运用整合组学研究HPV与宫颈癌
发布时间：2018-07-13 12:08
【摘要】：宫颈癌是女性生殖系统最常见的恶性肿瘤之一。每年全球新发病例超过50万,且其中80%来自发展中国家。根据中国卫计委官方网站公布的2011年卫生统计年鉴数据显示,我国2010年宫颈癌总患病率为15.1/10万人,与往年相比呈上升趋势。宫颈癌以鳞癌为主,其次为腺癌,有文献报道同期的宫颈腺癌与宫颈鳞癌相比生存率更低,预后更差。流行病学调查研究显示,高危型人乳头瘤病毒(Human papillomavirus, HPV)感染是宫颈癌发生的重要启动因子。乳头瘤病毒是增殖在皮肤和粘膜层状细胞上的一种小DNA病毒。按照临床分析,人乳头瘤病毒可以分为高危型和低危型两大类。高危型HPV或称致瘤HPV能够导致癌症；目前已经鉴定出了12种以上的高危型HPV,其中HPV16和HPV18与约70%HPV引起的宫颈癌有关。虽然过去的研究已经明确了高危型HPV与宫颈癌之间的相关性,然而对于高危型HPV的致癌机理,尚缺乏对HPV病毒影响宿主细胞的宏观认识。而伴随着测序技术和高分辨率质谱技术的日益成熟,人们有机会通过高通量测序和全蛋白质谱分析等手段对HPV的致癌机制进行更为全面的认识。据此,我们将上述两种新技术与其他分子生物学技术整合应用,获得了宫颈癌,HPV表达细胞和宫颈癌细胞系的转录组,蛋白组信息,并对HPV在以上两个层面所发挥的致癌作用进行了初步的探究。目的： 获得宫颈癌组织样本或HPV癌基因表达细胞/宫颈癌细胞系的转录组,蛋白组信息,比较它们与正常组织或正常细胞系之间的差异,并检测HPV与这些差异之间的相关性。 方法： 1.对HPV癌基因细胞系进行RNA测序：对稳定表达高危型HPVE7基因的NIKS-E7及其对照NIKS-Vector细胞进行RNA提取,对所得RNA进行测序并定量。 2.对RNA测序结果进行生物信息学分析：从RNA测序的结果中挑选出E7细胞与Vector细胞有明显表达差异,且能够表达为蛋白的基因。对这些基因进行GO (Gene Ontology)聚类。并用RTq-PCR验证RNA测序结果准确性。 3..对宫颈癌细胞系进行蛋白样本制备：分别从宫颈鳞癌SiHa和宫颈腺癌HeLa细胞系中提取细胞全蛋白,并用FASP方法进行蛋白酶解。 4.对宫颈癌细胞系进行蛋白组分析：将上述两种细胞系的肽段进行LCMS分析,并对所得质谱数据进行非标记定量,对定量数据进行生物信息学分析。 结果： 1.经过RNA测序,共发现236个基因在有HPV癌基因E7的细胞中与对照细胞相比存在明显差异表达,其中高表达基因150个,低表达基因86个 2.经过GO聚类分析,高表达基因中38%与细胞周期相关,30%与DNA代谢相关；低表达基因中约15%与细胞增殖调节相关,14%与细胞稳态相关。 3.经过质谱分析,在HeLa和SiHa之中共鉴定到2373种蛋白,其中在HeLa细胞中共鉴定到符合多肽可信度假阳性率(FDR)≤0.05的蛋白1401种。在SiHa细胞中共鉴定到符合多肽可信度FDR≤0.05的蛋白1620种。 4.在上述蛋白中,在两种细胞中有明显表达差异的蛋白,满足p≤0.05的蛋白共787种,其中HeLa高表达蛋白401种,HeLa低表达蛋白386种。HeLa高表达蛋白中有10.8%的蛋白与提高产能代谢相关,在Hela低表达的蛋白中,有14.3%的蛋白同调节细胞凋亡相关。 结论： 1.HPV癌基因能够促进细胞增殖和细胞代谢,从而促进癌症的发生。 2.HPV感染所导致的腺癌和鳞癌有明显的蛋白表达差异,这些差异很可能是导致腺癌预后不良的重要原因。
[Abstract]:Cervical cancer is one of the most common malignant tumors in the female reproductive system. More than 500 thousand of new cases in the world are emerging every year, and 80% of them come from developing countries. According to the 2011 Health Statistics Yearbook published by the official website of China's Health Planning Commission, the total prevalence rate of cervical cancer in 2010 is 15.1/10 million people, and the cervical cancer is on the rise compared with the past year. Cancer is mainly squamous cell carcinoma, followed by adenocarcinoma. It is reported that the survival rate of cervical adenocarcinoma is lower than that of cervical squamous cell carcinoma in the same period, and the prognosis is worse. Epidemiological study shows that high risk human papillomavirus (Human papillomavirus, HPV) infection is an important promoter of cervical cancer. Papillomavirus is proliferating in the skin and mucous layer. A small DNA virus on the form of a cell. According to clinical analysis, human papillomavirus can be classified into two major categories of high risk and low risk types. High risk HPV or tumorigenic HPV can lead to cancer; more than 12 high risk types of HPV have been identified, of which HPV16 and HPV18 are associated with cervical cancer caused by about 70%HPV. Although previous studies have shown that The correlation between high risk HPV and cervical cancer is confirmed. However, for the carcinogenic mechanism of high-risk HPV, there is still a lack of macro understanding of the HPV virus affecting host cells. With the growing maturity of sequencing and high resolution mass spectrometry, people have the opportunity to use high throughput sequencing and total protein mass spectrometry for HPV carcinogenic machines. On this basis, we integrate the two new technologies and other molecular biology techniques to obtain the cervical cancer, the HPV expression cells and the cervical cancer cell lines, the protein group information, and the preliminary exploration of the carcinogenic effect of HPV in the above two levels.
