新疆维吾尔族宫颈癌患者HPV宫颈细胞基因组整合情况
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摘要
目的采用高通量病毒整合检测方法(high throughput viral integration detection,HIVID)对20种亚型的HPV在维吾尔族宫颈癌患者基因组中的整合情况进行检测,探讨该人群中HPV宿主细胞基因组整合特点,为维吾尔族女性宫颈癌的防治提供分子流行病学依据。方法从新疆医科大学附属肿瘤医院肿瘤防治研究所获得40例既往显示HPV感染阳性的维吾尔族宫颈癌患者冷冻病理标本。从美国Agilent公司订制专门设计的针对20种HPV亚型的富集液相芯片,并结合二代基因测序技术检测标本中的HPV宫颈细胞基因组整合事件。结果 HIVID检测到的HPV多重感染率高达92.5%,远高于商品化试剂盒35.0%的检出率(χ~2=28.614,P<0.001)。在40例标本中存在13 423个整合事件,总共涉及到6 867个人类基因。这些整合事件在人类各条染色体上都有分布,其中2号染色体上分布的频率最多。本研究共检出PPP1R37、HECW2、EMBP1、ANKRD50、SPTBN4、LINC00895、LYRM4-AS1、LINC00374、RBFOX1、CSMD1、CDH13和KLHL4等12个高频整合位点。结论 HPV基因组整合到人类基因组存在普遍性和随机性,高频整合位点涉及基因可能为HPV感染导致新疆维吾尔族宫颈癌患者宫颈细胞恶变提供新的研究思路。
Objective To provide molecular epidemiology evidences for the prevention and treatment of cervical cancer through detecting the integration of 20 subtypes of HPV genes into host cell genome in Uygur cervical cancer patients by using high throughput viral integration detection(HIVID) method. Methods Forty frozen specimens of Uygur cervical cancer patients with positive HPV infection were obtained from the cancer prevention and treatment institute of the Tumor Hospital Affiliated to Xinjiang Medical University. The corresponding concentrated liquid-phase chips were designed and produced by Agilent company. Then the integration of HPV genes into host cell genome was detected using second-generation sequencing technology. Results The multiple infection rate of HPV detected by HIVID was as high as 92.5%, much higher than the 35.0% by the commercial kit(χ~2=28.614,P<0.001). There were 13,423 integration events in 40 specimens, involving a total of 6,867 human genes. These integration events were distributed on all human chromosomes, most of which were on chromosome 2. The study found 12 integration hotspots in human genome, including PPP1R37, HECW2, EMBP1, ANKRD50, SPTBN4, LINC00895, RBFOX1, LYRM4-AS1, LINC00374, CSMD1, CDH13 and KLHL4. Conclusion The integration of HPV genes into human genome is universal and random. The sites of HPV integration in Uygur cervical cancer with the highest frequency are related to cancer progression, which may provide some new ideas for the research on the occurrence and progress of cervical cancer in Xinjiang Uygur cervical cancer patients.
Objective To provide molecular epidemiology evidences for the prevention and treatment of cervical cancer through detecting the integration of 20 subtypes of HPV genes into host cell genome in Uygur cervical cancer patients by using high throughput viral integration detection(HIVID) method. Methods Forty frozen specimens of Uygur cervical cancer patients with positive HPV infection were obtained from the cancer prevention and treatment institute of the Tumor Hospital Affiliated to Xinjiang Medical University. The corresponding concentrated liquid-phase chips were designed and produced by Agilent company. Then the integration of HPV genes into host cell genome was detected using second-generation sequencing technology. Results The multiple infection rate of HPV detected by HIVID was as high as 92.5%, much higher than the 35.0% by the commercial kit(χ~2=28.614,P<0.001). There were 13,423 integration events in 40 specimens, involving a total of 6,867 human genes. These integration events were distributed on all human chromosomes, most of which were on chromosome 2. The study found 12 integration hotspots in human genome, including PPP1R37, HECW2, EMBP1, ANKRD50, SPTBN4, LINC00895, RBFOX1, LYRM4-AS1, LINC00374, CSMD1, CDH13 and KLHL4. Conclusion The integration of HPV genes into human genome is universal and random. The sites of HPV integration in Uygur cervical cancer with the highest frequency are related to cancer progression, which may provide some new ideas for the research on the occurrence and progress of cervical cancer in Xinjiang Uygur cervical cancer patients.
