HPV16/18 E7对宫颈癌细胞中HDAC1表达的影响
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摘要
研究目的:宫颈癌是女性多发的恶性肿瘤,而人类乳头瘤病毒(HPV)感染是早已被临床和流行病学研究证实的宫颈癌发生发展的主要因素。其中HPV16、18是临床检出率相对较高的两种HPV高危亚型。HPV病毒编码的E7是重要的原癌蛋白,在肿瘤发生中起着重要作用。表观遗传学改变是另一个与肿瘤发生相关的因素,很多肿瘤中都观察到异常水平的HDAC1的表达。而HPV感染类型不同与HDAC1表达的关系未见报道,因此我们设计了本实验观察不同HPV感染情况的宫颈癌细胞系中HDAC1的表达情况,以及E7对HDAC1表的的影响。另外,E7蛋白分为三个结构域,对HDAC1的表达改变有重要作用的分区尚不明确,因此我们构建了分别缺失各一个结构域的突变体,以期在后续实验中检测其功能改变。
方法及结果:
1.HDAC1在人宫颈癌细胞系C-33A中表达高于CaSki和HeLa
用RT-PCR和Western Blot的方法检测人宫颈癌细胞系C-33A、CaSki和HeLa中HDAC1的转录水平和蛋白水平差异。结果显示,HDAC1在C-33A(无HPV感染)中表达最高,CaSki(HPV16感染)中次之,HeLa(HPV18感染)中最低。
2.转染HPV16E7,HPV18E7后C-33A细胞系中HDAC1表达降低
向无HPV感染的C-33A细胞转染pEGFP-HPV18E7和pEGFP-N1空载体,通过RT-PCR和Western Blot观察转染后两组HDAC1表达量差异。结果显示,转染pEGFP-HPV18E7组的C-33A细胞中的HDAC1的表达明显低于转染pEGFP-N1组。
3.RNAi策略沉默CaSki细胞HPV16E7导致HDAC1表达上调
向HPV16感染的CaSki细胞转染siRNA沉默HPV16E7,对照组转染无关序列,通过RT-PCR观察转染后HDAC1表达改变。结果显示,转染siRNA组的HDAC1的表达明显高于转染无关序列组。
4.构建HPV16E7不同结构域缺失表达载体
用已克隆到pEGFP-N1载体上测序正确的HPV16E7的cDNA为模板,经常规PCR或一步反向PCR致突变策略扩增得到不同HPV16E7结构域缺失的表达载体:pEGFP-HPV16E7/CR1&2、pEGFP- HPV16E7/CR1&3和pEGFP- HPV16E7/CR2&3。
结论:
1.HDAC1在不同HPV感染的宫颈癌细胞系中表达有差异,在C-33A(无HPV感染)细胞中表达最高,在CaSki(HPV16感染)细胞中表达次之,在HeLa(HPV18感染)细胞中表达最低。
2.HPV病毒E7原癌蛋白可以下调宫颈癌细胞系中HDAC1表达。
Objective: Cervical cancer is one of the most common malignancies among women worldwide. Human papillomavirus (HPV) has been identified as the major etiological factor in cervical cancer through clinical medicine and epidemiological research. And the HPV16, 18 classified into high-risk group are associated with more than 90% of cervical cancer. E7 oncoprotein plays an important role in transformation. On the other hand, the abnormal expression of histone deacetylase1 (HDAC1) is observed in many cancer. Therefore, these reseaches were conducted to investigate the expression level of HDAC1 in HPV positive and negative cervical cells and regulation of HDAC1 by HPV E7 oncoprotein.
Methods and Results:
1. Studied using RT-PCR and western blot, the expression of HDAC1 was differently in the C-33A, CaSki and HeLa cervical cancer lines, highest in C-33A followed by CaSki and HeLa in turn.
2. After transient transfection of plasmid pEGFP-HPV18 E7, mRNA and protein of HDAC1 were observed downregulation.
3. siRNA targeting HPV16 E7 was transfected into CaSki human cervical cancer cells, then mRNA of HDAC1 was observed upregulation.
4. The encoding fragements of three different deleted mutants in HPV16 E7 were constructed by PCR or one-step opposite-direction PCR, which were lacking its CR1, CR2 and CR3 respectively. They were consistent with the sequence reported in GenBank. Conclusion: We concluded that the expression of HDAC1 may be associated with HPV infection and its subtypes, and downregulation by HPV E7 oncoprotein.
