小干扰RNA表达载体抑制子宫颈癌HPV16 E7基因的研究
|
摘要
背景与研究目的 高危型HPV感染在宫颈癌的发生发展中起着至关重要的作用,特别是E6/E7癌基因,已被证实有恶性转化能力,因此它们已成为宫颈癌基因治疗的理想靶点。RNA干扰(RNA interference,RNAi)是新近发展起来的一种封闭基因表达的有效方法,它比反义寡核苷酸和核酶技术能更高效的抑制目的基因的表达。已有研究证实化学合成的siRNA(small interfering RNA)能高效地介导RNAi作用,但作用持续时间短,而采用siRNA表达载体(small interfering RNA expression vector)的方法可能延长作用时间。因此本研究旨在探讨HPV16E7特异性siRNA表达载体对宫颈癌Caski和SiHa细胞E7基因的沉默作用及作用持续时间,以及体内外对肿瘤细胞生长的影响,探索siRNA表达载体作为宫颈癌基因治疗新策略的可行性。
方法 1.利用计算机辅助设计软件,设计合成了含不同HPV16E7特异性序列的56nt寡核苷酸片段,并将其定向克隆入真核表达载体psiRNA,得到重组质粒P1、P2、P3;2.利用脂质体将重组质粒转染CaSki和SiHa细胞,以荧光定量RT-PCR检测不同时间点转染前后E7 mRNA的变化,以免疫荧光标记法、western blot检测转染前后E7蛋
Background & Objective High-risk human papillomavirus (HPV) infection is believed to play a central in the carcinogenesis and development cervical carcinoma. Specifically, two viral oncogene, E6 and E7, possess cancer transforming ability, have been became ideal targets for gene therapy of cervical carcinoma. RNA interference is an effective method of blocking gene expression at the post-transcriptional level and is several orders of magnitude more efficient than antisense or ribozyme treatment. The study has identified that the objective gene expression could be selective silencing with synthetic siRNA and silencing was not sustained more than 7 days. But a vector-based siRNA expression system might exhibit a comparatively long-term RNA interference (RNAi) effect. So the study was designed to investigate the specificity and efficiency of HPV16 E7 gene silence in CaSki cells and SiHa cells of cervical cancer in vitro and in vivo by vector-based RNAi technique, and to research the effect on these cell proliferations, and to explore the possibility of siRNA expression vector in treatment of cervical carcinoma.
Methods 1. According to the computer aided design, 56nt oligonucleotide fragments containing different HPV16E7-specific sequences were synthesized and cloned into the expression vector psiRNA, and three recombinant vectors P1, P2 and P3 were constructed.2. The vectors were transfected into CaSki cells and SiHa cells by liposome. The expression level of HPV16E7 mRNA and protein were detected by real-time RT-PCR, western blot analysis and immunofluorescence using fluorescein isothiocyanate label at different times after transfection.3. The cell cycles and cell proliferations of CaSki cell and SiHa cell were measured with flow cytometry and MTT method after transfection.4. Human cervical cancer CaSki cells were transfected by vector P1 and were seeded hypopercutaneously on nude mice. The in vivo carcinogenic and growth activities of cancer cells were observed. E7 protein expression in tumor tissues was detected by immunohistochemisty.Results 1. PCR analysis and DNA sequencing confirmed that the HPV16 E7-specific siRNA expression vectors were constructed successfully.2. HPV16 E7 mRNA expression could be inhibited 92.86%, 54.24%, 87.22% respectively by vector P1, P2 and P3 at one week after the formation of CaSki cellular clone, and the HPV16 E7 protein was reduced by 84.21%, 44.66%, 83.46% respectively. So the inhibition effect of siRNA expression vector P1 was more effective.3. HPV16 E7 mRNA expression could be inhibited 92.15%, 54.51%, 87.22% respectively by vector P1, P2 and P3 at one week after the formation of SiHa cellular clone, and the HPV16 E7 protein was reduced by 84.30%, 41.65%, 82.91% respectively. So the inhibition effect of siRNA
expression vector PI was more effective.4. HPV16 E7 mRNA expression could be inhibited 68.95%, 65.69% respectively by vector PI into CaSki cells and SiHa cells at four weeks after the formation of cellular clone, and the HPV16 E7 protein was reduced by62.50%, 59.11% respectively.5. After transfected by vector PI, the ratio of Gl phase of CaSki cell andSiHa cell was improved and the ratio of S phase was descended significantly at one week after the formation of cellular clone, and the cell proliferations of CaSki cell and SiHa cell were inhibited.6. After transfected by vector PI, the carcinogenic activity of CaSki cell was decreased in nude mice, the volume and the weigh of tumors were reduced, the rate of inhibition was 50.61%, and E7 protein level in tumor tissues was reduced by 74.75%.Conclusions The expression of E7 gene in CaSki cells and SiHa cells can be inhibited specifically and high efficiently by HPV16E7-specific siRNA expression vector, and inhibition effect was sustained for at least four weeks after the formation of cellular clone. E7-specific siRNA expression vector could control the cell cycle, inhibit the cancer cell proliferation and partly reversed the malignant phenotype of tumor. So it might be one of the ideal strategies for gene therapy of cervical cancer.
