Effect Of KLF6 On The Growth And Metastasis Of Nasopharyngeal Carcinoma Cells And Its Mechanism

Objective: As a member of the KLFs family,KLF6 is a tumor suppressor gene that is widely expressed in organisms and participates in a variety of biological processes,including proliferation,apoptosis,differentiation,development,and signaling transduction.In a variety of tumors,due to the occurrence of heterozygous deletions and somatic mutations,the role of tumor suppressor genes cannot be exerted,leading to the occurrence and progression of tumors.In addition,the splicing variants of KLF6,KLF6-SV1,KLF6-SV2,and KLF6-SV3,are highly expressed in a variety of tumors,and promote tumorigenesis and development by affecting tumor cell proliferation,apoptosis,invasion and metastasis.However,so far,domestic and foreign researches on KLF6 in nasopharyngeal carcinoma(NPC)are scarce,and its specific role in the occurrence and development of nasopharyngeal carcinoma is not clear.To clarify the expression and localization of KLF6 in nasopharyngeal carcinoma and the effect of KLF6 on the proliferation,apoptosis,invasion and migration of nasopharyngeal carcinoma,we can further understand the molecular mechanism of nasopharyngeal carcinoma,provide theoretical basis for the diagnosis and prognosis assessment of nasopharyngeal carcinoma.and find new targets and directions for its prevention and treatment research.Methods: 1.Collect 43 cases of clinically diagnosed nasopharyngeal carcinoma tissue and 8 cases of non-cancerous control nasopharyngeal epithelial tissue,applying immunohistochemical staining to detect and analyze the expression and localization of KLF6 gene in nasopharyngeal carcinoma tissue cells.2.The specific designed siRNAs target KLF6 and its splicing variants KLF6-SV1,KLF6-SV2,KLF6-SV3 were synthesized and assessed by QT-PCR and western blotting in the previous period,were applied to transfect to nasopharyngeal carcinoma cells CNE2 and 5-8F,respectively.Western blotting was applied to verify and observe the interference effect of KLF6 protein.3.After KLF6-siRNA was transfected into nasopharyngeal carcinoma CNE2 and 5-8F cell lines,respectively.The cell growth was detected by MTT assay.The cell morphology was observed by Immunofluorescence assay under microscope.The cell apoptosis was detected by Hoechst 33258 staining and mitochondrialmembrane apoptosis kit.The cell invasion and migration ability were detected by Transwell test(coated and uncoated).The expression of KLF6,cell cycle related proteins,apoptosis related factors,cell invasion and migration related molecules and its potential role of proteins and signaling pathways were detected and analyzed by western blotting.Results: 1.Among the total 43 cases of nasopharyngeal carcinoma specimens,23(53.5%)cases showed plasma stained for KLF6 protein,17 cases showed nuclear stained but significantly lighter than the control or nonstaining(39.5%),only 3 cases showed nuclear staining.And all the 8 control nasopharyngeal epithelial tissues showed nuclear staining(P <0.01).These results suggested that KLF6 abnormally or ectopic in nasopharyngeal carcinoma tissues.2.Specific siRNAs targeting KLF6 and its splice variants can significantly down-regulate the expression of KLF6 and its splice variants in CNE2 and 5-8F cells.3.After nasopharyngeal carcinoma cells CNE2 and 5-8F transfected with KLF6-specific siRNA,the cell number decreased significantly and the cell morphology changed obviously under the microscope observation.The cells became thinner and longer,exhibited a long spindle shape,and the volume increased obviously.It is obviously that the cells spread more,the cytoplasm is richer,and the nuclear plasma ratio becomes smaller.Immunofluorescence detection showed the similar result about the cell appearence.4.The cell growth in the transfection group was significantly reduced(P <0.01)by MTT assay.Combined with the results by Western bloting,KLF6-siRNA may up-regulate the expression of cell cycledependent kinase inhibitors p21 and p27,and down-regulate the expression of cyclin B and cyclin D to prevent cell cycle progression and inhibit the growth of nasopharyngeal carcinoma cells.5.Apoptosis experiments showed that KLF6-siRNA can obviously induce apoptosis of nasopharyngeal carcinoma cells.Combined with the results of Western blot experiments,KLF6-siRNA may down-regulate the expression of Bcl-2 and Bcl-Xl and up-regulate the expression of Bax and Caspase-3 through endogenous apoptotic pathways to initiate cell apoptosis.6.The number of cells in the transfection group migrated or invaded to the transwell outside was significantly less than that in the control groups(P <0.01).Combined with the results of Western blot experiments,KLF6-siRNA may down-regulat the expression of Snail and Slug(both transcription inhibitor of E-cadherin),and N-cadherin and Vimentin(both mesenchymal markers),and down-regulate the expression of matrix metalloproteinases MMP2,MMP9,VEGF,and β-catenin,the key factor of the Wnt pathway,which promote invasion and migration,to reduce the ability of invasion and metastasis by reversing the ability of EMT.KLF6-siRNA transfection can also significantly reduce the expression of activated p-AKT and p-RSK.Conclusions: 1.KLF6 gene was abnormally expressed in nasopharyngeal carcinoma tissue cells.2.The KLF6 in nasopharyngeal carcinoma cells may promote the growth of nasopharyngeal carcinoma cells by regulating cell cycle and inhibit cell apoptosis through mitochondrial pathway.3.The KLF6 in nasopharyngeal carcinoma cells may induce EMT and affect the expression of key proteins that promote invasion and migration to promote the invasion and migration of nasopharyngeal carcinoma cells through AKT and RSK signaling pathways.The specific molecular mechanism still needs to be further studied and verified.