方法及结果:
1.HDAC1在人宫颈癌细胞系C-33A中表达高于CaSki和HeLa
用RT-PCR和Western Blot的方法检测人宫颈癌细胞系C-33A、CaSki和HeLa中HDAC1的转录水平和蛋白水平差异。结果显示,HDAC1在C-33A(无HPV感染)中表达最高,CaSki(HPV16感染)中次之,HeLa(HPV18感染)中最低。
2.转染HPV16E7,HPV18E7后C-33A细胞系中HDAC1表达降低
向无HPV感染的C-33A细胞转染pEGFP-HPV18E7和pEGFP-N1空载体,通过RT-PCR和Western Blot观察转染后两组HDAC1表达量差异。结果显示,转染pEGFP-HPV18E7组的C-33A细胞中的HDAC1的表达明显低于转染pEGFP-N1组。
3.RNAi策略沉默CaSki细胞HPV16E7导致HDAC1表达上调
向HPV16感染的CaSki细胞转染siRNA沉默HPV16E7,对照组转染无关序列,通过RT-PCR观察转染后HDAC1表达改变。结果显示,转染siRNA组的HDAC1的表达明显高于转染无关序列组。
4.构建HPV16E7不同结构域缺失表达载体
用已克隆到pEGFP-N1载体上测序正确的HPV16E7的cDNA为模板,经常规PCR或一步反向PCR致突变策略扩增得到不同HPV16E7结构域缺失的表达载体:pEGFP-HPV16E7/CR1&2、pEGFP- HPV16E7/CR1&3和pEGFP- HPV16E7/CR2&3。
结论:
1.HDAC1在不同HPV感染的宫颈癌细胞系中表达有差异,在C-33A(无HPV感染)细胞中表达最高,在CaSki(HPV16感染)细胞中表达次之,在HeLa(HPV18感染)细胞中表达最低。
2.HPV病毒E7原癌蛋白可以下调宫颈癌细胞系中HDAC1表达。
Objective: Cervical cancer is one of the most common malignancies among women worldwide. Human papillomavirus (HPV) has been identified as the major etiological factor in cervical cancer through clinical medicine and epidemiological research. And the HPV16, 18 classified into high-risk group are associated with more than 90% of cervical cancer. E7 oncoprotein plays an important role in transformation. On the other hand, the abnormal expression of histone deacetylase1 (HDAC1) is observed in many cancer. Therefore, these reseaches were conducted to investigate the expression level of HDAC1 in HPV positive and negative cervical cells and regulation of HDAC1 by HPV E7 oncoprotein.
Methods and Results:
1. Studied using RT-PCR and western blot, the expression of HDAC1 was differently in the C-33A, CaSki and HeLa cervical cancer lines, highest in C-33A followed by CaSki and HeLa in turn.
2. After transient transfection of plasmid pEGFP-HPV18 E7, mRNA and protein of HDAC1 were observed downregulation.
3. siRNA targeting HPV16 E7 was transfected into CaSki human cervical cancer cells, then mRNA of HDAC1 was observed upregulation.
4. The encoding fragements of three different deleted mutants in HPV16 E7 were constructed by PCR or one-step opposite-direction PCR, which were lacking its CR1, CR2 and CR3 respectively. They were consistent with the sequence reported in GenBank. Conclusion: We concluded that the expression of HDAC1 may be associated with HPV infection and its subtypes, and downregulation by HPV E7 oncoprotein.
引文
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9 Glaunsinger BA, Lee SS, Thomas M, et al. Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins[J]. Oncogene, 2000,19(46):5270-80.
10 Thomas M, Laura R, Hepner K, et al. Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation[J]. Oncogene, 2002,21(33):5088-96.
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29 Lee SS, Glaunsinger B, Mantovani F, et al. Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins[J]. J Virol, 2000,74(20):9680-93.
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31 Thomas M, Laura R, Hepner K, et al. Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation[J]. Oncogene, 2002,21(33):5088-96.
32 Handa K, Yugawa T, Narisawa-Saito M, et al. E6AP-dependent degradation of DLG4/PSD95 by high-risk human papillomavirus type 18 E6 protein[J]. J Virol,
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58 Schaeffer AJ, Nguyen M, Liem A, et al. E6 and E7 oncoproteins induce distinct patterns of chromosomal aneuploidy in skin tumors from transgenic mice[J]. Cancer Res, 2004,64(2):538-46.
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2 Munger K, Baldwin A, Edwards KM, et al. Mechanisms of human papillomavirus-induced oncogenesis[J]. J Virol, 2004,78(21):11451-60.
3 Wu SY, Chiang CM. The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regulation[J]. J Biol Chem, 2007,282(18):13141-5.
4 Gammoh N, Grm HS, Massimi P, et al. Regulation of human papillomavirus type 16 E7 activity through direct protein interaction with the E2 transcriptional activator[J]. J Virol, 2006,80(4):1787-97.
5 Ronco LV, Karpova AY, Vidal M, et al. Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity[J]. Genes Dev, 1998,12(13):2061-72.
6 Lee SS, Weiss RS,Javier RT. Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein[J]. Proc Natl Acad Sci U S A, 1997,94(13):6670-5.
7 Nakagawa S, Huibregtse JM. Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase[J]. Mol Cell Biol, 2000,20(21):8244-53.
8 Lee SS, Glaunsinger B, Mantovani F, et al. Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins[J]. J Virol, 2000,74(20):9680-93.
9 Glaunsinger BA, Lee SS, Thomas M, et al. Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins[J]. Oncogene, 2000,19(46):5270-80.
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