方法 1.利用计算机辅助设计软件,设计合成了含不同HPV16E7特异性序列的56nt寡核苷酸片段,并将其定向克隆入真核表达载体psiRNA,得到重组质粒P1、P2、P3;2.利用脂质体将重组质粒转染CaSki和SiHa细胞,以荧光定量RT-PCR检测不同时间点转染前后E7 mRNA的变化,以免疫荧光标记法、western blot检测转染前后E7蛋
Background & Objective High-risk human papillomavirus (HPV) infection is believed to play a central in the carcinogenesis and development cervical carcinoma. Specifically, two viral oncogene, E6 and E7, possess cancer transforming ability, have been became ideal targets for gene therapy of cervical carcinoma. RNA interference is an effective method of blocking gene expression at the post-transcriptional level and is several orders of magnitude more efficient than antisense or ribozyme treatment. The study has identified that the objective gene expression could be selective silencing with synthetic siRNA and silencing was not sustained more than 7 days. But a vector-based siRNA expression system might exhibit a comparatively long-term RNA interference (RNAi) effect. So the study was designed to investigate the specificity and efficiency of HPV16 E7 gene silence in CaSki cells and SiHa cells of cervical cancer in vitro and in vivo by vector-based RNAi technique, and to research the effect on these cell proliferations, and to explore the possibility of siRNA expression vector in treatment of cervical carcinoma.
Methods 1. According to the computer aided design, 56nt oligonucleotide fragments containing different HPV16E7-specific sequences were synthesized and cloned into the expression vector psiRNA, and three recombinant vectors P1, P2 and P3 were constructed.2. The vectors were transfected into CaSki cells and SiHa cells by liposome. The expression level of HPV16E7 mRNA and protein were detected by real-time RT-PCR, western blot analysis and immunofluorescence using fluorescein isothiocyanate label at different times after transfection.3. The cell cycles and cell proliferations of CaSki cell and SiHa cell were measured with flow cytometry and MTT method after transfection.4. Human cervical cancer CaSki cells were transfected by vector P1 and were seeded hypopercutaneously on nude mice. The in vivo carcinogenic and growth activities of cancer cells were observed. E7 protein expression in tumor tissues was detected by immunohistochemisty.Results 1. PCR analysis and DNA sequencing confirmed that the HPV16 E7-specific siRNA expression vectors were constructed successfully.2. HPV16 E7 mRNA expression could be inhibited 92.86%, 54.24%, 87.22% respectively by vector P1, P2 and P3 at one week after the formation of CaSki cellular clone, and the HPV16 E7 protein was reduced by 84.21%, 44.66%, 83.46% respectively. So the inhibition effect of siRNA expression vector P1 was more effective.3. HPV16 E7 mRNA expression could be inhibited 92.15%, 54.51%, 87.22% respectively by vector P1, P2 and P3 at one week after the formation of SiHa cellular clone, and the HPV16 E7 protein was reduced by 84.30%, 41.65%, 82.91% respectively. So the inhibition effect of siRNA
expression vector PI was more effective.4. HPV16 E7 mRNA expression could be inhibited 68.95%, 65.69% respectively by vector PI into CaSki cells and SiHa cells at four weeks after the formation of cellular clone, and the HPV16 E7 protein was reduced by62.50%, 59.11% respectively.5. After transfected by vector PI, the ratio of Gl phase of CaSki cell andSiHa cell was improved and the ratio of S phase was descended significantly at one week after the formation of cellular clone, and the cell proliferations of CaSki cell and SiHa cell were inhibited.6. After transfected by vector PI, the carcinogenic activity of CaSki cell was decreased in nude mice, the volume and the weigh of tumors were reduced, the rate of inhibition was 50.61%, and E7 protein level in tumor tissues was reduced by 74.75%.Conclusions The expression of E7 gene in CaSki cells and SiHa cells can be inhibited specifically and high efficiently by HPV16E7-specific siRNA expression vector, and inhibition effect was sustained for at least four weeks after the formation of cellular clone. E7-specific siRNA expression vector could control the cell cycle, inhibit the cancer cell proliferation and partly reversed the malignant phenotype of tumor. So it might be one of the ideal strategies for gene therapy of cervical cancer.
