| Objective:Thyroid cancer(TC)is the most common endocrine tumor in human,and its incidence is increasing year by year,especially in women,bringing heavy burden to patients’ lives.Therefore,it is of great significance to clarify the etiology and pathogenesis of TC.Ion channel/transporter is a hot topic in tumor research in recent years,and SLC26A9(Solute carrier family 26 Member 9)is one of them.SLC26A9 belongs to SLC26A family,and its main function is to participate in the transport of bicarbonate and chloride ions.Previous studies have confirmed that SLC26A9 can participate in the occurrence and development of gastric cancer,colorectal cancer and breast cancer.However,the functional role of SLC26A9 in thyroid cancer is still unclear.Therefore,the main purpose of this project is to clarify the functional role of SLC26A9 in the occurrence and development of thyroid cancer and preliminarily explore its molecular mechanism,in order to provide new potential targets for the diagnosis and treatment of thyroid cancer.Methods:1.Immunohistochemical staining was used to detect the expression difference of SLC26A9 in thyroid cancer tissues and normal thyroid tissues,and the correlation between SLC26A9 and clinicopathological features was analyzed.2.Western blotting was used to detect SLC26A9 expression difference between different thyroid cancer cell lines and normal thyroid epithelial cells.3.Lentivirus was used to overexpress SLC26A9 in TPC-1 cell line,and TPC-1 overexpressed stable strain was constructed.SLC26A9 was silenced in KTC-1 cell line,and KTC-1 stable strain was constructed.Western blotting and RT-qPCR were used to verify whether the overexpressed/silenced stable strain was successfully constructed.4.CCK8 and Ki67 staining were used to detect the proliferation of thyroid cancer cells after silencing/overexpression of SLC26A9.5.The change of migration and invasion ability of thyroid cancer cells after silencing/overexpressing SLC26A9 was observed by Wound-healing test and Transwell test.6.Apoptosis and cell cycle of thyroid cancer cells after silencing/overexpression of SLC26A9 were detected by flow cytology.7.Western blot was used to detect the differences in the expression of intracellular proliferation,migration,invasion,apoptosis and cell cycle related proteins between the overexpressed/silenced SLC26A9 stable strain and the control group,and to further explore the effect of SLC26A9 on EGFR/MEK/ERK signaling pathway.8.The localization of SLC26A9 and EGFR in cells was detected by immunofluorescence staining,and the combination of SLC26A9 and EGFR was verified by immunoprecipitation technique.9.Xenograft tumor model was constructed in nude mice to observe the effect of SLC26A9 on the occurrence and development of thyroid cancer in vivo.Results:Immunohistochemical staining showed that SLC26A9 expression in thyroid carcinoma tissues was significantly higher than that in normal thyroid tissues.Correlation analysis of clinicopathological features suggested that SLC26A9 expression was correlated with gender of thyroid cancer patients,and the expression of SLC26A9 in female patients was higher than that in male patients.The expression of SLC26A9 in five thyroid cancer cell lines(BCPAP,TPC-1,KTC-1,FTC133,8305C)was significantly higher than that in normal thyroid epithelial cell lines(Nthyori-3-1),and the expression of SLC26A9 in KTC-1 cell line was the highest in papillary carcinoma.TPC-1 cell line SLC26A9 had the lowest expression.Silencing SLC26A9 in KTC-1 cells significantly inhibited the proliferation,migration and invasion of thyroid cancer cells,blocked the cell cycle in G0/G1 phase and promoted apoptosis.Overexpression of SLC26A9 in TPC-1 cells can significantly promote the proliferation,migration and invasion of thyroid cancer cells,promote cell cycle transformation from GO/G1 phase to S phase and G2 phase,and inhibit cell apoptosis.In vivo experiments suggested that silencing SLC26A9 could significantly inhibit the growth of transplanted tumor,while overexpression of SLC26A9 could promote the growth of transplanted tumor.Molecular mechanism studies suggest that SLC26A9 can induce changes in downstream molecules by activating EGFR/RAS/RAF/MEK/ERK signaling pathway,including CyclinD1 and CyclinE2.Apoptosis related proteins Caspase3,Caspase8,Caspase9,Bcl2,Bax are changed.In addition,SLC26A9 can also induce changes in epithelial mesenchymal transformation-related proteins,including E-cadherin,N-cadherin,Vimentin and Snail.Conclusion:SLC26A9 promotes the development of thyroid cancer by activating the EGFR/Ras/Raf/MEK/ERK signaling pathway.SLC26A9 plays an important role in the progression of thyroid cancer. |
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