拷贝数变异在肝癌性别差异中的作用及形成机制研究

发布时间：2018-06-22 11:19

本文选题：肝细胞肝癌 + 性别差异　；参考：《第二军医大学》2016年博士论文

【摘要】：原发性肝细胞肝癌(Hepatocellular carcinoma,HCC),作为最常见的肝癌类型,是一种世界范围内的致死性恶性肿瘤,具有高复发、高转移的特点。流行病学调查资料显示,肝癌具有显著的性别差异,其中男性与女性的肝癌发病比例约为2:1~4:1,并且在不同原因导致的肝癌中,男性患者的比例也均高于女性患者。不仅在发病率上,男性明显高于女性,在预后方面,男性的生存时间与生存率也显著低于女性。对于肝癌性别差异的解释,目前主要集中于性激素和细胞因子,然而针对雌雄激素受体通路和炎症干预的治疗方案在临床上并未取得满意的结果。一方面归因于现有的很多研究成果来源于化学诱导的动物模型,并不能完全模拟临床上的肝癌,尤其是由于HBV和HCV感染导致的肝癌;另一方面是由于肝癌性别差异的原因和机制并不完全清楚,尤其是基因组层面的变化并不了解。拷贝数变异(Copy number variations,CNVs)作为最重要的基因组变异之一,与各种遗传性和发育性疾病密切相关,是肿瘤形成的重要原因。拷贝数变异可以通过影响变异区域内重要的癌基因和抑癌基因的基因组含量,导致这些重要调控基因的表达异常,从而导致肿瘤的发生,而拷贝数变异在肝癌发生发展中所发挥的重要作用也越来越受到人们的重视。然而在肝癌的性别差异方面,对于男女基因组层面的差异却很少有研究涉及。对于性别相关的基因组变异区域的确定,对性别相关基因组变异的意义研究,以及对基因组变异的性别差异原因的探索,将会增加我们对于肝癌性别差异的认识,补充完善肝癌性别差异的机制,为肝癌的预防和治疗提供新的思路。在本研究中,我们重新分析了网上公共数据库中一组大规模的肝癌基因组芯片数据,将肝癌的基因组数据与病人的性别资料进行了匹配,筛选得到性别特异性的拷贝数变异区域,首先确定了肝癌中确实存在基因组变异的性别差异。我们进一步将性别相关的拷贝数变异区域与我们的一组RNA测序结果进行了联合分析,筛选得到了性别相关的拷贝数变异基因,作为我们研究拷贝数变异性别差异意义的候选基因集。之后我们在一组肝癌队列中对芯片结果进行了q PCR的验证,发现其中UBE2D1在基因组水平和转录水平均存在显著的性别差异。因此我们靶定UBE2D1作为研究拷贝数变异性别差异的关键基因。我们首先确定了UBE2D1的临床意义,发现UBE2D1在肝癌中存在基因组的复制增加,并且基因组的复制增加导致了其在肝癌组织中的高表达。UBE2D1的高表达与肝癌病人较差的总体生存率相关。进一步探索UBE2D1的生物学功能发现,UBE2D1可以促进正常肝细胞和肝癌细胞的增殖,增强克隆形成能力,抑制细胞凋亡,并且促进肝癌细胞的体内成瘤能力。机制研究发现,UBE2D1是通过增强p53的泛素化,降低了p53的蛋白水平,进而抑制了p53依赖的细胞衰老过程。在确定肝癌中性别相关拷贝数变异的意义基础上,我们进一步探索了拷贝数变异性别差异的形成原因。我们发现IL-6、IL-8、IL-1β和TNF-α在肝癌的癌旁组织中存在显著的性别差异。之后我们利用复制压力和染色体不稳定性作为评判拷贝数变异形成的指标,发现IL-6可以最为明显的诱导细胞的复制压力反应和染色体不稳定性,而持续的IL-6刺激可以导致染色体不稳定性的显著增强,并增加UBE2D1的基因组含量。不同于短期IL-6刺激,我们发现长期IL-6刺激会导致不依赖于外源细胞因子刺激的自分泌增加,说明一定时间的IL-6刺激对于这一过程是必须的。机制研究发现,长期IL-6刺激是通过Stat3抑制了Rad51b的转录激活,进而维持了复制压力反应,积累了染色体不稳定性。长期的IL-6刺激可以显著促进细胞增殖,抑制细胞凋亡,增强肝癌细胞的体内成瘤能力和正常肝细胞的恶性转化。而Stat3和Rad51b的干预会逆转对复制压力和染色体不稳定的影响,逆转长期IL-6的生物学功能。我们进一步探讨了IL-6-Stat3-Rad51b这一调控轴的临床意义,发现Rad51b在肝癌中显著低表达,并且这种低表达在癌前病变就已出现。根据病人血清中IL-6水平进行分组,发现高的血清IL-6水平组的病人Rad51b下调更为明显,而UBE2D1的基因组复制增加也更为显著。而Rad51b在癌旁组织中低表达是肝癌病人预后的危险因素,其表达量与Stat3显著负相关,与UBE2D1的表达水平也存在负相关。通过我们的研究,我们得到了以下的结论:首先,在肝癌中确实存在着基因组层面的性别差异,确定了男女在拷贝数变异水平的不同;其次,UBE2D1作为性别相关拷贝数变异的关键基因,在男性中的基因组水平显著高于女性,并且可以通过调控p53泛素化下调p53蛋白水平,进而促进肝癌发生发展;第三,肝癌中,拷贝数变异的性别差异是由于肿瘤微环境中IL-6的差异,长期IL-6刺激可以通过Stat3抑制Rad51b,维持复制压力反应,增强染色体不稳定性,导致了UBE2D1基因组水平的增加,并且这一过程是个长期作用结果,较长时间的IL-6刺激和Rad51b的作用是这一过程所必需的。通过对肝癌中拷贝数变异性别差异的研究,可以加深我们对于肝癌性别差异在基因组层面的认识,并且完善了肝癌中拷贝数变异性别差异的形成机制,作为性别相关拷贝数变异的关键基因UBE2D1和形成这一差异的关键因子Rad51b,可以作为诊断肝癌的标志物和治疗肝癌的潜在药物靶定。
[Abstract]:Primary hepatocellular carcinoma (Hepatocellular carcinoma, HCC), as the most common type of liver cancer, is a worldwide fatal malignant tumor, characterized by high recurrence and high metastasis. Epidemiological data show that liver cancer has significant gender differences, and the incidence of liver cancer in males and females is about 2:1~4:1, and The proportion of male patients in different causes of liver cancer is also higher than that of female patients. Not only in the incidence rate, men are obviously higher than women, but also in the prognosis, the survival time and survival rate of men are also significantly lower than that of women. No satisfactory results have been achieved in the treatment of the vegetarian receptor pathway and inflammatory intervention. One reason is that many of the existing research results are derived from chemical induced animal models and can not fully simulate the clinical liver cancer, especially the liver cancer caused