引文
1. Herrero R. Epidemiology of cervical cancer [J]. J Nali Cancer Inst Monogr, 1996, 21: 1-6
2.郎景和。子宫颈上皮内瘤变的诊断与资料[J].中华妇产科杂志,2001,36(5):261-263
3. Walbooner JM, Jacobs MV, Manes MM, et al. Human papillomavirus is a necessary cause of invasive cervicd cancer worldwide [J]. J Pathol, 1999, 189: 12-19
4. Rion G, Faver M, Jeannel D, et al. Association between poor prognosis in early stage invasion cervical carcinomas and non-detection of HPV DNA [J]. Lancet, 1990, 335: 1171-1174
5. Cheah PL, Looi LM. P53 immunohistochemical expression: messages in cervical carcinogenesis [J]. Pathology, 2002, 34(4): 326-331
6. Scheffner M, Huibregtse JM, Viestra RD, et al. The HPV16E6 and E6-AP complex functions as a ubiquitin protein ligase in the ubiquitination of p53 [J]. Cell, 1993, 7(3): 495-505
7. Sherr C. J. The Pezcoller lecture: Cancer cell cycles revisited [J]. Cancer Res, 2000, 60: 3689-3695
8. Martin L. G., Demers W., Galloway D. A., Disruption of the G_1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin [J]. E. J. Virol, 1998, 72: 975-985
9. Alvarez-Salas LM, Benitez-Hess ML, and DiPaolo JA. Advances in the development of ribozymes and antisense oligodeoxynucleotides as antiviral agents for human papillomaviruses. Antivir Ther. 2003, 8(4): 265-278
10. Cho CW, Poo H, Cho MC, et al. HPV E6 antisense induces apoptosis in Caski cells via suppression of E6 splicing. Exp Mol Med. 2002, 34(2):159-166
11. C. K. Choo, M. T.Ling, C. K.M. Suen, et al. Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells[J]. Gynecologic Oncology, 2000, 78(3 pt 1), 293-301
12. Zheng Y, Zhang J, Qu L. Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line. Chin Med J (Engl). 2002, 115(10):1501-1506
13. Zheng Y, Zhang J, Rao Z. Ribozyme targeting HPV 16 E6E7 transcripts in cervical cancer cells suppresses cell growth and sensitizes cells to chemotherapy and radiotherapy [J]. Cancer Biol Ther, 2004 , 3(11): 1129-34
14. Hannon GJ. RNA interference [J]. Nature, 2002, 418(6894): 244-251
15. MacManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs [J]. Nat Rev Genet, 2002, 3: 737-747
16. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells [J]. Genes Dev,2002, 16: 948-958
17. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296:550-553
18. Yu JY, DeRuiter SL, and Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells [J]. Proc Natl Acad Sci, 2002, 99: 6047-6052
19. Kawasaki H, and Taira K. Short hairpin type of dsRNAs that are controlled by tRNA (Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells [J]. Nucleic Acids Res, 2003, 31: 700-707
20. Tuschl T. Expanding small RNA interference [J]. Nature Biotechnol, 2002, 20: 446-448
21. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669): 806-811
22. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J]. Nat Rev Genet, 2002, 3(10): 737-747
23. Elbashir SM, Harorth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs [J]. Methods, 2002, 26(2): 199-213
24. Miyagishi M, Taira K. U6 promoter driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat Biotechnol, 2002, 20(5): 497-500
25. Tuschl T. Expanding small RNA interference [J]. Nat Biotechnol, 2002, 20(5): 446-448
26. Ming Jiang, Jo Milner. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference[J]. Oncogene, 2002, 21: 6041-6048
27.牛晓宇,彭芝兰,王何。RNA干涉抑制子宫颈癌CaSki细胞株HPV16 E6基因的研究。癌症,2004,23(11):1257-62
28. Hall AH, Alexander KA. RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells [J]. J Virol, 2003, 77(10): 6066-9
29. Hasuwa H, Kaseda K, Einarsdottir T, et al. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett. 2002, 532: 227-230
30. Carmell MA, Zhang L, Conklin DS, et al. Germline transmission of RNAi in mice. Nat Struct Biol. 2003, 10: 91-92
31. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003, 9: 347-351
32. Filleur S, Courtin A, Ait-Si-Ali S, et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth . Cancer Res. 2003, 63: 3919-3922
2.郎景和。子宫颈上皮内瘤变的诊断与资料[J].中华妇产科杂志,2001,36(5):261-263
