Downregulation Of C-FLIP Induce JNK Activion In Malignant Melanoma Cell Line

Partâ… Expression of c-FLIP in malignant melanoma and itsrelation to the clinicopathologic featuresObjective:The aim of this work was to examine the expression patterns of c-FLIP in MM in vivo and explore the relationship of its expression with the clinicopathologic featuresMethods:Immunohistochemical staining with anti-c-FLIP antibody was performed in 77 tissue samples obtained from MM and 23 tissue samples from naevi. Immunoperoxidase staining method was applied to analyze the location of c-FLIP expressions in 34 CA and 16 normal foreskin tissues. Real time quantitative reverse transcriptase-polymerase chain reactions (RT-PCR) and western blotting were performed to further identify the expression of c-FLIP in CA. c-FLIP expression in A375, A875, SK-Mel-land SK-Mel-28 cell lines were examined by WB and FCM.Results:The expression of c-FLIP was increased in MM tissue compared with the detectable levels in the matched pigmented naevi lesions. It was significantly associated with the histological type and Clark's level of malignant melanoma. In four MM cell lines, c-FLIP expression was the highest in A875 cell lines. c-FLIP expression at either its mRNA or protein level was significantly higher in CA than that in normal foreskin.Conclusion:Exprect the role of anti-apoptosis, c-FLIP could promote the proliferation of cells. It might play an important role in the obtaining of aggressive biologic behaviors and be useful in predicting prognosis of patients with MM. Overexpression of c-FLIP might be involved in the hyperproliferation of keratinocytes in CA. Partâ…¡Construction and Identification of c-FLIP shRNAsObjectives:Short interfering RNA (siRNA) eukaryotic expression vector for c-FLIPT/L/S shRNA was constructed and transfected into MM A875 cell lines. To verify its effection of interference for c-FLIP in MM cell lines in vitro.Methods:â‘ c-FLIPT/L/S shRNA targeting human c-FLIP isoforms common sequence was synthesized and it was inserted into Bam HI-Hindâ…¢linearized Pgenesile-1 vector. The sequence of c-FLIPT/L/S shRNA plasmid was analyzed by DNA sequencer and restrict endonuclease cutting.â‘¡To screen the best silencing effect c-FLIPT/L/S shRNA, the alteration of c-FLIP mRNA and protein was checked by Rt-PCR, western blot and FCM after c-FLIPT/L/S shRNAs were transfected in MM cell lines.â‘¢The monoclone A875 cells with stable expression of best effect c-FLIPshRNAs were obtained by G418 selection and were identified with checking GFP expression by FCM.Results:â‘ It was verified that the sequence of constructed recombinant plasmids were correct by DNA sequencing and restrict endonuclease cutting.â‘¡Among the c-FLIPT/L/S shRNAs we tested, c-FLIPT1shRNAå’Œc-FLIPL1shRNA possessed the strongest inhibitory effect against c-FLIP.â‘¢We screened and obtained A875 cell lines with stable expressions of c-FLIPT/L/S shRNAs as A875 stable clone could observed under Fluorescence microscope and 96% A875 stable clone expressed GFP detected by FCM.Conclusions:It indicated that hairpin siRNA eukaryotic expression vector for c-FLIP isoforms would be successfully establishedand. It played a specific inhibitory role in A875 cell lines. This study laid theoretical foundation for further research of the therapy of c-FLIPT/L/S shRNAs vector in MM. Partâ…¢Downregulation of c-FLIP induce JNK activion in malignant melanoma cell linesObjective:To observe the influence of difference isoforms of c-FLIP shRNA on JNK pathway in MM A875 cell lines.Methods:To detecte the expression of p-JNK protein in stable clone A875 transfected with c-FLIPT/L/S shRNAs by western blot.Results:Comparing with untranstected and stable transtected with c-FLIPC shRNA A875 cell lines, p-JNK protein was increased significantly in A875 cell lines which were transtected with c-FLIPT/LshRNA. And in transtected with c-FLIPS shRNA A875 cell lines, p-JNK protein was unchanged.Couclusions:Downregulation of c-FLIP can induce the activion of JNK pathway in MM A875 cell lines. Partâ…£The influence of difference isoforms of c-FLIP shRNA on bionomics of MM cell linesObjective:To observe the influence of difference isoforms of c-FLIP shRNA on bionomics of MM A875 cell lines such as growth, proliferation and apoptosis.Methods:To count cells of A875cell lines which were transtected with c-FLIPT/L/S/C shRNA and obtain the cells'growth curve, and then to compare the difference among those type of cells. To detecet the proliferational activity of those cells by MTT; Using annexin V and propidium iodide(PI) double staining, we observe the rate of apoptosis by Flow cytometry(FCM).Results:Comparing with untranstected and stable transtected with c-FLIPc shRNA A875 cell lines, the growth were slower in A875 cell lines which were transtected with c-FLIPT/LshRNA. And the time they enter the log phase prolonged and platform were cut down. And optical density decreased significantly. Using annexin V and propidium iodide(PI) double staining, we observed that the rate of apoptosis of A875 cell lines which were transtected with c-FLIPT/LshRNA comparing with untranstected and stable transtected with c-FLIPc shRNA A875 cell lines by FCMConclusions:Difference isoforms of c-FLIP shRNA can not only suppress the growth and proliferation of MM A875 cell lines but also promote their apoptosis.