Research Communication mir-4299 Mediates the Invasive Properties and Tumorigenicity of Human Follicular Thyroid Carcinoma by Targeting ST6GALNAC4

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1 Research Communication mir-4299 Mediates the Invasive Properties and Tumorigenicity of Human Follicular Thyroid Carcinoma by Targeting ST6GALNAC4 Xiaolong Miao 1 Li Jia 2 Huimin Zhou 3 Xiaobo Song 4 Ming Zhou 1 Jinchao Xu 1 Lifen Zhao 2 Xiaobin Feng 2 Yongfu Zhao 1 * 1 Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, Liaoning Province, China 2 College of Laboratory Medicine, Dalian Medical University, Dalian, Liaoning Province, China 3 Department of Microbiology, Dalian Medical University, Dalian, Liaoning Province, China 4 Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway Abstract Altered sialylation is closely associated with tumor progression and invasiveness. Micro-RNAs endogenous regulators of gene expression have been implicated in human thyroid carcinoma invasiveness. The objective of this study is to examine the alterations of mir-4299 and ST6GALNAC family in human follicular thyroid carcinoma during metastatic process. qrt- PCR showed the differential expressional profiles of mir-4299 and ST6GALNAC family in three kinds of thyroid cell lines (FTC-133,FTC-238, Nthy-ori 3-1) and clinical tissue specimens(malignant and borderline). The altered expression levels of ST6GALNAC4 were corresponding to invasive phenotypes of FTC-133 and FTC-238 cells both in vitro and in vivo. Further date indicated that mir-4299 regulated tumor progression and invasiveness by directly targeting ST6GALNAC4. This study implies the potential therapeutic application of mir-4299 and ST6GALNAC4 in modulating the invasion and tumorigenicity of follicular thyroid carcinoma cell. VC 2015 IUBMB Life, 68(2): , 2016 Keywords: ST6GALNAC family; invasive properties; tumorigenicity; human follicular thyroid carcinoma cell line; mir-4299 Introduction Thyroid cancers are the most common malignant tumors of the endocrine system, which led to 1 to 1.5% of cancer-related deaths. Although most thyroid cancers are easily curable with surgery, there are no curative treatments for metastatic thyroid cancers. Specific changes in the glycosylation patterns of cell surface glycoproteins play a physiological role in regulating metastatic efficiency of tumor cells (1). Sialyltransferases have been recognized to be involved in various diseases by catalyzing the biosynthesis of different glycoconjugates and Abbreviations: FTC, follicular thyroid carcinoma; ST, sialyltransferase; PCR, polymerase chain reaction; PBST, PBS containing 0.1% Tween 20 phosphate-buffered saline VC 2015 International Union of Biochemistry and Molecular Biology Volume 68, Number 2, February 2016, Pages *Address correspondence to: Yongfu Zhao, Department of General Surgery, The Second Affiliated Hospital of Dalian Medical University, 465 Zhongshan Road, Dalian , Liaoning Province, China. Tel: Fax: zyf0386@sina.com Received 6 July 2015; Accepted 7 December 2015 DOI /iub.1467 Published online 30 December 2015 in Wiley Online Library (wileyonlinelibrary.com) 136 IUBMB Life

2 saccharide structures (2). It is well known that the alteration of cell surface sialylated antigens may affects many cellular biological properties: cell-cell adhesion, cell-extracellular matrix adhesion, immune evasion, cell metastasis, and invasion abilities etc. (3 6). In recent years, much research has been devoted to exploring the relationship between glycan alterations and invasive properties of malignant cells. Changes in oligosaccharides structures are reported in association with tumor invasion in thyroid cancer (7). Therefore, it has attracted great interest in studying metastasis suppression of thyroid cancer through regulation of glycosyltransferases and corresponding glycogenes. Sialyltransferases (ST) are a group of enzymes, which catalyze the transfer of sialic acid residues to terminal positions of glycoprotein and glycolipid carbohydrate groups by using CMP-Neu5Ac as an activated sugar donor. Human ST have been characterized and classified according to the specific sialic acid linkage [alpha-2, 3-sialyltransferases, alpha-2, 6- sialyltransferases, alpha-2, 8-sialyltransferases (8)]. Alpha 2, 6-sialyltransferases mediate the transfer of sialic acid with an alpha 2,6-linkage to it with terminal Gal (ST6Gal I-II) (9) or GAlNAc residues (ST6GalNAc I-VI). Changes in expression of specific ST6GALNAC family have been reported in several tumors. For example, high-level expression of ST6GalNAcI was associated with the tumourigenicity of MDA-MB-231 breast cancer cells (10). ST6GalNAcIV promoted lung cancer metastasis through adhesion to galectins (11). ST6GalNAc V has recently