3. Walbooner JM, Jacobs MV, Manes MM, et al. Human papillomavirus is a necessary cause of invasive cervicd cancer worldwide [J]. J Pathol, 1999, 189: 12-19
4. Rion G, Faver M, Jeannel D, et al. Association between poor prognosis in early stage invasion cervical carcinomas and non-detection of HPV DNA [J]. Lancet, 1990, 335: 1171-1174
5. Cheah PL, Looi LM. P53 immunohistochemical expression: messages in cervical carcinogenesis [J]. Pathology, 2002, 34(4): 326-331
6. Scheffner M, Huibregtse JM, Viestra RD, et al. The HPV16E6 and E6-AP complex functions as a ubiquitin protein ligase in the ubiquitination of p53 [J]. Cell, 1993, 7(3): 495-505
7. Sherr C. J. The Pezcoller lecture: Cancer cell cycles revisited [J]. Cancer Res, 2000, 60: 3689-3695
8. Martin L. G., Demers W., Galloway D. A., Disruption of the G_1/S transition in human papillomavirus type 16 E7-expressing human cells is associated with altered regulation of cyclin [J]. E. J. Virol, 1998, 72: 975-985
9. Alvarez-Salas LM, Benitez-Hess ML, and DiPaolo JA. Advances in the development of ribozymes and antisense oligodeoxynucleotides as antiviral agents for human papillomaviruses. Antivir Ther. 2003, 8(4): 265-278
10. Cho CW, Poo H, Cho MC, et al. HPV E6 antisense induces apoptosis in Caski cells via suppression of E6 splicing. Exp Mol Med. 2002, 34(2):159-166
11. C. K. Choo, M. T.Ling, C. K.M. Suen, et al. Retrovirus-mediated delivery of HPV16 E7 antisense RNA inhibited tumorigenicity of CaSki cells[J]. Gynecologic Oncology, 2000, 78(3 pt 1), 293-301
12. Zheng Y, Zhang J, Qu L. Effects of anti-HPV16E6-ribozyme on phenotype and gene expression of a cervical cancer cell line. Chin Med J (Engl). 2002, 115(10):1501-1506
13. Zheng Y, Zhang J, Rao Z. Ribozyme targeting HPV 16 E6E7 transcripts in cervical cancer cells suppresses cell growth and sensitizes cells to chemotherapy and radiotherapy [J]. Cancer Biol Ther, 2004 , 3(11): 1129-34
14. Hannon GJ. RNA interference [J]. Nature, 2002, 418(6894): 244-251
15. MacManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs [J]. Nat Rev Genet, 2002, 3: 737-747
16. Paddison PJ, Caudy AA, Bernstein E, et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells [J]. Genes Dev,2002, 16: 948-958
17. Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells [J]. Science, 2002, 296:550-553
18. Yu JY, DeRuiter SL, and Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells [J]. Proc Natl Acad Sci, 2002, 99: 6047-6052
19. Kawasaki H, and Taira K. Short hairpin type of dsRNAs that are controlled by tRNA (Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells [J]. Nucleic Acids Res, 2003, 31: 700-707
20. Tuschl T. Expanding small RNA interference [J]. Nature Biotechnol, 2002, 20: 446-448
21. Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans [J]. Nature, 1998, 391(6669): 806-811
22. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs[J]. Nat Rev Genet, 2002, 3(10): 737-747
23. Elbashir SM, Harorth J, Weber K, et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs [J]. Methods, 2002, 26(2): 199-213
24. Miyagishi M, Taira K. U6 promoter driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells [J]. Nat Biotechnol, 2002, 20(5): 497-500
25. Tuschl T. Expanding small RNA interference [J]. Nat Biotechnol, 2002, 20(5): 446-448
26. Ming Jiang, Jo Milner. Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference[J]. Oncogene, 2002, 21: 6041-6048
27.牛晓宇,彭芝兰,王何。RNA干涉抑制子宫颈癌CaSki细胞株HPV16 E6基因的研究。癌症,2004,23(11):1257-62
28. Hall AH, Alexander KA. RNA interference of human papillomavirus type 18 E6 and E7 induces senescence in HeLa cells [J]. J Virol, 2003, 77(10): 6066-9
29. Hasuwa H, Kaseda K, Einarsdottir T, et al. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett. 2002, 532: 227-230
30. Carmell MA, Zhang L, Conklin DS, et al. Germline transmission of RNAi in mice. Nat Struct Biol. 2003, 10: 91-92
31. Song E, Lee SK, Wang J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003, 9: 347-351
32. Filleur S, Courtin A, Ait-Si-Ali S, et al. SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth . Cancer Res. 2003, 63: 3919-3922