引文
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[23]Missaoui N,Hmissa S,Trabelsi A,et al.Promoter hypermethylation of CDH13,DAPK1and TWIST1genes in precancerous and cancerous lesions of the uterine cervix[J].Pathol Res Pract,2011,207(1):37-42.
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[25]WANG Zhijun,TU Wei,LIU Fang.Effect of siRNA againstβ2-spectrin on proliferation of AML-12cells[J].Acta Med Univ Sci Technol Huazhong(Med Sci),2013,42(1):8-11.(in Chinese)汪志军,涂炜,刘芳.β2-spectrin的siRNA对AML-12肝细胞增殖特性的影响[J].华中科技大学学报(医学版),2013,42(1):8-11.
[26]ZENG Qiyan,QIN Xue,ZHANG Hong.Effect of inhibitor-1of protein phosphataseⅠon apoptosis of human cervical carcinoma cell line HeLa[J].Chin J Cancer,2007,26(11):1177-1182.(in Chinese)曾麒燕,秦雪,张红.Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞凋亡的影响[J].癌症,2007,26(11):1177-1182.
[27]ZENG Qiyan,CUI Bo.Research progress on regulation of Ser/Thr phosphatase in cell cycle and tumor proliferation[J].J Guangxi Acad Sci,2011,27(3):263-268.(in Chinese)曾麒燕,崔博.蛋白丝氨酸/苏氨酸磷酸酶对细胞周期的调节作用及其与肿瘤关系的研究进展[J].广西科学院学报,2011,27(3):263-268.
[28]Wu PK,Hong SK,Park JI.Steady-state levels of phosphorylated mitogen-activated protein kinase kinase 1/2determined by mortalin/HSPA9and protein phosphatase 1 Alpha in KRASand BRAF tumor cells[J].Mol Cell Biol,2017,37(18):pii:e00061-17.
[2]WANG Senyu,MIJITI Rena,FENG Yangchun,et al.Human papilloma virus infected in Xinjiang Han women[J].J Southeast Univ(Med Sci),2015,34(6):902-905.(in Chinese)王森钰,热娜·米吉提,冯阳春,等.新疆地区汉族女性人乳头状瘤病毒基因型及感染情况分析[J].东南大学学报(医学版),2015,34(6):902-905.
[3]Feng YC,Yang J,Liu CM,et al.DNA ploidy of cervical epithelial cells should be a cure criterion of high-risk HPV infection in Xinjiang Uygur women[J].Oncol Targets Ther,2015,8:827-833.
[4]Feng YC,Zhang Y,Liu CM,et al.Increased HPV L1gene methylation and multiple infection status Lead to the difference of cervical epithelial cell lesion in different ethnic women of Xinjiang,China[J].Medicine,2017,96(12):e6409.
[5]Feng YC,Cheng ZZ,Huang YC,et al.Association between PD-L1and HPV status and the prognostic value for HPVtreatment in premalignant cervical lesion patients[J].Medicine,2017,96(25):e7270.
[6]Hu Z,Zhu DA,Wang W,et al.Genome-wide profiling of HPVintegration in cervical cancer identifies clustered genomic hot spots and a potential microhomology-mediated integration mechanism[J].Nat Genet,2015,47(2):158-163.
[7]XU Aimin,LIU Wen,ZHANG Liping,et al.Analytical research on HPV infection among women in Xingjiang Kashgar area[J].Int J Lab Med,2017,38(16):2232-2233.(in Chinese)许爱敏,刘雯,张丽萍,等.新疆喀什地区维吾尔族女性人乳头状瘤病毒感染分析研究[J].国际检验医学杂志,2017,38(16):2232-2233.
[8]ABLIMIT Tangnar,TURGAN Muyassaer,ABLIZ Guzalnur,et al.Study on the distribution of HPV subtypes in Uighur people living in the Karsay township,Moyu county,Xinjiang[J].Chin J Epidemiol,2011,32(5):477-480.(in Chinese)唐努尔·阿布力米提,穆也沙尔·吐尔干,古扎丽努尔·阿不力孜,等.新疆宫颈癌高发区维吾尔族人群人乳头瘤病毒亚型的研究[J].中华流行病学杂志,2011,32(5):477-480.
[9]Groves IJ,Coleman N.Human papillomavirus genome integration in squamous carcinogenesis:what have next-generation sequencing studies taught us?[J].J Pathol,2018,245(1):9-18.
[10]Andreas G,Alexandre M,Jared M,et al.Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing[J].Nat Biotechnol,2009,27(2):182-189.