been found to mediate brain metastasis of breast cancer cells (12). MicroRNAs (mirnas) are a class of noncoding RNAs with highly conserved noncoding RNA sequences and functions as regulators of gene expression by targeting mrnas (13). A number of studies have demonstrated the regulation role of mirnas in physiologic and pathologic conditions, such as mir- NAs participate in the regulation of cell differentiation (14,15), cancer prognosis (16), tumorigenesis (17). Down-regulation of mir-181b expression led to cell growth inhibition and cell apoptosis by targeting CYLD in human thyroid papillary cancer (18). mir-101 inhibited thyroid cancer cell migration and invasion by targeting the Rac1 gene (19).However, the biological role of mir-4299 in regulating the invasive properties by targeting ST3GAL4 has been unrevealed. The aims of this study are to determine the expression level of mir-4299 and ST6GALNAC family in three kinds of thyroid cell lines (FTC-133, FTC-238, Nthy-ori 3-1) and clinical tissue specimens isolated from the diagnosed thyroid cancer patients, to identify its function in thyroid cancer cells in vitro and in vivo, and to explore the relationship between mir-4299 and ST6GALNAC4. FTC-133 cell lines and FTC-238 cell lines are two follicular thyroid cancer cell lines derived from the same patient. FTC-133 was established in culture from a lymph node and FTC-238 from a lung metastasis (20). FTC- 238 was shown to be more invasive than FTC-133 (21,22). Nthy-ori 3-1 cell lines was human thyroid follicular epithelial cells (23). Materials and Methods Cell Culture Human thyroid cell lines FTC-133, FTC-238, Nthy-ori 3-1, were purchased from GuangZhou Jennio Biotech Co (China). The three cell lines were cultured in 90% DMEM (Gibco) supplemented with antibiotics (13 penicillin/streptomycin 100 U/mL, Gibco) and 10% heat-inactivated fetal bovine serum (Gibco). Cells were incubated at 37 8C in a humidified atmosphere containing 5% CO 2. Tissue Samples After obtaining informed consent, thyroid cancer and transitional tissues (3 cm from the tumor edge) were collected from 101 patients who underwent surgical resections at the Second Affiliated Hospital of Dalian Medical University from July 2012 to May No patients had received chemotherapy or radiation therapy. The investigation project and the informed consent have been approved by the Ethics Committee of the Second Affiliated Hospital of Dalian Medical University. The extracted specimens were confirmed to be thyroid cancer tissue with pathological diagnosis according to the International Union against Cancer (UICC). These tissues were snap-frozen in liquid nitrogen and stored at 280 8C until used. Medical records were used to extracted clinicopathological data. RNA Isolation and Real-Time RT-PCR The total RNA was extracted from four thyroid carcinoma cell lines by using the RNeasy Mini Kit (QIAGEN, Valencia, CA). The cdna was synthesized by using QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA) according to the manufacturer s instruction. The expression level of mir-4299 was determined by using mirvana TM qrt-pcr microrna Detection Kit according to the manufacturer s protocol (Ambion Inc., Austin, TX) and normalized by using the 2 2DDCT method relative to U6-small nuclear RNA. ST6GALNAC mrnas were quantified by SYBR-Greenquantitative real-time PCR (Takara, Otsu, Shiga, Japan) and normalized to GAPDH. The sequences of the upstream and downstream primers were as follows: 5 0 -CTGGTCTTCTTTCTCTTCG-3 0 and 5 0 -GTTGAGGGCATTGTTCTCT-3 0 for ST6GalNAC1; CTTTGCCCTGTACTTCTCG-3 0 and 5 0 -CAGCACTGGAATGGA GAGA-3 0 for ST6GalNAc2; 5 0 -GGACAACCTGGTACAAAGT-3 0 and 5 0 -TATCTCA TTTCCCACCTTC-3 0 for ST6GalNAc3; ACCTGCCTGGACCACCACT-3 0 and 5 0 -TCGGCACTGTCGATCTCAG- 3 0 for ST6GalNAc4; 5 0 -TGGACGGATACCTCG GAGT-3 0 and GTCTGGTCAATCTGGGAGC-3 0 for ST6GalNAc5; 5 0 -ACCTACC CCTCAGCAGACG-3 0 and 5 0 -CTTGAGGTTGACAGGTCGG-3 0 for ST6GalNAc6; 5 0 -CTCCTCCACCTTTGACGCTG-3 0 and TCCTCTTGTGCTCTTGCTGG-3 0 for GAPDH. The expression level of target genes was determined relatively to GAPDH and calculated as 2 2(CtTarget gene2ctgapdh). All PCRs were done in triplicates. Western Blot Analysis Whole cell protein was electrophoresed under reducing conditions in 10% polyacrylamide gels. The separated proteins were MIAO ET AL. 137

