MCPH1 shRNA逆转录病毒载体构建及其稳定表达的宫颈癌CaSki细胞系的建立
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摘要
子宫颈癌(cervical cancer, CC)是女性常见的恶性肿瘤之一,居我国女性肿瘤第一位,每年新发现的病例为13.15万,近年来年轻妇女患宫颈癌的越来越多,已由20世纪50年代的9%上升到现在的24%。所以关于宫颈癌的研究越来越引起人们的重视。
近年来,随着人们对宫颈癌病因及分子机制等研究的深入,分子生物学技术的发展,宫颈癌的治疗有了一种新策略。研究者们提出可以将目的基因用基因转移技术导入靶细胞,在分子水平上阻断宫颈癌的发生发展。此方法具有治疗特异性强、效果显著、基本不损伤正常组织的优点,是一项非常有前景的宫颈癌治疗方案。
本研究拟使用逆转录病毒介导的MCPH1 shRNA建立RNA干扰表达体系,并观察其在宫颈癌CaSki细胞中对MCPH1表达的影响,为宫颈癌基因治疗的深入研究奠定必要的实验基础。
研究方法:
1.MCPH1 siRNA有效序列的筛选。设计siRNA序列,人工化学合成后,用RNAiMax将其转入宫颈癌CaSki细胞中,①利用RT-PCR技术检测处理后细胞中MCPH1 mRNA的表达情况。②使用Western-blotting技术检测处理后细胞中MCPH1蛋白的表达情况。
2.逆转录病毒载体的构建及鉴定。根据筛选出的有效siRNA序列,设计并合成shRNA双链DNA片段,将该片段重组到逆转录病毒质粒pSUPER Retro中,进行测序分析鉴定,构建携带人MCPH1基因RNA干扰的逆转录病毒载体pSUPER- shRNA- MCPH1。
3.稳定表达的宫颈癌CaSki细胞系的建立。①用PT67细胞包装构建好的逆转录病毒载体。②使用产生的重组逆转录病毒感染宫颈癌细胞株CaSki细胞,用puromycin筛选产生稳定的细胞克隆。③用RT-PCR与real time PCR技术检测细胞中MCPH1 mRNA表达的变化。④用Western-Blotting技术检测细胞中MCPH1蛋白表达的变化。
实验结果:
1.设计出两条siRNA的序列,转入细胞72h后提取总RNA和蛋白,经RT-PCR及Western-blot技术发现MCPH1的表达均受到抑制。其中1号序列的干扰效果最为明显。
2.成功构建重组逆转录病毒质粒,经测序鉴定正确。
3.成功包装出逆转录病毒,感染CaSki细胞后用puromycin筛选出稳定的单细胞克隆;RT-PCR, real time PCR和Western-Blotting检测人MCPH1 mRNA和蛋白表达水平明显低于阴性对照组和正常对照组。
结论:
1.成功筛选出有效的siRNA序列。
2.成功构建逆转录病毒载体pSUPER-shRNA-MCPH1,并包装出逆转录病毒,感染CaSki细胞,建立了稳定的细胞系。在该细胞系中,MCPH1基因的mRNA和蛋白水平明显下降,说明构建MCPH1的RNA干扰体系初步成功,为进一步研究MCPH1在宫颈癌中的作用奠定了基础。
Cervical cancer is one of common malignant tumors in women, worldwide, it is the second leading cause of cancer deaths among women. Experts say more than 200,000 women die from cervical cancer every year. These deaths are most common in developing countries. Therefore, research on cervical cancer has drawn increasing attention.
Current mainstream treatments of cancer are surgery, radiotherapy and chemotherapy. Even though scientists and medical staff have continuously find ways to improve these three treatments, many patients are still not healed by these treatments. Gene therapy, which is the introduction of genetic material into a patient's tissues with the intent to achieve therapeutic benefit, is the only way to tackle cancer in the 21st century.
MCPH1 is one of six genes causing primary microcephaly when non-functional mutations exist in the homozygous state. The gene encodes the BRCT-domain containing protein microcephalin /BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage.
RNA interference (RNAi) is a conserved biologic response to double-stranded RNA that results in the sequence-specific silencing of target gene expression. Over the past 5 years, an intensive research effort has facilitated the rapid movement of RNAi from a relatively obscure biologic phenomenon to a valuable tool used to silence target gene expression and perform large-scale functional genomic screens.
In this study, we have investigated the efficacy of preformed shRNA to modulate the expression of MCPH1 gene in CaSki cells, and established the cell lines with stable silencing MCPH1 gene expression in cervix cancer cell line.
