In addition, TK1 knockdown increased the protein levels of active caspase-3, caspase-9, and E-cadherin, but decreased the protein levels of vimentin and N-cadherin (Figure 4D). Open in a Selamectin separate window Figure 4 Thymidine kinase 1 (TK1) knockdown suppressed tumor growth of thyroid carcinoma cells. Silencing of TK1 suppressed cell proliferation, invasion, migration, and DKFZp781B0869 epithelialCmesenchymal transition, and also induced cell apoptosis in the thyroid carcinoma cell lines. Animal studies showed that TK1 knockdown inhibited tumor growth of thyroid carcinoma cells. Importantly, miR-34a-5p was found to be downregulated in the thyroid carcinoma cells. Furthermore, miR-34a-5p targeted the 3 untranslated region of TK1 and suppressed the expression of TK1 in thyroid carcinoma cell lines. In summary, first, these results exhibited the upregulation of TK1 in thyroid nodules and thyroid carcinoma tissues; second, TK1 promoted thyroid carcinoma cell proliferation, invasion, and migration; lastly, TK1 was negatively regulated by miR-34a-5p. Our study may provide novel insights into the role of TK1 in regulating thyroid carcinoma progression. functional studies showed that TK1 silencing suppressed thyroid malignancy cell proliferation, invasion, migration, epithelialCmesenchymal transition (EMT) and induced cell apoptosis. Furthermore, the upregulation of TK1 in the thyroid malignancy may be related to the downregulation the tumor-suppressive miR-34a-5p. Materials and Methods Clinical Samples The serum samples were collected from 1, 112 subjects who underwent the physical examination at First Affiliated Hospital of Southern University or college of Science and Technology, Second Clinical College of Jinan University or college between 2015 and 2018. Among the subjects, 431 patients were positive for thyroid nodules by ultrasound examination, and 681 patients were unfavorable for thyroid nodules. The protein levels of TK1 in the serum were detected using the enzyme-linked immunosorbent assay (ELISA) assay kit (#ab223595, Abcam, Cambridge, USA). All the experimental protocols were approved by the Ethics Committee of the First Affiliated Hospital of Southern University or college of Science and Technology, and all the patients signed the written informed consent. Cell Lines and Cell Culture The normal human main thyroid follicular epithelial cells (Nthy-ori 3-1, #90011609) and thyroid carcinoma cell collection (TPC-1, #SCC147) were obtained from Merck (Darmstadt, USA). The thyroid carcinoma cell lines (BC-PAP, #ACC273) were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were cultured in RMPI-1640 medium (Sigma-Aldrich, St. Louis, USA) supplemented with 10% fetal bovine serum (FBS; #10100154, Life Technologies, Waltham, USA) and were kept in a humid atmosphere of 5% Selamectin (Tumor Growth Assay A total of 12 male BALB/nude mice (6C8 weeks aged) were obtained from Guangzhou Laboratory Animal Center (Guangzhou, China). All animal experiments were approved by the Animal Ethics Committee of First Affiliated Hospital of Southern University or college of Science and Technology. TPC-1 cells (5 106 cells) with stably expressing TK1 shRNA (sh_TK1) or scrambled unfavorable control shRNA (sh_NC) were subcutaneously injected into the right flank of the nude mice and six animals in each group. After injection of carcinoma cells, the tumor volume of the nude mice was measured every 7 days for 42 days. At Selamectin the end of the experiments, the mice were killed, and the tumor tissues were collected for further analysis. Dual-Luciferase Reporter Assay To construct the reporter vectors, the 3 untranslated region (UTR) of TK1 made up of the putative binding sites of miR-34a-5p was amplified by PCR and cloned into downstream of the luciferase gene of the pGL3 vector (#E1751, Promega, Madison, USA). The mutant reporter Selamectin vectors were generated by Selamectin mutating three nucleotides in the binding region. Thyroid carcinoma cells were cotransfected with reporter vectors and miRNAs using Lipofectamine 2000 reagent (Invitrogen). At 24 h after transfection, luciferase activity in the thyroid carcinoma cells was decided using the Dual-Luciferase Reporter Assay System (#E1910, Promega). Statistical Analysis All data analysis was performed using GraphPad Prism (Version 5.0; GraphPad Software, La Jolla, USA). Summary data are offered as the imply standard deviation. Significant differences between different groups were evaluated using Student’s test or one-way ANOVA followed by Bonferroni’s test. Statistical significance was set at < 0.05. Results TK1 Was Upregulated in Serum From Patients With Thyroid Nodules and Was Upregulated in the Thyroid Carcinoma Tissues We first analyzed the serum TK1 protein levels from the subjects who underwent physical examination in our hospital and found that serum TK1 levels were significantly higher in the subjects with thyroid nodules compared to the normal subjects (Physique 1A). A further analysis using data mining tool (http://gepia.cancer-pku.cn/) showed that TK1 was markedly upregulated in the thyroid carcinoma tissues when compared to normal thyroid tissues (Figure.