Experimental Study On Construction Of SiRNA Eukaryotic Expression Vector Specific To Protein Kinase CK2ɑ And Its Inhibitory Effects In Laryngeal Carcinoma

Part I Expression of protein kinase CK2ɑin laryngeal carcinoma Hep-2 cells and laryngeal Squmons Cell CarcinomaParagraph one Expression of protein kinase CK2ɑin human laryngeal carcinoma Hep-2 cellsObjective:To study the expression of Protein kinase CK2ɑin human laryngeal carcinoma Hep-2 cells and its significance.Methods:In this study, Hep-2 cells and Hacat cells were cultured in vitro and the expression level of Protein kinase CK2ɑmRNA in Hep-2 cells and Hacat cells were measured with reverse transcription polymerase chain reaction (RT-PCR).The expression of Protein kinase CK2ɑprotein in the Hep-2 cells and Hacat cells were measured with immunocytochemical technique.Results:It was showed that the expression level of Protein kinase CK2ɑmRNA and Protein kinase CK2ɑprotein were significantly stronger in Hep-2 cells as compared with Hacat cells (P < 0.05).Conclusion: Formation and development of laryngeal carcinoma could be in connection with the overexpression of Protein kinase CK2ɑ. Paragraph two The expressions of protein kinase CK2ɑin laryngeal squamous cell carcinomaObjective:To study the expression of Protein kinase CK2ɑin squamous cell carcinoma of larynx(LSCC) and to evaluate its clinical significance.Methods: The expressions of protein kinase CK2ɑprotein in 50 cases of LSCC tissues and 10 cases of normal mucous membrane of Larynx were studied by using immunohistochemical SP staining method and quantitative analysis of image.Results:The positive rate of Protein kinase CK2ɑstaining was 64.00%(32/50)in LSCC tissues,while only 30.00%(3/10)in normal mucous membrane of Larynx.A significant difference was observed for the average absorbance value of protein kinase CK2ɑpositive expression in two groups(P﹤0.01).No correlation was found between the average absorbance value of protein kinase CK2ɑpositive expression and sex,age,tumor sites,TNM stage and lymphatic matastasis,but there was significant correlation between the average absorbance value of protein kinase CK2ɑpositive expression and differentiation grading(G1→G2+G3)(P﹤0.01).Conclusion:The over expression of Protein kinase CK2ɑis closely related to the formation and development of LSCC. Protein kinase CK2ɑmay be one of the predictors for the malignant grade of LSCC.To inhibit the over expression might be new therapeutic methods for LSCC. PartⅡConstruction of siRNA eukaryotic expression vector specific to protein kinase CK2ɑand its inhibitory effects on the growth and invasion of laryngeal carcinoma Hep-2 cellsObjective: To study the effect of small interfering RNA(siRNA) specific to protein kinase CK2ɑon proliferation,apoptosis and invasion of laryngeal carcinoma Hep-2 cells.Methods: siRNA expression plasmid psiRNA-hH1neo-CK2 specific to protein kinase CK2ɑand non-specific siRNA expression plasmid psiRNA-hH1neo-cont were constructed respectively ,and then were transfected into Hep-2 cells by lipofectamine methods. Protein kinase CK2ɑmRNA and protein of the transfected cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. The invasion of the transfected Hep-2 cells wes measured by Boyden chamber. Results: The siRNA eukaryotic expression vector specific to protein kinase CK2ɑwas constructed successfully.Protein kinase CK2ɑmRNA and protein expressions were significantly decreased in the cells transfected with psiRNA-hH1neo-CK2(P﹤0.05). The proliferation of Hep-2 cells was efficiently inhabited after transfected with psiRNA-hH1neo-CK2(P﹤0.05 ) . Obvious subdiploidy peaks were found in the cells transfected with psiRNA-hH1neo-CK2. the invasion of Hep-2 cells were efficient inhabited in the psiRNA-hH1neo-CK2 group(P﹤0.01). Conclusion: The siRNA expression plasmid specific to protein kinase CK2ɑmay suppress the proliferation and the invasion of Hep-2 cells,and induce its apoptosis. PartⅢInhibitory effects of siRNA expression plasmid specific to protein kinase CK2ɑon human laryngeal carcinoma xenograft in nude miceObjective: To study the effect on the expression of Protein kinase CK2ɑand growth of human laryngeal carcinoma xenograft in nude mice by applying small interfering RNA(siRNA) specific to protein kinase CK2ɑ.Methods:Human laryngeal carcinoma Hep-2 cells were implanted under the skin of nude mice.After the tumors grew to a definite size,the tumors were injected with siRNA expression plasmid specific to protein kinase CK2ɑ.The weight and volume of subcutaneous tumors were measured. the expression level of Protein kinase CK2ɑmRNA and protein of tumors were measured with reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical technique, respectively.Results: Protein kinase CK2ɑmRNA and protein expressions were significantly decreased in tumors transfected with siRNA expression plasmid specific to protein kinase CK2ɑ(P﹤0.05). The tumor grew slowly after transfected with siRNA expression plasmid specific to protein kinase CK2ɑ(P﹤0.01).Conclusion: The siRNA expression plasmid specific to protein kinase CK2ɑmay suppress the growth and the protein kinase CK2ɑexpression of subcutaneous tumors. RNA interfering technology may be a new strategy for the treatment laryngeal cancer.