1. To screen effective siRNA sequences targeting MCPH1 gene. METHODS: We generated two siRNA sequences targeting against MCPH1. then the annealed duplexes were were transfected into CaSki cells. The effect of siRNA on the transcription and translation of MCPH1 gene was analyzed by RT-PCR and Western blot.
RESULTS: Transcription level of MCPH1 gene decreased significantly, the amount of Microcephalin protein also decreased significantly. The siRNA sequence 1 group decreased more than siRNA sequence 2 group. CONCLUSION: siRNA can inhibit the expression of MCPH1 gene, and the silence effect of siRNA sequence 1 was better than siRNA sequence 2.
2.To construct the recombinant retroviral vector with MCPH1 gene targeted shRNA, and establish its stable CaSki cell line.
METHODS: For preparation of duplexes, sense- and antisense-stranded oli- gonucleotides were mixed and annealed, the annealed duplexes were cloned into the sites of BglII and HindⅢof the pSUPER retroviral vector for constructing the recombinant retrovirus plasmid. Then the plasmid was packaged through PT67 cells in order to prepare the recombinant retrovirus expressing shRNA of MCPH1 gene. Retrovirus serum was collected and used to directly transfect CaSki cells. After puromycin screening, cells with stable expression of shRNA were cultured. The silencing effect of MCPH1 gene were determined by RT-PCR, real time PCR and Western blot assay. RESULTS: Sequencing revealed that shRNA was successfully cloned into the pSUPER retrovirus vector. We got two vectors called pSUPER- shRNA-MCPH1 and Negative control. After being transfected into CaSki cells, the pSUPER-shRNA-MCPH1 was found to inhibit the MCPH1 gene effectively.
CONCLUSION: The pSUPER-shRNA-MCPH1 retrovirus vector was successfully constructed. It shows effective inhibition on the expression of MCPH1 at mRNA and protein levels, which is the basis for further study of molecular functions of MCPH1, may be potentially useful in cervix cancer gene therapy.
近年来,随着人们对宫颈癌病因及分子机制等研究的深入,分子生物学技术的发展,宫颈癌的治疗有了一种新策略。研究者们提出可以将目的基因用基因转移技术导入靶细胞,在分子水平上阻断宫颈癌的发生发展。此方法具有治疗特异性强、效果显著、基本不损伤正常组织的优点,是一项非常有前景的宫颈癌治疗方案。
本研究拟使用逆转录病毒介导的MCPH1 shRNA建立RNA干扰表达体系,并观察其在宫颈癌CaSki细胞中对MCPH1表达的影响,为宫颈癌基因治疗的深入研究奠定必要的实验基础。
研究方法:
1.MCPH1 siRNA有效序列的筛选。设计siRNA序列,人工化学合成后,用RNAiMax将其转入宫颈癌CaSki细胞中,①利用RT-PCR技术检测处理后细胞中MCPH1 mRNA的表达情况。②使用Western-blotting技术检测处理后细胞中MCPH1蛋白的表达情况。
2.逆转录病毒载体的构建及鉴定。根据筛选出的有效siRNA序列,设计并合成shRNA双链DNA片段,将该片段重组到逆转录病毒质粒pSUPER Retro中,进行测序分析鉴定,构建携带人MCPH1基因RNA干扰的逆转录病毒载体pSUPER- shRNA- MCPH1。
3.稳定表达的宫颈癌CaSki细胞系的建立。①用PT67细胞包装构建好的逆转录病毒载体。②使用产生的重组逆转录病毒感染宫颈癌细胞株CaSki细胞,用puromycin筛选产生稳定的细胞克隆。③用RT-PCR与real time PCR技术检测细胞中MCPH1 mRNA表达的变化。④用Western-Blotting技术检测细胞中MCPH1蛋白表达的变化。
实验结果:
1.设计出两条siRNA的序列,转入细胞72h后提取总RNA和蛋白,经RT-PCR及Western-blot技术发现MCPH1的表达均受到抑制。其中1号序列的干扰效果最为明显。
2.成功构建重组逆转录病毒质粒,经测序鉴定正确。
3.成功包装出逆转录病毒,感染CaSki细胞后用puromycin筛选出稳定的单细胞克隆;RT-PCR, real time PCR和Western-Blotting检测人MCPH1 mRNA和蛋白表达水平明显低于阴性对照组和正常对照组。
结论:
1.成功筛选出有效的siRNA序列。
2.成功构建逆转录病毒载体pSUPER-shRNA-MCPH1,并包装出逆转录病毒,感染CaSki细胞,建立了稳定的细胞系。在该细胞系中,MCPH1基因的mRNA和蛋白水平明显下降,说明构建MCPH1的RNA干扰体系初步成功,为进一步研究MCPH1在宫颈癌中的作用奠定了基础。
Cervical cancer is one of common malignant tumors in women, worldwide, it is the second leading cause of cancer deaths among women. Experts say more than 200,000 women die from cervical cancer every year. These deaths are most common in developing countries. Therefore, research on cervical cancer has drawn increasing attention.
