人类乳腺癌与病毒感染相关性以及基因治疗靶点的研究

发布时间：2018-04-27 21:32

  本文选题：乳腺癌 + HPV　； 参考：《中国疾病预防控制中心》2016年博士论文

【摘要】：乳腺癌已经成为全球范围女性最常见的恶性肿瘤之一,其发病率逐年增高,数据显示,在美国女性中2014年新增癌症病例数中乳腺癌占比达到29%。我国近年来乳腺癌发病率正以每年3%的速度递增,在所有癌症中乳腺癌的发病率位居第一,成为城市中死亡率增长最快的癌症。乳腺癌的发病原因复杂,至今尚不能用已知的单因素或多因素模型来完全解释其发生原因与发展过程。流行病学研究认为乳腺癌的发生与生活环境、生活方式,膳食结构,内分泌等因素有关。近年来,对病毒感染与乳腺癌病因关系的研究成为研究的热点。1995年国际癌症组织发布高风险的人乳头瘤病毒(Human papillomavirus简称HPV)16型和18型具有导致人类癌症的作用,有研究证实HPV的E6和E7基因导入乳腺导管上皮细胞导致细胞永生化或癌变。由于HPV亚型众多,不同的亚型致病力不同,以及检测方法、标本类型、人群地域分布等差异致使文献报道检测结果阳性率的差异,使HPV感染与乳腺癌发生的相关性存在着争议。小鼠乳腺肿瘤病毒(Mouse mammary tumor virus简称MMTV)是一种p逆转录病毒,含有9kb的RNA基因组,其中包含GAG基因、POL基因以及编码病毒进入宿主细胞时需要的包膜蛋白的ENV基因。研究证实90%以上的小鼠乳腺肿瘤与MMTV相关。Indik等学者发现MMTV可感染人类乳腺癌细胞系,这一研究证实了MMTV跨物种传播的可能性。但是,至今没有MMTV感染人类乳腺细胞致瘤的直接证据,更没有以HPVE6和E7病毒基因为靶点的特异性基因治疗的先例。明确病毒基因在乳腺癌组织中的存在以及表达状况,探索以病毒基因为靶点的乳腺癌基因治疗和疫苗研究的新策略,对于预防乳腺癌的发生降低乳腺癌的死亡率具有重要的意义。本研究分为两部分：第一部分是对乳腺癌石蜡包埋组织标本以及乳腺癌细胞系SKBR3进行了人乳头瘤病毒即HPV和小鼠乳腺肿瘤病毒即MMTV的基因筛查；第二部分在筛查结果的基础上应用RNA干扰技术沉默HPV致癌基因,探索以病毒基因为靶点对乳腺癌细胞株的抑制作用。第一部分乳腺癌细胞中人乳头瘤病毒和小鼠乳腺肿瘤病毒基因的筛查收集76例病理学确诊的乳腺癌石蜡包埋组织提取DNA,以乳腺癌细胞系SKBR3为对照,分别设计合成HPV L1、HPV16 E6、HPV16 E7、HPV18 E6、HPV18 E7以及MMTV-ENV基因的引物,聚合酶链式反应(PCR)扩增基因片段。在76例乳腺癌石蜡包埋组织样本中,HPV16型E6、E7皆为阴性。HPV L1阳性7例(9.21%)。HPV18 E6阳性5例(6.58%),HPV18 E7阳性18例(23.68%),在SKBR3细胞系中HPV16E6、E7均为阴性,HPV18E6、E7为阳性,并有HPV18E6、E7的基因表达。对照样本的临床病理资料显示HPV18E6和E7在乳腺癌细胞基因的检出率与年龄无关,而与乳腺癌分级以及乳腺细胞的分化程度有关；在19例HPV18E6、E7阳性的患者中均为浸润性导管癌,随着浸润程度级别的增加,HPV18E6、E7的检出率增加。其中6例为低分化的乳腺细胞癌。小鼠乳腺肿瘤病毒基因的筛查阳性23例,阳性率为30.26%。这项研究为人乳头瘤病毒感染与乳腺癌的发生存在相关性提供了依据,为探索以病毒基因为靶点的乳腺癌基因治疗和疫苗研究的新策略提供依据,从HPV18E6、E7基因在不同类型乳腺癌中的检出率可以预测以病毒基因为靶点的基因治疗对不同类型乳腺癌的有效性,为临床的应用提供了依据。第二部分探索以病毒基因为靶点对乳腺癌细胞株抑制作用的研究在第一部分研究结果的基础上,以HPV18E6 E7为靶点应用bioinformatics research computing提供的在线siRNA设计工具设计合成siRNA,构建入pSUPER RNAi System,筛选出最优的siRNA,检测以HPV18 E6、E7为靶点的RNA干扰对乳腺癌细胞的生长活力、克隆形成能力、侵袭能力、细胞周期以及细胞周期调控基因的表达情况分析对乳腺癌细胞的抑制作用。细胞生长活力的检测试验显示,与对照组相比48h时针对HPV18 E6、E7的RNA干扰组抑制率分别是12%和9%(差异显著,P0.05)。克隆形成试验结果显示,与对照组相比针对HPV18E6 E7的RNA干扰组抑制率分别是61%和64%(差异显著,P0.01)。细胞侵袭试验显示,与对照组相比针对HPV18 E6、E7的RNA干扰组抑制率分别是59%和65%(差异显著,P0.01)；应用流式试验测定细胞周期变化,与对照组相比HPV18 E6E7的RNA干扰组的S%降低65%和80%(差异显著,P0.01),GO/G1升高26%和35%(差异显著,P0.01),G2/M降低50%和72%(差异显著,P0.01)；应用荧光定量PCR测定细胞株中HPV18 E6、 E7靶基因以及细胞周期调控基因的表达差异,与对照组相比,HPV18 E6的RNA干扰组对E6的抑制率为35%,HPV18 E7的RNA干扰组对E7的抑制率为78%,与对照组相比HPV18 E6、E7的RNA干扰组的TP53-mutant表达降低55%和39%, MDM2表达降低63%和36%, CCNA1表达降低7%和15%, CCND1表达降低18%和23%,BCL-2表达降低39%和77%,RB表达增高94%和109%, VEGFA表达没有发生明显变化。以HPV18E6 E7为靶点的RNA干扰对乳腺癌细胞的抑制作用明显,为乳腺癌基因治疗提供了新的靶点。
[Abstract]:Breast cancer has become one of the most common malignant tumors in the world. The incidence of breast cancer is increasing year by year. The data show that in 2014, the number of new cases of cancer in the United States has reached 29%., the incidence of breast cancer in China is increasing at a rate of 3% in recent years, and the incidence of breast cancer is the first in all cancers. It has become the fastest growing cancer in the city. The causes of breast cancer are complex. So far, a known single factor or multi factor model can not be used to fully explain the cause and development process. Epidemiological studies suggest that the occurrence of breast cancer is related to the living environment, lifestyle, dietary