix in the form of Exocyst or the extracellular segment after the hydrolysis of the extracellular segment (shedding). In recent years, it has been found that the characteristics of the malignant progression of the tumor are the phenotype of the tumor, and this characteristic has been recognized, but what is the significance of the CD147 internalization cycle for the progression of the tumor? At the same time, are there any physiological significance of the remaining part after the CD147 hydrolysis off the extracellular segment? These questions help us To further uncover the mystical veil that plays an important role in CD147 and to provide a theoretical basis for the research and development of drugs targeting CD147. Based on this, we launched the following three experiments: Part 1, we start from the close relationship between CD98 and the poor prognosis of liver cancer, and first confirmed that the CD98 of cell membrane expressed through beta 1-integrin and The downstream signal pathway plays an important role in the spreading and tumorigenesis of the hepatoma cells, and then based on the flow results of CD147 forward regulation of the expression level of the cell membrane of CD98, and the co localization of the interaction between CD147 and CD98, pull-down and SPR and other experimental results, we speculate that CD147 can regulate the cell membrane location of CD98; and then we use K77. The 21 cells overexpressed the fluorescent protein labeled CD98, and the laser confocal results showed that in the absence of CD147, CD98 blocked the endoplasmic reticulum and Rab5 positive early endosomes, and after the recovery of CD147, CD98 could be relocated to the cell membrane; and further experiments confirmed that the steps of the internalization of CD147 and CD98, that is, the steps of the vesicle cycle. It is mediated by Flotilin-1 and is also regulated by the Arf6 molecule. Thus, CD147 can be transported by direct interaction with the CD98 extracellular segment, presenting the phenomenon of CO membrane expression, and then promoting the activation of the beta 1-integrin and its downstream signal pathway, and plays an important role in the spreading and tumorigenesis of the hepatoma cells. Therefore, CD147 can be adjusted. The cell membrane localization and vesicular circulation mechanism of CD98 explains the synergy between CD147 and CD98, and is of great significance in the progression of liver cancer. The second part of the study is based on the internalization of CD147 molecules. After the use of the cholesterol inhibitor M beta CD to block the CD147 internalization, the flow results show that the expression level of CD147 on the cell membrane is reversed. After further study, we found that after the decrease of the cholesterol level of the cell membrane, ADAM10 enhanced the extracellular cutting effect of CD147 (shedding), and the use of HAb 18 antibody could block the process. After blocking the proteasome degradation pathway and the lysosome degradation path, we found the remaining fragments after the CD147 extracellular cutting. The exosomal cleavage of CD147 and the subsequent degradation of lysosome may lead to a CD147 degradation pathway. However, it is possible that the intracellular segment of CD147 may play a more important physiological role after the continuous hydrolysis. The third part of the study is that we first found that there is one of the CD147 intracellular segments. The classical nuclear localization signal sequence, and the CD147 intracellular segment can guide the fusion fluorescent protein to the nucleus; then, we construct the GFP-CD147-mCherry vector and transfect into the HEK293T cell, and find that the CD147 intracellular segment can guide the fused mCherry to the nucleus because of the continuous hydrolysis of the CD147; by autophagy induction, autophagy is blocked. We found that the production of CD147 cells is likely to be related to the autophagy process of hepatoma cells. After overexpression of the CD147 intracellular segment, the autophagy process of the hepatoma cells is further enhanced; the fluorescence report gene screening, transcriptional gene chip analysis, and RT-PCR and Western blot verification, the intracellular segment of CD147 may inhibit the activity of NF- kappa B and NF- kappa. B-TRAIL-caspase8-ATG3 pathway makes hepatoma cells autophagic, and autophagic cells with autophagy tend to give liver cancer cell chemotherapeutic drugs cisplatin resistance to some extent. In the light of HAb18 blocking the sequential hydrolysis of CD147 to a certain extent, we found that cisplatin combined with HAb18 can significantly inhibit the growth of hepatoma cells. Our study confirms that CD147 promotes the membrane localization of CD98 through the internalization cycle, as well as the downstream integrin signaling pathway, which plays an important role in the adhesion of hepatoma cells and the subcutaneous tumor formation of nude mice. By exploring the sequential hydrolysis of CD147, we found that CD147 is combined with ADAM10 and lysosomes. In addition, step cutting is realized, which may be the mechanism of cell protein degradation to maintain homeostasis. Finally, through overexpression of CD147 intracellular segment analysis in vitro, we found that the CD147 intracellular segment with nuclear localization enhanced the cisplatin resistance of hepatoma cells through autophagy, and HAb18 was used to inhibit the sequential hydrolysis of CD147 by inhibiting the sequential hydrolysis of the chemotherapeutic drugs. It plays a significant role in the sensitization effect.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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本文编号：1820121