Current mainstream treatments of cancer are surgery, radiotherapy and chemotherapy. Even though scientists and medical staff have continuously find ways to improve these three treatments, many patients are still not healed by these treatments. Gene therapy, which is the introduction of genetic material into a patient's tissues with the intent to achieve therapeutic benefit, is the only way to tackle cancer in the 21st century.
MCPH1 is one of six genes causing primary microcephaly when non-functional mutations exist in the homozygous state. The gene encodes the BRCT-domain containing protein microcephalin /BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage.
RNA interference (RNAi) is a conserved biologic response to double-stranded RNA that results in the sequence-specific silencing of target gene expression. Over the past 5 years, an intensive research effort has facilitated the rapid movement of RNAi from a relatively obscure biologic phenomenon to a valuable tool used to silence target gene expression and perform large-scale functional genomic screens.
In this study, we have investigated the efficacy of preformed shRNA to modulate the expression of MCPH1 gene in CaSki cells, and established the cell lines with stable silencing MCPH1 gene expression in cervix cancer cell line.
1. To screen effective siRNA sequences targeting MCPH1 gene. METHODS: We generated two siRNA sequences targeting against MCPH1. then the annealed duplexes were were transfected into CaSki cells. The effect of siRNA on the transcription and translation of MCPH1 gene was analyzed by RT-PCR and Western blot.
RESULTS: Transcription level of MCPH1 gene decreased significantly, the amount of Microcephalin protein also decreased significantly. The siRNA sequence 1 group decreased more than siRNA sequence 2 group. CONCLUSION: siRNA can inhibit the expression of MCPH1 gene, and the silence effect of siRNA sequence 1 was better than siRNA sequence 2.
2.To construct the recombinant retroviral vector with MCPH1 gene targeted shRNA, and establish its stable CaSki cell line.
METHODS: For preparation of duplexes, sense- and antisense-stranded oli- gonucleotides were mixed and annealed, the annealed duplexes were cloned into the sites of BglII and HindⅢof the pSUPER retroviral vector for constructing the recombinant retrovirus plasmid. Then the plasmid was packaged through PT67 cells in order to prepare the recombinant retrovirus expressing shRNA of MCPH1 gene. Retrovirus serum was collected and used to directly transfect CaSki cells. After puromycin screening, cells with stable expression of shRNA were cultured. The silencing effect of MCPH1 gene were determined by RT-PCR, real time PCR and Western blot assay. RESULTS: Sequencing revealed that shRNA was successfully cloned into the pSUPER retrovirus vector. We got two vectors called pSUPER- shRNA-MCPH1 and Negative control. After being transfected into CaSki cells, the pSUPER-shRNA-MCPH1 was found to inhibit the MCPH1 gene effectively.
CONCLUSION: The pSUPER-shRNA-MCPH1 retrovirus vector was successfully constructed. It shows effective inhibition on the expression of MCPH1 at mRNA and protein levels, which is the basis for further study of molecular functions of MCPH1, may be potentially useful in cervix cancer gene therapy.
引文
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[2] Turkcuoglu I, Tezcan S, Kaygusuz G, et a l. The role of p53, Bcl2 and Ki67 in p remalignant cervical lesions and cervical cancer[ J ].Eur J Gynecol O ncol, 2007, 28 (4) : 290-293.
[3] Bernd K, Sven P, Michael B, et a l. Gene expression profiles reveal an up regulation of E2 F and down regulation of interferon targets by HPV18 but no changes between keratinocytes with integrated or episomal viral geneomes [ J ]. V irology, 2006, 353 (1) : 2002209
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[7] Wood JL, Singh N, Mer G, et al. MCPH1 functions in an H2AX-dependent but MDC1-in dependent pathway in response to DNA damage [J]. Biol Chem,2007,282(48):35416-35423.
[8] Bartkova J, Horejsi Z, Koed K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis [J]. Nature, 2005, 434(7035):864-870.
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[11] Lin SY, Rai R, Li K, et al. BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1-Chk1 pathway, implicating checkpoint dysfunction in microcephaly [J]. Proc. Natl. Acad. Sci. 102, 15105-15109.
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