Excessive Iodine Intake Promotes Ptc Development Through Down-regulating Tumor Suppressor Gene RSK4

Aim:The relationship between iodine intake and the occurrence of papillary thyroid cancer(PTC)has attracted much attention.However,it is still undefined whether excessive iodine intake is a risk factor for PTC,and the molecular mechanism remains to be elucidated.In this study,based on our previous findings that long-term excessive iodine intake affects the expression of many genes such as RSK4 in PTC cells,we aim to investigate the mechanism of long term effect of high iodine on the development PTC in vitro,in vivo and in clinical cases.Method:We collected 424 fasting urine samples of thyroid nodule patients who were admitted to the Affiliated Hospital of Qingdao University from June 2016 to June 2018,including 359 with PTC and 65 with benign tumor.Iodine in the urinary was determined by mass-spectrometry,and clinical data of the patients was recorded in detail.The benign and malignant thyroid nodules and para-nodular tissues of the patients who underwent thyroidectomy in our hospital were collected,including 284 cases of PTC and 65 cases of benign nodules.Total RNAs were extracted from the tissues.The expression of RSK4 was detected by RT-qPCR and western blot,and the relationship of RSK4 expression and clinicopathology was analyzed.Meanwhile,based on human blood iodine concentration,thyroid papillary cancer cell lines BCPAP and TPC-1 were cultured with 80 ?M potassium iodide(KI)solution for 4 months to simulate the long-term excessive iodine intake,and the expression of RSK4 was detected.BCPAP and TPC-1 cells were infected with lentivirus RSK4 or lentivirus RSK4 shRNA respectively.Cell proliferation,migration and invasion were analyzed using CCK8,transwell and gel invasion assay respectively.Tumor xenografts were established by subcutaneous injection of cancer cells into nude mice.The effect of RSK4 overexpression on p21 expression was detected by RT-qPCR.Western blot was used to detect the effect of iodine at a high concentration on MAPK activity in thyroid cancer cells,as well as the effect of RNA interference RSK4 or overexpression on MAPK activity.SPSS 23.0 and Graphpad Prism 7 were used for statistical analysis and mapping.Results:Urinary iodine concentration of PTC patients with tumor size of>1 cm(n=134)was significantly higher than those with tumor size of ?1 cm(n=225).In addition,the diameter of PTC tumors in patients with urine iodine level ?300 ?g/L was significantly larger than that in patients with urine iodine concentration between 100-299 ?g/L and with urine iodine concentration < 100 ?g/L,indicating that excessive iodine intake is closely related to PTC tumor growth.RSK4 was significantly down-regulated after long-term exposure to 80?M KI in TPC-1 and BCPAP cells.The proliferation,migration and invasion activity of TPC-1 and BCPAP was significantly increased after RSK4 was knocked down.On the contrary,when RSK4 was overexpressed,cell proliferation,migration and invasion was inhibited.The xenografts model confirmed the role of RSK4 in tumor growth.The mRNA level of RSK4 in PTC was remarkably lower than in adjacent tissues and in benign thyroid nodules.RSK4 expression was lower in tumor with diameter>1 cm(n=108)than in those with diameter?1 cm(n=176),and was lower in tumor with capsule invasion than in those without capsule invasion.Conclusion:Our results suggest that excessive iodine intake promotes PTC tumor development.Long-term excessive iodine intake may affect MAPK pathway activity and cell senescence by down-regulating the expression of RSK4 to promote the development of PTC.