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Effects of HPV18-E6 Gene siRNA on Cultured Hep-2 Cells

Author: WuYaZuo
Tutor: ChengShiYin；ZhangHuiZhong
School: Fourth Military Medical University
Course: Otorhinolaryngology
Keywords: HPV18 - E6 gene survivin siRNA Laryngeal Gene therapy
CLC: R739.65
Type: Master's thesis
Year: 2008
Downloads: 179
Quote: 0
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Abstract


Objective: human papilloma virus (HPV) E6 gene in the early region is one of the most important coding cancer oncogene protein, and reported in the literature, related to HPV-E6 gene and laryngeal carcinoma cell immortalization. This study was closed laryngeal carcinoma cell line Hep-2 HPV-E6 gene expression using RNA interference to explore the downregulation of the activity of laryngeal carcinoma cell proliferation, further analysis of the gene with laryngeal cancer occurrence and development of the relationship, in order to laryngeal cancer of gene therapy screening of new targets. Method: (1) PCR amplification of survivin promoter, the recycling amplification product and replace the Ambion pSilencer-1 vector cmv promoter. Design two pairs of siRNA against HPV18-E6 and HPV18-E6 gene were constructed siRNA eukaryotic expression vector, restriction enzyme digestion and sequencing. (2) to carry HPV18-E6 gene laryngeal cancer Hep-2 cell system as target cells by PCR method HPV18-E6 gene, recycling its fragment and ligated into pGEM-T-Easy vector and sequencing, named as E6-T-Easy. Then this plasmid as a template to build a real-time PCR system required standard quality grain, named E6'-T-Easy. (3) to carry HPV18-E6 gene laryngeal carcinoma Hep-2 cell system as target cells by cationic liposome transfection with siRNA expression vector and stably transfected cell lines using G418 selection. (4) Realtime PCR and Western blot detection after transfection, cells HPV18-E6 gene mRNA and protein expression levels. (5) MTT cell proliferation was detected; flow cytometry analysis of cell cycle changes. Results: (1) successfully constructed HPV18-E6 gene of RNA interference eukaryotic expression vector was named pSilencer4.1-Surp neo-E1, and pSilencer4.1-the Surp neo-E2. Correct by restriction enzyme digestion and sequencing; (2) successfully cloned from laryngeal cancer Hep-2 cell lines HPV18-E6 full length cDNA fragment by restriction enzyme digestion and sequencing with Gene Bank sequence; (3 ) filter out interference vector-transfected cells, HPV18-E6 mRNA and protein expression of cut throat cancer cell lines stably transfected; (4) E6 gene expression down-induced laryngeal cancer cell proliferation activity was significantly decreased; (5) E6 The genes downregulated induced G0/G1 phase cells increased up to 30.4%, and G2 / M phase cells to reduce a 20% increase. Conclusion: the the two interferometers carrier laryngeal cancer Hep-2 cell lines can cut the HPV18-E6 gene expression and pSilencer4.1-Surp neo-E1 strongest inhibition. HPV18-E6 gene downregulated significantly reduced the growth of Hep-2 cell proliferation activity, growth inhibition may be related to the blockade and cell cycle G0/G1. Closed HPV18-E6 gene expression compared to a new target for the development of laryngeal cancer targeting laboratory foundation.

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CLC: > Medicine, health > Oncology > Department of Otolaryngology tumor > Laryngeal tumors
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