| Objective:Serum midkine（MK）high expresses in various malignant tumors and plays an important biological role in the occurrence and development of tumors.There are few studies on MK in thyroid cancer.This article aims to investigate the predictive value of MK on the metastasis of papillary thyroid cancer;inhibit the expression of MK in anaplastic thyroid cancer cells（ARO）and study its effect on apoptosis and proliferation and related mechanisms.Methods:（1）A retrospective analysis of 241 postoperative papillary thyroid carcinoma（PTC）patients who underwent 131I ablation treatment in the Department of Nuclear Medicine,General Hospital of Tianjin Medical University from January 2011to January 2015 were followed up for at least 2 years.The serum MK concentration was determined by enzyme-linked immunosorbent assay before and after the first 131I ablation treatment in PTC patients.According to the comparison between serum MK and thyroid stimulating hormone（TSH）stimulated thyroglobulin（Tg）concentrations at the time of 10-12 months after the first 131I ablation treatment,the PTC patients were divided into 3 groups when compared with the first 131I ablation treatment.The predictive value of serum MK and Tg on the risk of metastasis in PTC patients was statistically analyzed.（2）Western blotting was used to detect the expression of MK in thyroid cancer and paracancerous tissues and thyroid cancer cell lines（FRO,ARO,WRO,KTC,and TPC）。Five MK-specific siRNAs were used to transfect the anaplastic thyroid cancer（ATC）cells（ARO cells）by liposamal transfection and down-regulation of MK expression in the cells.To detect expression changes of phosphorylation of ERK1/2 and Akt,two key proteins of MAPK and PI3K pathways,and downstream apoptosis-related proteins caspase-3 and survivin;ARO cells transfected with different concentrations of MK-siRNA-3,and then the effect of MK down-regulation on cell proliferation was observed by cell MTT assay.Results:（1）The 241 patients had undergone thyroidectomy or subtotal thyroidectomy and/or cervical lymph node dissection at least 1 month before the first 131I ablation treatment.Postoperative pathological diagnosis confirmed PTC and the diameter of cancer stove and lymph node metastasis were clearly defined.When Tg decreased during TSH stimulation at 10-12 months after the first 131I ablation treatment(compared with TSH stimulation Tg at the first 131I ablation treatment)but not at the ideal Tg level（Tg below 1.0 ng/ml under TSH stimulation）,the risk of metastasis in PTC patients was 12.5-fold higher than the risk of metastasis in PTC patients with ideal Tg level（P<0.01）.When Tg increased(compared with Tg under TSH stimulated state in the first 131I ablation treatment),the risk of metastasis in PTC patients was 19.5 times than that of ideal Tg（P<0.01）.Serum MK levels decreased at 10-12 months after the first 131I ablation treatment(compared to serum MK before the first 131I ablation treatment),but did not reach the generally accepted normal level（255.01±126.78 pg/ml）,the risk of metastasis in PTC patients was 3.0 times（P<0.01）than that reduced to a generally accepted normal level（255.01±126.78 pg/ml）.When serum MK levels increased(compared to serum MK before the first 131I ablation treatment),the risk of metastasis was 5.0 times（P<0.01）than that reached the generally accepted normal level（255.01±126.78 pg/ml）.In the third group of patients,disease-free survival was significantly lower than in any other two groups.（2）Western blotting results showed that MK was expressed in thyroid cancer tissues and paracancer tissues,and the expression of MK in thyroid cancer tissues was significantly higher than that in paracancer tissues（5.51 times）;In thyroid cancer 5cell lines（FRO,ARO,In WRO,KTC,and TPC）,MK was highly expressed in thyroid cancer cell lines ARO,WRO,and KTC,and the expression of MK in ARO was higher than that in WRO and KTC,while no obvious MK expression was seen in the cell lines FRO and TPC.Five specific siRNAs targeting MK（at a working concentration of 50 nM）transfected for 48 hours were able to effectively inhibit the expression of MK in ARO cells;siRNA were transfected into ARO cells for 72 hours.The siRNA-1,siRNA-2,siRNA-3 and siRNA-4 test group can inhibit the phosphorylation of Akt in the PI3K pathway and down-regulate the levels of the downstream anti-apoptotic protein Survivin and up-regulate the pro-apoptotic protein Caspase-3（p<0.05）.In the MAPK pathway,only siRNA-4 test group inhibited Erk1/2 phosphorylation levels（p<0.05）.After 24 hours of siRNA-3 transfected ARO cells,cell survival experiments showed that when the concentration of MK-siRNA-3was higher than 500 nM,the proliferation of thyroid cancer cells was inhibited.Conclusion:Monitoring the dynamic changes of serum MK has potential clinical value in predicting the risk of metastasis in patients with DTC.MK is highly expressed in thyroid cancer.By inhibiting the expression of MK,the phosphorylation of MAPK and PI3K pathway and apoptosis-related protein levels can be affectted.Downward adjustment of MK can inhibit cell proliferation. |
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