Effect Of FAK Gene Silencing On Proliferation And Invasion In Hep-2 Cells

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of tumors worldwide , 7.9% to 35 % of them is laryngocarcinoma . Despite recent advancements in diagnosis and treatment, the overall survival has undergone little improvement over the past few decades. The major cause of the lethal progression of this type of cancer is the spreading of the malignant cells to regional lymph nodes, which represents a major prognostic indicator and serves as a guide for therapeutic strategies. Although many efforts had been devoted to make better understand the molecular mechanisms involved in the progression of this type of cancer, accurate and reliable biomarkers that can predict patients at high risk of lymphatic metastases have not yet to be defined. Loss of adhesion of the malignant cells to the extracellular matrix is one of the fundamental pathways that promote tumor cell migration, invasion, and metastasis. A key factor involved in the control of cell-extracellular matrix interactions is the focal adhesion kinase (FAK), an intracellular tyrosine kinase protein that is localized to cellular focal contact sites.Evidences have suggest that FAK overexpression correlates with the proliferation and invasive potential of a tumor and patient prognosis ,such as carcinomas of the breast, colon, thyroid, liver, prostate . However, the researches in laryngeal carcinoma are rare. This study was to evaluate FAK protein expression in laryngeal carcinoma (laryngeal squamous cell carcinoma tissue specimens) and Hep-2 cells by methods of immunohistochemical (SP) and immunocytochemical, respectively. And a recombinant plasmid generating FAK short hairpin RNA in mammalian cells was constructed to investigate the effects of FAK silencing on proliferation and invasion in transfected l Hep-2 cells.Part One FAK protein expression in laryngeal carcinoma and Hep-2 cellsObjective: FAK protein expression were analyzed by method of immunohistochemical (SP) and immunocytochemical in laryngeal carcinoma and Hep-2 cells, respectively.We also studied the possible relationship of FAK protein deregulation with clinicopathologic variables.Methods: Immunohistochenical (SP) method was used to examine the expression of FAK protein in 42 patients with laryngeal carcinoma and 40 patients with polyp of cord (control group). Expression of FAK protein was detected by immunocytochemical in Hep-2 cells. Positive rates of FAK were evaluated by microscopy .The difference was analyzed by statistics. Results: (1) FAK protein expression level in aryngeal carcinoma:The positive rates of FAK protein was 78.57%(33/42)and 12.50%(5/40) in human laryngeal carcinoma and control group,respectively,with significant difference(P<0.05).Moreover, the expression of FAK protein were higher in the specimens with invasion than that these without invasion and lymph node metastasis (P<0.05). (2) The FAK protein was positive in all of Hep-2 cells.Conclusion:FAK protein plays a key role in the invasion and metastasis of laryngeal carcinoma and might prognosticate invasion and metastasis.Part Two Effect of FAK gene silencing on proliferation and invasion in Hep-2 cellsObjective: To construct recombinant plasmids generating short hairpin RNA in mammalian cells,and investigate its effect on FAK mRNA and protein in laryngeal carcinoma Hep-2 cells, as well as the effects of recombinant vector FAK-pGenesil on proliferation and invasion of Hep-2 cells.Methods: Construct recombinant plasmids generating short hairpin RNA, including two interferential sequences FAK-pGenesil-CH1, FAK-pGenesil-CH2 and one negative control sequence FAK-pGenesil-HK. Hep-2 cells were transfected with recombinant plasmids.FAK mRNA and FAK protein were analyzed by RT-PCR and Western blot, respectively.Cell proliferation,cell cycle and invasion were observed by MTT assay,flow cytometry and transwell invasion assay, respectively.Results:(1)The transfected conditions and the rate of transfection:the most highly efficiency was 55% when the ratio of recombinant plasmids to lipofectamineTM2000 is 1:2.5(.2)RT-PCR,Western blot results: recombinant plasmids FAKpGenesil-CH1,FAK-pGenesil-CH2 downregulate the expression of FAK mRNA and protein. The inhibition rate of FAK mRNA was 71.54%,67.89% respectively, with significant difference to the control(P<0.05);The inhibition rate of FAK protein was 41.74%,39.38% respectively, with significant difference to the control(P<0.05). (3)MTT assay,flow cytometry(FCM) and transwell invasion assay results : the proliferation inhibition ratio reached to 58.34%,52.78%, 48 hours after transfection of recombinant plasmids FAKpGenesil-CH1,FAK-pGenesil-CH2 ,while the control was 8.34%(P<0.05); FCM analysis showed that more cells stay at G0-G1 phase in the transfected group than control; the numbers cells of through Matrigel membrane were 56.0±6.7,51.0±5.1, respectively, with significant difference to the control group (P<0.05).Conclusion:The constructed recombinant plasmid can suppress the expression of FAK mRNA and protein in Hep-2 cells.Transfection with the recombinant vector FAK-pGenesil may inhibit the cloning and invasion of Hep-2 cells.