structure, endocrine and other factors. The research on the relationship between virus infection and the cause of breast cancer has become a hot spot of research in.1995..1995 international cancer organization published high risk human papillomavirus (Human papillomavirus for short HPV) type 16 and type 18 have the effect on human cancer. It has been proved that HPV's E6 and E7 genes are introduced into mammary gland ductal epithelial cells to lead to cell immortalization or cancer Because of the diversity of HPV subtypes, different subtypes of pathogenicity, detection methods, specimen types, and regional distribution of the population, the difference in the positive rate of the results of the literature report results in the correlation between the HPV infection and the incidence of breast cancer. The Mouse mammary tumor virus (MMTV) is a P inverse. The transcriptional virus, which contains the RNA genome of 9KB, contains the GAG gene, the POL gene, and the ENV gene of the envelope protein needed to encode the virus into the host cell. The study confirmed that more than 90% of the mice breast tumor and MMTV related.Indik have found that MMTV can infect human breast cancer cell lines. This study confirmed the spread of MMTV across species. But there is no direct evidence of MMTV infection in human breast cells, and there is no precedent for specific gene therapy targeting HPVE6 and E7 virus genes. The strategy is of great significance for preventing the occurrence of breast cancer and reducing the mortality of breast cancer. This study is divided into two parts: the first part is the screening of paraffin embedded tissue specimens and breast cancer cell line SKBR3 for human papillomavirus (HPV) and mouse mammary tumor virus (MMTV) gene screening; the second part is screened. On the basis of the results, the RNA interference technique was used to silence the HPV oncogene and explore the inhibitory effect of the virus gene on the breast cancer cell lines. In part 1, the screening of human papillomavirus and mouse breast tumor virus gene in breast cancer cells was used to collect 76 cases of pathological diagnosis of breast cancer with paraffin embedded tissue and extract DNA for breast cancer. HPV L1, HPV16 E6, HPV16 E7, HPV18 E6, HPV18 E7 and MMTV-ENV gene primers, polymerase chain reaction (PCR) amplification gene fragment were designed, respectively. In 76 cases of paraffin embedded tissue samples of breast cancer, 7 cases (9.21%) positive were positive (6.58%), 18 cases (23) positive (23). .68%), in the SKBR3 cell line, HPV16E6, E7 were negative, HPV18E6, E7 were positive, and HPV18E6, E7 gene expression. The clinicopathological data of the control samples showed that the detection rate of HPV18E6 and E7 in the breast cancer cell genes was not related to age, but related to the classification of breast cancer and the degree of differentiation of mammary cells; in 19 HPV18E6, E7 positive patients All of them were infiltrative ductal carcinoma. With the increase of degree of infiltration, the detection rate of HPV18E6 and E7 increased. 6 of them were low differentiated breast cancer. 