by HBV and HCV infection, and the other is the origin of the gender differences in liver cancer. Copy number variations (CNVs), one of the most important genomic variations, is closely related to various genetic and developmental diseases, and is an important cause of tumor formation. The genomic content of oncogene and tumor suppressor gene leads to the abnormal expression of these important regulatory genes, which leads to the occurrence of tumor, and the important role of the copy number variation plays a more and more important role in the development and development of liver cancer. However, there is little difference in gender difference between male and female genomes. There are studies involved. The identification of genomes related genomes, the significance of sex related genome variation and the reasons for gender differences in genome variation will increase our understanding of the gender differences in liver cancer, complement the mechanisms of the gender differences in liver cancer, and provide new prevention and treatment for the liver cancer. In this study, we reanalyzed a group of massive liver cancer genome chip data in the public database of the Internet, matching the genome data of the liver cancer with the patient's sex data, screening the sex specific copy number variation region, and first determined the gender difference of the genomic variation in the liver cancer. We further analyzed the sex related copy number variation region and our group of RNA sequencing results, and screened the sex related copy number variant gene, as a candidate gene for our study of the gender differences in the copy number variation. After that, we conducted a Q PC for the results of the chip in a group of liver cancer groups. R proved that there was a significant gender difference between the genomic level and the transcriptional level of UBE2D1. Therefore, we targeted UBE2D1 as the key gene to study the gender differences in the copy number variation. We first identified the clinical significance of UBE2D1, and found that the replication of the gene group in the liver cancer was increased in UBE2D1 and the replication of the genome was increased. The high expression of the high expression of.UBE2D1 in the liver cancer tissues is associated with the poor overall survival rate of the liver cancer patients. Further exploring the biological function of UBE2D1, UBE2D1 can promote the proliferation of normal hepatocytes and hepatoma cells, enhance the clone formation ability, inhibit the apoptosis of the cell and promote the tumor formation in the liver of the liver cancer cells. Ability. The mechanism study found that UBE2D1, by enhancing the ubiquitination of p53, reduced the protein level of p53 and then inhibited the cell aging process of p53 dependent. On the basis of determining the significance of the mutation of the neutrophilic copy number of liver cancer, we further explored the causes of the gender differences in the copy number variation. We found that IL-6, IL-8, IL-1 beta and IL-1 beta. TNF- alpha has significant gender differences in the adjacent tissues of the carcinoma of the liver. Then we use the replication pressure and chromosome instability as an indicator of the copy number variation. It is found that IL-6 can most