| Backgrounds:Follicular thyroid carcinoma(FTC)is one of the most common malignant tumor of endocrine system.Its incidence is increasing yearly all over the world.Follicular thyroid carcinoma is one of the most malignant thyroid cancers,which is prone to distant metastasis.Currently,it is mainly diagnosed by pathological staining membrane.There is no sensitive and specific tests for the diagnosis,and also lack of the treatment and prognosis markers.Voltage-gated sodium channels(VGSCs)are proteins formed by α-subunit(Nav1.1-Nav1.9)and β-subunit.It mainly distributes in neuromuscular excitable cells.Its main function is to mediate the transmembrane in flow of sodium ions to produce and transmit action potentials.Recently,several studies have shown that Navs have abnormally high expression in many tumors,which promotes the proliferation,migration,invasion and metastasis of tumor cells.However,the expression,function and molecular mechanism of Navs in follicular thyroid carcinoma cells are not clear.Here,the expression of Nav1.1-Nav1.9 was screened and analyzed in normal thyroid cells and tissues,as well as FTC-133 cells and cancer tissues.By downregulating the expression of the confirmed Nav subtype to further explore its role and molecular mechanism,as well as its influence on the drug susceptibility.The study provides a new molecular target for the diagnosis and treatment of follicular thyroid carcinoma.Methods:1.Real-time PCR and western blot were used to detect the expression levels of Navs in normal thyroid cell line Nthy-ori 3-1 and follicular thyroid cancer cell line FTC-133.2.Immunohistofluorescence staining showed the expression and localization of Nav1.6 in normal and cancer tissues.3.mRNA and protein levels of Nav1.6 in FTC133 cells were detected after Nav1.6-siRNA transfection by Real-time PCR and western blot.4.The effect of Nav1.6-siRNA and general sodium ion blocker(TTX)on cell proliferation was detected by Real-time PCR,western blot,CCK-8 assay and Ed U incorporation assay.5.The effect of Nav1.6-siRNA and TTX on cell migration ability was examined by Real-time PCR,western blot,transwell cell invasion assay and gelatin zymography assay.6.The effect of Nav1.6-siRNA and TTX on cell stemness was detected by Realtime PCR,western blot and colony formation assay.7.The effect of Nav1.6-siRNA,over expression of FAK and FAK inhibitor on cell signaling pathway was detected by western blot,gelatin zymography assay and colony formation assay.8.Explore the optimal concentration of Ubenimex by CCK-8 assay.The apoptosis markers were detected by western blot,TUNEL and DAPI staining.Results:1.The results of Real-time PCR showed the expression levels of Nav1.6 gene in follicular thyroid carcinoma cells were higher than Nthy-ori 3-1 cells.2.Immunohistofluorescence staining showed that Nav1.6 was expressed in the plasmic membrane of thyroid follicular cancer cells.The expression levels of Nav1.6 in follicular thyroid carcinoma were higher than those in normal thyroid tissues.3.Real-time PCR and western blot showed that the expression levels of Nav1.6 mRNA and protein were reduced after transfection with Nav1.6-siRNA.4.Real-time PCR and western blot showed that knocking-down of Nav1.6 expression decreased the mRNA and protein levels of PCNA,Cyclin D1 and Cyclin E1 in FTC-133 cells.CCK-8 assay and Ed U incorporation assay showed TTX inhibited the proliferation of FTC-133 cells.5.Western blot showed that knocking-down of Nav1.6 decreased the expressions of MMP9 in FTC-133 cells,while the expressions of TIMP1 and TIMP2 were increased.Transwell cell invasion assay showed that Nav1.6-siRNA transfection and TTX inhibited the invasion ability of FTC-133.6.Western blot showed that knocking-down of Nav1.6 decreased the expressions of Nanog and SOX2 in FTC-133 cells.Colony formation assay showed Nav1.6-siRNA transfection and TTX inhibited the stemness of FTC-133.7.Knocking-down of Nav1.6 decreased the phosphorylation levels of FAK and PAX in FTC-133 cells.Western blot,gelatin zymography assay and colony formation assay showed that over expression of FAK increased the phosphorylation levels of FAK and PAX,as well as the downstream molecules such as PCNA,Nanog and MMP9.Nav1.6-siRNA transfection and FAK inhibitor reduced the expression of those.8.CCK-8 cell proliferation assay showed Ubenimex(2 mg/ml)inhibited the proliferation of FTC-133 cells.Ubenimex decreased the expressions of bcl-2 in FTC-133 cells,while the expressions of Cleaved PARP and Cleaved Caspase 8 were increased.Nav1.6-siRNA enhanced the inhibitoring effect.TUNEL staining showed that Ubenimex promoted apoptosis of FTC-133 cells,and downregulation of Nav1.6 expression enhanced the drug susceptibility.Conclusions:1.The expression levels of Nav1.6 were increased in the cells and tissues of follicular thyroid carcinoma compared with the normal thyroid cells and tissues.2.Nav1.6 promoted the proliferation,stemness and invasion abilities of follicular thyroid cancer cells through activating FAK signaling pathway.3.Nav1.6 reduced the drug susceptibility of follicular thyroid cancer cells. |
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