The transcriptional group of the cervical cancer tissue or the HPV oncogene expression cell / cervical cancer cell line, the protein group information, were compared with the normal or normal cell lines, and the correlation between the HPV and these differences was detected.
Method:
1. RNA sequencing of the HPV oncogene cell line: RNA extraction of NIKS-E7 and its control NIKS-Vector cells, which stably expressed high risk HPVE7 gene, were sequenced and quantified.
2. bioinformatics analysis of the results of RNA sequencing: from the results of RNA sequencing, the difference between E7 cells and Vector cells was clearly expressed and the genes expressed as proteins were expressed as proteins. GO (Gene Ontology) was used to cluster these genes. And the accuracy of RNA sequencing was verified with RTq-PCR.
3.. Was used to prepare the protein samples from the cervical cancer cell lines: the total protein was extracted from the cervical squamous cell carcinoma SiHa and the HeLa cell line of the cervical adenocarcinoma, and the proteolysis was performed by the FASP method.
4. the protein group analysis of cervical cancer cell lines: the peptide segments of the two cell lines were analyzed by LCMS, and the mass spectrometry data were unlabeled, and the quantitative data were analyzed by bioinformatics.
Result:
1. after RNA sequencing, a total of 236 genes were found to be significantly different from the control cells in the cells with the HPV oncogene E7, including 150 high expression genes and 86 low expression genes.
2. after GO cluster analysis, 38% of the highly expressed genes were associated with cell cycle, and 30% was associated with DNA metabolism, and about 15% of the low expression genes were associated with cell proliferation regulation, and 14% was associated with cell homeostasis.
3. by mass spectrometric analysis, 2373 proteins were identified in HeLa and SiHa, of which 1401 proteins were identified in HeLa cells that were less than 0.05 of the positive rate of polypeptides (FDR) less than 0.05. In SiHa cells, 1620 kinds of proteins were identified to be in accordance with the reliability of the polypeptide less than 0.05.
4. in the above protein, there were significant differences in the protein expression in the two kinds of cells, which met 787 proteins of P less than 0.05, of which 401 were HeLa high expression proteins, 10.8% of the 386.HeLa high expression proteins of HeLa low expression protein were associated with the increase of productivity metabolism. In the low expression of Hela protein, 14.3% protein was associated with the regulation of apoptosis. Close.
Conclusion:
1.HPV oncogene can promote cell proliferation and cell metabolism, thereby promoting cancer.