23 cases of mouse breast tumor virus gene screening were positive, and the positive rate was 30.26%.. The study provided the basis for the correlation of human papillomavirus infection and breast cancer. Explore new strategies for gene therapy and vaccine research that target virus genes. The detection rate of HPV18E6 and E7 gene in different types of breast cancer can predict the effectiveness of gene therapy targeting different types of breast cancer for different types of breast cancer, and provide the basis for clinical application. The second part explores the disease. On the basis of the results of the first part of the study, the research on the inhibitory effect of the target on the breast cancer cell line is based on the results of the first part of the study. HPV18E6 E7 is used as an on-line siRNA design tool provided by the bioinformatics research computing as a target. The pSUPER RNAi System is constructed and the optimal siRNA is screened. The inhibitory effects of interference on breast cancer cell growth activity, clonogenic ability, invasion ability, cell cycle and cell cycle regulation gene expression analysis on breast cancer cells. Detection test of cell growth activity showed that the inhibition rates of RNA interference group for HPV18 E6 compared with the control group were 12% and 9% respectively (the difference was 12% and 9%, respectively). Significant, P0.05). The results of clone formation test showed that the inhibition rates of RNA interference group for HPV18E6 E7 were 61% and 64% respectively compared with the control group (significant difference, P0.01). Cell invasion test showed that the inhibition rates of RNA interference group of E7 were 59% and 65%, respectively compared with the control group, compared with the control group (the difference was significant, P0.01); the flow test was used to determine the cells. Compared with the control group, the S% of the RNA interference group in the HPV18 E6E7 group decreased by 65% and 80% (the difference was significant, P0.01), the GO/G1 increased by 26% and 35% (P0.01), the G2/M decreased by 50% and 72% (the difference was significant, P0.01). The differential expression of the HPV18 E6, the target gene and the cell cycle regulation gene in the cell lines was measured by the fluorescent quantitative PCR, and the control group was compared with the control group. Compared with the RNA interference group of HPV18 E6, the inhibition rate of E6 was 35%, and the inhibition rate of RNA interference group of HPV18 E7 was 78%, compared with the control group, the expression decreased by 55% and 39%, the expression decreased by 63% and 36%, the expression decreased by 7% and 15%, the expression decreased 18% and 23%, and the expression decreased 39% and 77% expression. The expression of VEGFA was increased by 94% and 109%, and there was no obvious change in expression. The inhibitory effect of RNA interference targeting HPV18E6 E7 on breast cancer cells was obvious, which provided a new target for the gene therapy of breast cancer.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9;R450
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本文编号：1812405