| Objective: This thesis mainly conducts a research on Real-time PCR to detect the expressions of miR-193b in laryngeal squamous cell carcinoma and its clinical significance. Construction of hsa-miR-193b-3p lentiviral expression vector, preparation and packaging of lentivirus.It establishes the stable target gene about expression of laryngeal squamous cell carcinoma in Hep-2 cell strain. It investigates the effect of antisense oligodeoxynucleotides recombinant lentivirus (ASO-miR-193b) effect on the behavior of laryngeal squamous cell carcinoma cell biology with this experimentation.Methods: The expression of miR-193b was detected by real-time fluorescent quantitative PCR in laryngeal carcinoma and adjacent tissues, and to research its relationship between staging, pathological grading between laryngeal carcinoma. The design and construction of miR-193b plasmid expression vector, were transfected into 293T cells by lentiviral vector in the down-regulation of miR-193b expression. The experiment was divided into three groups: experimental group (miR-193b), blank control group, negative control group (NC), then transfected into laryngeal squamous cell carcinoma Hep-2 cells, real-time fluorescence quantitative PCR (Q-PCR) to detect the expression level of miR-193b to verify its transfection efficiency. Laryngeal squamous cell carcinoma Hep-2 cells after transfection by CCK-8 proliferation assay, colony formation assay and to observe its effect on cancer cell proliferation.Results: Q-PCR detected that the expression level of laryngeal squamous cell carcinoma tissue than in adjacent tissue miR-193b were significantly increased. The down-regulation of hsa-miR-193b expression lentivirus expression vector was successfully constructed, titer of lentivirus 1×108TU/ml. Downregulation of miR-193b lentiviral vector of laryngeal cancer cells express high levels of miR-193b activity, Hep-2 cell clone formation rate compared with the control group decreased significantly, P<0.05,48-72h group compared with the control group, ASO-miR-193b was significantly reduced in Hep-2 cells (P＜0.05), which found that can inhibit the proliferation of laryngeal carcinoma cells.Conclusions: This research found that laryngeal cancer tissue miR-193b expression was significantly up-regulated, suggesting that miR-193b expression may be related to the occurrence and development of laryngeal carcinoma than in adjacent tissue. Transfection of ASO-miR-193b can significantly inhibit the proliferation of cancer cells, which indicates that miR-193b play a role of cancer; It provides gene therapy for laryngeal carcinoma and other malignant tumors is an effective target and theoretical basis. |
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