| Background:Thyroid cancer is the most common malignant tumor of the endocrine system and its incidence has most rapidly increased in recent decades.Thyroid cancer is a result of a series of gene mutations and molecular abnormalities which activate multiple enzymes and signaling pathways.Meanwhile,molecular detection technology is an important tool in thyroid cancer.Most differentiated thyroid carcinomas have a good prognosis,but there were still a part of refractory differentiated thyroid carcinomas,poorly differentiated and undifferentiated thyroid carcinomas died from local invasion or distant metastasis.Telomerase activation is a basic step in tumorigenesis.More than 85 to 90percent of cancer cells were found to up-regulate telomerase expression,conferring them with the potential to proliferate indefinitely.Telomerase reverse transcriptase?TERT?promoter mutation is one of the most frequent mutations in progressive thyroid cancer in which the anaplastic thyroid cancer?ATC?mutation rate of TERT promoter is as high as46.3%.Studies have shown that thyroid cancer with mutation of TERT promoter was closely associated with extraglandular invasion,vascular invasion and distant metastasis.The prognosis of thyroid cancer patients with mutation of TERT was extremely poor.Both in vivo and in vitro experiments showed that TERT increased cancer metastasis and recurrence by promoting cell epithelial metaplasia?EMT?.The study of drugs targeting TERT mutation is of great significance for the treatment of aggressive thyroid cancer.cAMP/PKA pathway,as one of the classic intracellular signaling pathways,is also known to signal downstream from thyroid stimulating hormone?TSH?and abnormally activated in various cancer and cancer cell lines.cAMP/PKA pathway has been shown,under some conditions,played a key role in the regulation of cytoskeleton rearrangement and cell epithelial–mesenchymal transition,migration,and invasion in cancer cells.The inhibition of PKA using a PKA inhibitor also led to a significant cell growth and migration inhibition in several cancer including cholangiocarcinoma and colonic carcinoma.Currently,there are no studies on the relationship between PKA pathway and TERT expression in thyroid cancer,and the study on the relationship between PKA pathway and TERT expression is expected to provide clues for clinical treatment.Objective:The mutations of TERT promoter region and the polymorphism of rs2853669 site in 12 thyroid cancer cell lines were detected systematically,which provided a reference for choosing appropriate cell model for experimental study in vitro about the function of TERT in thyroid cancer.To investigate the effect and mechanism of PKA inhibitors on TERT expression in human thyroid cancer cell lines,we aim to provide clues for molecular targeted therapy and TSH suppression therapy of thyroid cancer in clinic.Methods:By amplifying BRAF gene and TERT promoter region gene in 12 human thyroid cancer cell lines,the genotypes of BRAF,TERT mutation site and single nucleotide polymorphism site of rs2853669 were detected.The human thyroid cancer cell lines were treated with PKA inhibitor H89.The migration effect of H89 on thyroid cancer cells was detected by wound scratch assay,and the effect of PKA inhibitor on TERT expression of thyroid cancer was detected.According to whether the TERT promoter mutation C228 or C250T and rs2853669 SNP TT/CC were present,6 different combinations of luciferase plasmids were constructed,respectively named as WT+SNP TT,WT+SNP CC,C228T+SNP TT,C228T+SNP CC,C250T+SNP TT and C2250T+SNP CC.Then luciferase reporter assay was analyzed to explore the relationship between regulation of TERT by H89 and TERT promoter genotype.Finally,the protein levels of c-fos,ETS2,GABPA and GABPB were detected by western bloting to explore the mechanism of regulation of TERT expression by H89.Results:?1?Among the 12 common cell lines of thyroid cancer,BRAFV600E mutation was found in BCPAP,SW1736,OCUT1 and MDA-T41.TERT promoter mutations were found in BCPAP,SW1736,OCUT1,TPC1,FTC133 and C643.Among them,C228T mutation was found in BCPAP,SW1736,TPC1,FTC133 and C643,and only C250T mutation existed in OCUT1.For SNP rs2853669,SNP CC was found in FB1,WRO and HTORI3,SNP TT was found in 4 cells including BCPAP,OCUT1,FTC133 and T351,and SNP TC existed in the remaining 5 cells.The incidence of C in these 12 cell lines was45.8%.?2?Wound healing assay showed PKA inhibitor H89 could reduce the migration rate of FTC133,C643,T351 and Cal62 in thyroid cancer cell lines,but has no effect on FB1,HTORI3 cell lines.?3?PKA inhibitor H89 could decrease the TERT expression of thyroid cancer cell lines BCPAP,T351,C643,Cal62,MDA-T41,SW1736,OUCT1,FTC133 and TPC1 but not in FB1,HTORI3 and WRO.?4?Luciferase reporter gene results showed that TERT promoter activity in TERT SNP CC group was lower than that in TERT SNP TT group.?5?Luciferase reporter gene results showed that PKA inhibitor H89 could reduce TERT activity in cell lines harboring TERT promoter mutation or SNP TT,but not in cell lines harboring TERT WT combined with TERT SNP CC.?6?PKA inhibitor H89 could decrease the c-fos,ETS2 and GABPB protein level but not GABPA protein in thyroid cell lines.Conclusion:Our study found for the first time that PKA inhibitors decrease TERT expression in a TERT promoter mutation or TERT SNP site rs2853669 TT/TC dependent way,which is of great significance for precise treatment of thyroid cancer patients with different TERT genotype.The mechanism of PKA inhibitors to decrease TERT expression mainly involves two aspects:H89 could decrease the TERT expression by inhibiting the pCREB pathway and down-regulating c-fos,followed by reduced binding of GABP and TERT promoter mutation sites and decreased TERT expression;on the other hand,H89could decrease the expression of hTERT by reducing the binding of ETS2 to the rs2853669TT/TC site. |
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