Study On Effect Of Lentivirus-mediated RNA Interference Of E2F-1 In Oral Squamous Cell Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is one of malignancies from epithelium tissue, which is the sixth most common cancer, and frequently occurs in floor of oral cavity, buccal division, gingiva, lip, palate and especially in tongue. During past years, the approaches in diagnosis and treatment of OSCC have been improved by endeavor of researchers and development of technology, but the overall and disease-free survival rates of OSCC have not been improved significantly. In the researches for mechanisms of tumorgenesis, transcriptional factor E2F family has been found that is involved in regulating cells'proliferation and differentiation. E2F-1 is one of the eight members in E2F family, however, it may have a unique role compared to other E2Fs, showing characteristics of both an oncogene and a tumour suppressor. In lesion precancerous and OSCC, phosphorylated pRb deregulates E2F-1, and the levels of E2F-1 expression refer to prognosis of OSCC closely. Thus, E2F-1 may act as an oncogene in development of OSCC. RNA interference (RNAi) is an evolutionarily conserved process that inhibits gene expression primarily by targeting mRNAs. In this study, lentivirus-mediated shRNA was employed to downregulate endogenous E2F-1 expression to study the function of E2F-1 in the pRb/E2F-1 pathway in OSCC cell line Tca8113 cells, and investigate the alteration of Tca8113 cells in proliferation and apoptosis.Part I Construction and identification of lentiviral vector of RNA interference of E2F-1 gene Objective: To construct a lentiviral vector-mediated RNA interference (RNAi) of targeting gene E2F-1.Methods: Three shRNA sequences targeting to E2F-1 mRNA were designed on Sigma-Aldrich website, then synthesized and cloned to pLKO.1-TRC cloning vector. The pLKO.1-TRC cloning vectors were transfected to E.coli TG1, and positive clones were selected and tested the sequences. The plasmids consisted of pLKO.1-TRC cloning vector loaded shRNAs, pCMV-dR8 and pVSVG were amplified by E.Coli Top10, and recombined lentivirus particles. Tca8113 cells transfected with recombinant lentivirus particles contained E2F-1-shRNAs were selected by puromycin, their total RNAs and proteins were extracted for PCR, Real-time PCR, Western-blot to detected expressions of E2F-1, respectively, and the most efficient interference effect of three E2F-1-shRNAs were selected.Result: The bands of 300bp were displayed by agarose gel electrophoresis with PCR products and the sequences of these shRNAs were accordant with design. The fragments of shRNA were cloned in pLKO.1-TRC cloning vectors. Tca8113 cells transfected with lentivirus particles contained three shRNAs were selected by puromycin, and cultured. The total RNAs and proteins of these cells were extracted, and the expression of E2F-1 in these cells were detected by PCR and Western-blot. The expression of E2F-1 was downregulated significantly in E2F-1-shRNA-1 group cells.Conclusions: It indicates the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of E2F-1 mRNA,and those provides the precondition for the further study of E2F-1 in OSCC (oral squamous cell carcinoma) pathogenesis by using lentivirus system to construct stably silencing E2F-1 in OSCC cell lines.Part II Study on effect of lentivirus-mediated RNA interference of E2F-1 in oral squamous cell carcinoma cells and the molecular mechanisms Objective: To observe the effect of lentivirus-mediated RNA interference of E2F-1 in oral squamous cell carcinoma cells and explore the underlying molecular mechanisms.Methods: Tca8113 cells transfected by lentivirus particles contained the most efficient interference effect E2F-1-shRNA were cultured, and their proliferation and the rate of apoptosis were detected by MTT and flow cytometry, respectively. Real-time PCR and western-blot were used to detect alteration of some of genes in pRb/E2F-1 pathway in these Tca8113 cells, such as E2F-1, pRb, Cyclin E, p14, p53.Result: In Tca8113 cells in group E2F-1-shRNA-1, the capability of proliferation was suppressed remarkably. The rate of apoptosis in these cells increased significantly (P<0.05). The expressions of Cyclin E, p14, p53 have been seen to decrease(P<0.05), while the expression of pRb has not been found alteration (P>0.05). The propotion of G1 phase was decreased, while G2 phase was accunmulated.Conclusion: Downregulation of E2F-1 may inhibit the proliferation of Tca8113 cells, while it can induce apoptosis and cell arrest at G2 phase via multiple network of cell cycle, that may refer to Cyclin E, p14, p53.