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Study on Effect of Lentivirus-mediated RNA Interference of E2F-1 in Oral Squamous Cell Carcinoma Cells

Author: JiangFei
Tutor: ChenNing
School: Nanjing Medical University
Course: Clinical Stomatology
Keywords: Tongue squamous cell carcinoma cells Tca8113 E2F-1 Recombinant lentivirus RNAi Gene therapy Cell cycle
CLC: R739.8
Type: Master's thesis
Year: 2011
Downloads: 17
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Abstract

Oral squamous cell carcinoma (orals squamous cell carcinoma, OSCC) is a class of epithelial-derived malignancies, systemic six common cancers. Common in the tongue, floor of the mouth, cheek, gums, lips and palate, which went mostly tongue. Although in recent years scholars from various countries and various efforts clinicians continue to promote oral squamous cell carcinoma, clinical diagnosis and treatment techniques, and postoperative overall 5-year survival rate was no significant improvement. E2F family of transcription factors in the regulation of cell proliferation and differentiation has an important role in the study of tumor occurrence and development mechanism. E2F-1 is one of the eight members of the E2F family, two characteristics of oncogenes and tumor suppressor genes, suggesting that E2F-1 in tumor formation played a the oncogenes role or from the tumor suppressor gene role depends on the state of the two effects, and organ-specific. E2F-1 high expression in oral premalignant lesions and oral squamous cell carcinoma, and there is a close correlation between the expression level of the patient's prognosis. RNA interference (RNA interference, RNAi) is a revolutionary technology to interference targeting mRNA thereby inhibiting the expression of the corresponding genes. Through interference of mRNA of the target gene expression can be effectively broken ring of mRNA reached so that the gene \This study combines lentiviral vector-mediated targeting E2F-1 mRNA the shRNA fragments interference tongue squamous cell carcinoma cell line Tca8113 E2F-1 mRNA expression, explore the pathway downstream gene of E2F-1 mRNA interference after pRb/E2F-1 The expression Tca8113 cell proliferation, apoptosis biological changes provide a theoretical basis and experimental basis for the study of the pathogenesis and gene therapy in OSCC. . The first part of the E2F-1 gene RNAi lentiviral vector and identification purposes: Construction and identification of lentiviral vector of RNA interference targeting the E2F-1 gene. Methods: This topic through Sigma-Aldrich website design targeting the E2F-1mRNA three shRNA fragment, according to the carrier in the lentiviral packaging system required restriction sites (Age I / EcoR I), plus synthetic shRNA fragment on the corresponding base, the shRNA fragment connected to the vector, transforming competent cells, and positive clones sequenced. The the the sequencing results consistent with the design, transferred E.ColiTop10 mass preparation two helper plasmid containing the shRNA fragments pLKO.1-TRC cloning vector plasmid and recombinant lentiviral packaging systems, namely the pCMV-dR8, pVSVG, reorganization of E2F-1 gene RNAi lentiviral particles. Tongue squamous cell carcinoma of the three kinds of E2F-1-shRNA Lentiviral Particles infection of Tca8113. The infection completed, Tca8113 cells successfully infected with puromycin. Tca8113 cells in PCR, Real time PCR, Western blot infection in E2F-1 expression screening effective shRNA. Results: shRNA fragments connected to the carrier, PCR amplification products by agarose gel electrophoresis showed specific bands shRNA fragments successfully loaded to pLKO.1-TRC cloning vector. The sequencing results consistent with the design. Three kinds of recombinant lentiviral particles Tca8113 cells after infection, the PCR amplification products by agarose gel electrophoresis visible strip E2F1-shRNA-1 significantly weakened; Real time PCR detection display of E2F-1 expression of E2F-1-shRNA-1 group reduce the level of about 70%; Western blot E2F-1-shRNA-1 group E2F-1 protein levels were also significantly reduced. E2F-1-shRNA-1 has obvious interference efficiency. Conclusion: shRNA lentiviral vector targeting E2F-1 gene was successfully constructed, and the specific silencing of E2F-1 gene expression, for the study of E2F-1 in oral squamous cell carcinoma development provides the basis of preliminary experiments. The second part of the specific silencing of E2F-1 gene on human tongue squamous cell carcinoma cell line Tca8113 cells and its molecular mechanism research purposes: observation of E2F-1 gene silencing in human tongue squamous carcinoma cell line Tca8113 cell proliferation, apoptosis and cell cycle , and to explore the molecular mechanisms. METHODS: Cultured Tca8113 cell interference E2F-1 expression by Real time PCR, Western blot, respectively, from gene and protein expression levels detected E2F-1, pRb, Cyclin E, p14 and p53 expression. Application of flow cytometry cell cycle and apoptosis rate of change. MTT assay The change in cell proliferation. Results: E2F-1-shRNA group Tca8113 cells in MTT assay results show: the value-added ability of the cells compared with the negative control group and blank group were significantly lower. The results of flow cytometry: apoptosis than column E2F-1-shRNA-1 group was significantly higher than the negative control group and blank group was statistically significant (P lt; 0.05), and E2F-1- shRNA-1 cells, cell cycle, reduce the proportion of G1 phase, G2 phase was increased compared with the control group was statistically significant (P lt; 0.05), the S period no significant change. E2F-1-shRNA-1 cells reduced expression of Cyclin E, p14, p53 (P lt; 0.05), no significant changes and Rb. Conclusion: Tca8113 cells in E2F-1 levels decreased, it is able to inhibit their proliferation, and can induce apoptosis, change Tca8113 cell cycle distribution. The mechanism may be downstream proliferation (Cyclin E) the ways and p14/MDM2/p53 apoptosis pathway. Tip E2F-1 downregulation Tca8113 cell changes involving multi-gene, multi-level regulation.

Study on Effect of Lentivirus-mediated RNA Interference of E2F-1 in Oral Squamous Cell Carcinoma Cells

Abstract

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