[11]Sulonen AM,Ellonen P,Almusa H,et al.Comparison of solution-based exome capture methods for next generation sequencing[J].Genome Biol,2011,12(9):R94.
[12]Zhao LH,Liu X,Yan HX,et al.Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma[J].Nat Commun,2016,7:12992.
[13]Li W,Qi Y,Cui X,et al.Characteristic of HPV integration in the genome and transcriptome of cervical cancer tissues[J].Biomed Res Int,2018,2018:6242173.
[14]Gao G,Smith DI.Role of the common fragile sites in cancers with a Human papillomavirus etiology[J].Cytogenet Genome Res,2016,150(3/4):217-226.
[15]Xie H,Lee L,Scicluna P,et al.Novel functions and targets of miR-944in human cervical cancer cells[J].Int J Cancer,2014,136(5):E230-E241.
[16]Muralikrishna B,Chaturvedi P,Sinha K,et al.Lamin misexpression upregulates three distinct ubiquitin ligase systems that degrade ATR kinase in HeLa cells[J].Mol Cell Biochem,2012,365(1/2):323-332.
[17]Thomas G,Jacobs KB,Kraft P,et al.A multistage genomewide association study in breast cancer identifies two new risk alleles at 1p11.2and 14q24.1(RAD51L1)[J].Nat Genet,2009,41(3):579-584.
[18]Eun YG,Lee D,Lee YC,et al.Clinical significance of YAP1activation in head and neck squamous cell carcinoma[J].Oncotarget,2017,8(67):111130-111143.
[19]Urashima M,Hama T,Suda T,et al.Distinct effects of alcohol consumption and smoking on genetic alterations in head and neck carcinoma[J].PLoS One,2013,8(11):e80828.
[20]Koneva LA,Zhang Y,Virani S,et al.HPV integration in HNSCC correlates with survival outcomes,immune response signatures,and candidate drivers[J].Mol Cancer Res,2017,9:MCR-17-0153.
[21]van Kempen PM,van Bockel L,Braunius WW,et al.HPV-positive oropharyngeal squamous cell carcinoma is associated with TIMP3 and CADM1 promoter hypermethylation[J].Cancer Med,2014,3(5):1185-1196.
[22]Vasiljevic′N,Scibior-Bentkowska D,Brentnall AR,et al.Credentialing of DNA methylation assays for human genes as diagnostic biomarkers of cervical intraepithelial neoplasia in highrisk HPV positive women[J].Gynecol Oncol,2014,132(3):709-714.
[23]Missaoui N,Hmissa S,Trabelsi A,et al.Promoter hypermethylation of CDH13,DAPK1and TWIST1genes in precancerous and cancerous lesions of the uterine cervix[J].Pathol Res Pract,2011,207(1):37-42.
[24]Andreeva AV,Kutuzov MA.Cadherin 13in cancer[J].Genes Chromosomes Cancer,2010,49(9):7757-90.
[25]WANG Zhijun,TU Wei,LIU Fang.Effect of siRNA againstβ2-spectrin on proliferation of AML-12cells[J].Acta Med Univ Sci Technol Huazhong(Med Sci),2013,42(1):8-11.(in Chinese)汪志军,涂炜,刘芳.β2-spectrin的siRNA对AML-12肝细胞增殖特性的影响[J].华中科技大学学报(医学版),2013,42(1):8-11.
[26]ZENG Qiyan,QIN Xue,ZHANG Hong.Effect of inhibitor-1of protein phosphataseⅠon apoptosis of human cervical carcinoma cell line HeLa[J].Chin J Cancer,2007,26(11):1177-1182.(in Chinese)曾麒燕,秦雪,张红.Ⅰ型磷酸酶抑制亚基-1对人宫颈癌HeLa细胞凋亡的影响[J].癌症,2007,26(11):1177-1182.
[27]ZENG Qiyan,CUI Bo.Research progress on regulation of Ser/Thr phosphatase in cell cycle and tumor proliferation[J].J Guangxi Acad Sci,2011,27(3):263-268.(in Chinese)曾麒燕,崔博.蛋白丝氨酸/苏氨酸磷酸酶对细胞周期的调节作用及其与肿瘤关系的研究进展[J].广西科学院学报,2011,27(3):263-268.
[28]Wu PK,Hong SK,Park JI.Steady-state levels of phosphorylated mitogen-activated protein kinase kinase 1/2determined by mortalin/HSPA9and protein phosphatase 1 Alpha in KRASand BRAF tumor cells[J].Mol Cell Biol,2017,37(18):pii:e00061-17.