運用整合組學研究HPV與宮頸癌
發(fā)布時間：2018-07-13 12:08
【摘要】：宮頸癌是女性生殖系統(tǒng)最常見的惡性腫瘤之一。每年全球新發(fā)病例超過50萬,且其中80%來自發(fā)展中國家。根據(jù)中國衛(wèi)計委官方網(wǎng)站公布的2011年衛(wèi)生統(tǒng)計年鑒數(shù)據(jù)顯示,我國2010年宮頸癌總患病率為15.1/10萬人,與往年相比呈上升趨勢。宮頸癌以鱗癌為主,其次為腺癌,有文獻報道同期的宮頸腺癌與宮頸鱗癌相比生存率更低,預后更差。流行病學調(diào)查研究顯示,高危型人乳頭瘤病毒(Human papillomavirus, HPV)感染是宮頸癌發(fā)生的重要啟動因子。乳頭瘤病毒是增殖在皮膚和粘膜層狀細胞上的一種小DNA病毒。按照臨床分析,人乳頭瘤病毒可以分為高危型和低危型兩大類。高危型HPV或稱致瘤HPV能夠?qū)е掳┌Y；目前已經(jīng)鑒定出了12種以上的高危型HPV,其中HPV16和HPV18與約70%HPV引起的宮頸癌有關(guān)。雖然過去的研究已經(jīng)明確了高危型HPV與宮頸癌之間的相關(guān)性,然而對于高危型HPV的致癌機理,尚缺乏對HPV病毒影響宿主細胞的宏觀認識。而伴隨著測序技術(shù)和高分辨率質(zhì)譜技術(shù)的日益成熟,人們有機會通過高通量測序和全蛋白質(zhì)譜分析等手段對HPV的致癌機制進行更為全面的認識。據(jù)此,我們將上述兩種新技術(shù)與其他分子生物學技術(shù)整合應用,獲得了宮頸癌,HPV表達細胞和宮頸癌細胞系的轉(zhuǎn)錄組,蛋白組信息,并對HPV在以上兩個層面所發(fā)揮的致癌作用進行了初步的探究。目的： 獲得宮頸癌組織樣本或HPV癌基因表達細胞/宮頸癌細胞系的轉(zhuǎn)錄組,蛋白組信息,比較它們與正常組織或正常細胞系之間的差異,并檢測HPV與這些差異之間的相關(guān)性。 方法： 1.對HPV癌基因細胞系進行RNA測序：對穩(wěn)定表達高危型HPVE7基因的NIKS-E7及其對照NIKS-Vector細胞進行RNA提取,對所得RNA進行測序并定量。 2.對RNA測序結(jié)果進行生物信息學分析：從RNA測序的結(jié)果中挑選出E7細胞與Vector細胞有明顯表達差異,且能夠表達為蛋白的基因。對這些基因進行GO (Gene Ontology)聚類。并用RTq-PCR驗證RNA測序結(jié)果準確性。 3..對宮頸癌細胞系進行蛋白樣本制備：分別從宮頸鱗癌SiHa和宮頸腺癌HeLa細胞系中提取細胞全蛋白,并用FASP方法進行蛋白酶解。 4.對宮頸癌細胞系進行蛋白組分析：將上述兩種細胞系的肽段進行LCMS分析,并對所得質(zhì)譜數(shù)據(jù)進行非標記定量,對定量數(shù)據(jù)進行生物信息學分析。 結(jié)果： 1.經(jīng)過RNA測序,共發(fā)現(xiàn)236個基因在有HPV癌基因E7的細胞中與對照細胞相比存在明顯差異表達,其中高表達基因150個,低表達基因86個 2.經(jīng)過GO聚類分析,高表達基因中38%與細胞周期相關(guān),30%與DNA代謝相關(guān)；低表達基因中約15%與細胞增殖調(diào)節(jié)相關(guān),14%與細胞穩(wěn)態(tài)相關(guān)。 3.經(jīng)過質(zhì)譜分析,在HeLa和SiHa之中共鑒定到2373種蛋白,其中在HeLa細胞中共鑒定到符合多肽可信度假陽性率(FDR)≤0.05的蛋白1401種。在SiHa細胞中共鑒定到符合多肽可信度FDR≤0.05的蛋白1620種。 4.在上述蛋白中,在兩種細胞中有明顯表達差異的蛋白,滿足p≤0.05的蛋白共787種,其中HeLa高表達蛋白401種,HeLa低表達蛋白386種。HeLa高表達蛋白中有10.8%的蛋白與提高產(chǎn)能代謝相關(guān),在Hela低表達的蛋白中,有14.3%的蛋白同調(diào)節(jié)細胞凋亡相關(guān)。 結(jié)論： 1.HPV癌基因能夠促進細胞增殖和細胞代謝,從而促進癌癥的發(fā)生。 2.HPV感染所導致的腺癌和鱗癌有明顯的蛋白表達差異,這些差異很可能是導致腺癌預后不良的重要原因。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33
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