significantly induce the replicative stress response and chromosome instability of the cells, and the persistent IL-6 stimulation can lead to chromosome instability. A significant increase in sex and an increase in the genomic content of UBE2D1. Unlike short-term IL-6 stimuli, we found that long-term IL-6 stimulation can lead to an autocrine increase that is not dependent on exogenous cytokines stimulation, indicating that a certain time of IL-6 stimulation is necessary for this process. Mechanism research is developed, and long-term IL-6 stimulation is the inhibition of Rad51b through Stat3. The transcriptional activation, which then maintains the replicative stress response, accumulates chromosome instability. Long-term IL-6 stimulation can significantly promote cell proliferation, inhibit cell apoptosis, enhance the tumorigenicity of hepatoma cells and malignant transformation of normal liver cells. Stat3 and Rad51b may reverse the replication pressure and chromosome instability. Effect, reversing the biological function of long-term IL-6. We further explored the clinical significance of the IL-6-Stat3-Rad51b regulation axis, and found that Rad51b was significantly lower in the liver cancer, and this low expression appeared in precancerous lesions. According to the level of IL-6 in the serum of the patients, the patients with high serum IL-6 level were found to be down regulated. It is more obvious that the genomic replication of UBE2D1 is more significant. And the low expression of Rad51b in the para cancerous tissue is a risk factor for the prognosis of the patients with liver cancer, and the expression is negatively correlated with the Stat3, and there is a negative correlation with the expression level of UBE2D1. The gender differences at the genome level determine the difference in the level of copy number variation between men and women. Secondly, UBE2D1 is the key gene of the sex related copy number variation, the genome level of the male is significantly higher than that of the female, and it can reduce the level of p53 protein by regulating the p53 ubiquitination, and then promote the development and development of liver cancer; third, liver cancer. The sex difference of the copy number variation is due to the difference in the IL-6 in the tumor microenvironment. The long-term IL-6 stimulation can inhibit Rad51b through Stat3, maintain the replicative stress response and enhance the chromosome instability, which leads to the increase of the UBE2D1 genome level, and this process is a long-term result, the longer IL-6 stimulation and the role of Rad51b. It is necessary for this process. Through the study of the gender differences in the copy number variation in the liver cancer, we can deepen our understanding of the gender difference at the genome level and improve the formation mechanism of the sex difference of the copy number variation in the liver cancer, as the key gene UBE2D1 of the sex related copy number variation and the difference in the formation of the sex related copy number. The key factor Rad51b can be used as a marker for the diagnosis of liver cancer and a potential drug target for the treatment of liver cancer.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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