The difference in protein expression between adenocarcinoma and squamous cell carcinoma caused by 2.HPV infection is likely to be an important cause of poor prognosis of adenocarcinoma.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
本文编号：2119321
[Abstract]:Cervical cancer is one of the most common malignant tumors in the female reproductive system. More than 500 thousand of new cases in the world are emerging every year, and 80% of them come from developing countries. According to the 2011 Health Statistics Yearbook published by the official website of China's Health Planning Commission, the total prevalence rate of cervical cancer in 2010 is 15.1/10 million people, and the cervical cancer is on the rise compared with the past year. Cancer is mainly squamous cell carcinoma, followed by adenocarcinoma. It is reported that the survival rate of cervical adenocarcinoma is lower than that of cervical squamous cell carcinoma in the same period, and the prognosis is worse. Epidemiological study shows that high risk human papillomavirus (Human papillomavirus, HPV) infection is an important promoter of cervical cancer. Papillomavirus is proliferating in the skin and mucous layer. A small DNA virus on the form of a cell. According to clinical analysis, human papillomavirus can be classified into two major categories of high risk and low risk types. High risk HPV or tumorigenic HPV can lead to cancer; more than 12 high risk types of HPV have been identified, of which HPV16 and HPV18 are associated with cervical cancer caused by about 70%HPV. Although previous studies have shown that The correlation between high risk HPV and cervical cancer is confirmed. However, for the carcinogenic mechanism of high-risk HPV, there is still a lack of macro understanding of the HPV virus affecting host cells. With the growing maturity of sequencing and high resolution mass spectrometry, people have the opportunity to use high throughput sequencing and total protein mass spectrometry for HPV carcinogenic machines. On this basis, we integrate the two new technologies and other molecular biology techniques to obtain the cervical cancer, the HPV expression cells and the cervical cancer cell lines, the protein group information, and the preliminary exploration of the carcinogenic effect of HPV in the above two levels.
The transcriptional group of the cervical cancer tissue or the HPV oncogene expression cell / cervical cancer cell line, the protein group information, were compared with the normal or normal cell lines, and the correlation between the HPV and these differences was detected.
Method:
1. RNA sequencing of the HPV oncogene cell line: RNA extraction of NIKS-E7 and its control NIKS-Vector cells, which stably expressed high risk HPVE7 gene, were sequenced and quantified.
2. bioinformatics analysis of the results of RNA sequencing: from the results of RNA sequencing, the difference between E7 cells and Vector cells was clearly expressed and the genes expressed as proteins were expressed as proteins. GO (Gene Ontology) was used to cluster these genes. And the accuracy of RNA sequencing was verified with RTq-PCR.
3.. Was used to prepare the protein samples from the cervical cancer cell lines: the total protein was extracted from the cervical squamous cell carcinoma SiHa and the HeLa cell line of the cervical adenocarcinoma, and the proteolysis was performed by the FASP method.
4. the protein group analysis of cervical cancer cell lines: the peptide segments of the two cell lines were analyzed by LCMS, and the mass spectrometry data were unlabeled, and the quantitative data were analyzed by bioinformatics.
Result:
1. after RNA sequencing, a total of 236 genes were found to be significantly different from the control cells in the cells with the HPV oncogene E7, including 150 high expression genes and 86 low expression genes.
2. after GO cluster analysis, 38% of the highly expressed genes were associated with cell cycle, and 30% was associated with DNA metabolism, and about 15% of the low expression genes were associated with cell proliferation regulation, and 14% was associated with cell homeostasis.
3. by mass spectrometric analysis, 2373 proteins were identified in HeLa and SiHa, of which 1401 proteins were identified in HeLa cells that were less than 0.05 of the positive rate of polypeptides (FDR) less than 0.05. In SiHa cells, 1620 kinds of proteins were identified to be in accordance with the reliability of the polypeptide less than 0.05.
4. in the above protein, there were significant differences in the protein expression in the two kinds of cells, which met 787 proteins of P less than 0.05, of which 401 were HeLa high expression proteins, 10.8% of the 386.HeLa high expression proteins of HeLa low expression protein were associated with the increase of productivity metabolism. In the low expression of Hela protein, 14.3% protein was associated with the regulation of apoptosis. Close.
Conclusion:
1.HPV oncogene can promote cell proliferation and cell metabolism, thereby promoting cancer.
The difference in protein expression between adenocarcinoma and squamous cell carcinoma caused by 2.HPV infection is likely to be an important cause of poor prognosis of adenocarcinoma.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33
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