3 IUBMB LIFE transferred to a polyvinylidene difluoride membrane. After blocking with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBST), the membrane was incubated with antibody (1/1,000, diluted; Abcam, Cambridge, UK) overnight at 4 8C and then with peroxidase-conjugated anti-rabbit IgG (1/10,000 diluted; GE Healthcare UK Ltd., Little Chalf-ont, UK). A GAPDH antibody (1/200 diluted; Santa Cruz Biotech) was used as an internal marker for control. All bands intensities were evaluated with ECL Western blot kit (Amersham Biosciences, Buckinghamshire, UK) and were analyzed with LabWorks (TM ver4.6, UVP, BioImaging systems). Deregulation of ST6GALNAC4 by RNAi FTC-238 cells were incubated in appropriate antibiotic-free medium with 10% fetal bovine serum, and were transferred to a six-well tissue culture plate incubating in a 37 8C CO 2 incubator to obtain 60 to 80% confluens. The cell cultures were transfected with ST6GALNAC4 specific shrna, and scrambled shrna used as the negative control. ST6GALNAC4 shrna was mixed with Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA). The transfected cells were cultured and incubated at 37 8C for6h,followedby incubation with complete medium for additional 24 h. Then the cells were harvested for further study. The cell transfection efficiency was 78% by fluorescent microscope investigation and the cell viability was 87% by trypan blue dye exclusion assay. Overexpression of ST6GALNAC4 The human ST6GALNAC4 coding sequences obtained from TaKaRa company (Dalian, China) were inserted into the pegfp-n2 vector (Invitrogen, Carlsbad, CA) at the sites of EcoRI and XhoI. FTC-133 cells were transfected with 5 lg of the target gene expression vector or empty vector (EV) in 100- mm dishes with PolyFect Transfection Reagent (QIAGEN, valencia, CA). After 4 weeks of screening, the cell lines stably expressing ST6GALNAC4 (FTC-133/ST6GALNAC4), and empty vector (FTC-133/mock) were established. The cell transfection efficiency was 78% and the survival rate was 86%. In Vitro ECM Invasion Assay The cell invasion in vitro was investigated by using 24-well transwell units (Corning, NY) with 8 lm pore size polycarbonate filter coated with ECMatrix gel (Chemicon) to form a continuous thin layer. FTC-238 and FTC-133 cells ( ) were harvested in serum-free medium containing 0.1% BSA and added to the upper chamber. The lower chamber contained 500 ll DMEM. The cells were incubated for 24 h at 37 8C, 5% CO 2 incubator. At the end of incubation, the cells on the upper surface of the filter were completely removed by wiping with a cotton swab. Then the filters were fixed in methanol and were stained with Wright-Giemsa. The cells that had invaded the Matrigel and reached the lower surface of the filter were counted under a light microscope at a magnification of Transfection and Luciferase Activity Assay Mimics, antagomir and negative control oligonucleotides for hsa-mir-4299 were obtained from RiboBio Co. Ltd (GenePharma, Shanghai, China). Plasmids containing wild-type FIG 1 Differential expression of ST6GALNAC gene family in three kinds of thyroid cell lines and tissue samples. A. The mrna level of ST6GALNAC gene family was analyzed in three kinds of thyroid cell lines (FTC-133,FTC- 238, Nthy-ori 3-1) by real-time PCR. Increase of ST6GALNAC2, ST6GALNAC4, ST6GALNAC5 were observed in three thyroid carcinoma cells compared with FTC-133 cells and Nthy-ori 3-1 cells (*P < 0.05). Only slight difference was observed in the expression level of ST6GALNAC6. No statistically significant differences were found in the expression of ST6GAL- NAC1, ST6GALNAC3.ST6GALNAC4 showed a remarkable increase expressional levels (*P < 0.05). The relative amount of gene mrna level was normalized to the GAPDH level. Data are the means 6 SD of triplicate determinants. B. The mrna level of ST6GALNAC gene family was analyzed in tissue samples. A significant difference was observed in the level of ST6GALNAC4 mrna. (*P < 0.05). No statistically significant differences were found in the rest enzymes of ST6GALNAC family. The relative amount of gene mrna level was normalized to the GAPDH level. Data are the means 6 SD of triplicate determinants. Luc-ST6GALNAC4, mutant Luc-ST6GALNAC4, lenti-mir-4299 were synthesized. Target FTC-238 and FTC-133 cells were transfected with the oligonucleotides using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer s instructions. Luciferase activities were measured 48 h after transfection by using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. In Vivo Tumorigenicity Assay Athymic nude mice (5 weeks old) were obtained from Animal Facility of Dalian Medical University, and were fed with sterilized food and water. Approximately cells were 138 mir-4299 MEDIATES

4 FIG 2 Suppression of ST6GALNAC4 gene inhibits the invasive ability of FTC-238 cells both in vitro and in vivo. A. Suppression of ST6GALNAC4 in FTC-238 cells was analyzed by RNAi approach. B. After shrna transfection, distinct reduction of ST6GAL- NAC4 was observed at protein levels by real-time PCR and Western blot analysis. C. The average number of cells that invaded through the filter was assessed by In vitro ECMatrix gel analysis. FTC-238-ST6GALNAC4 shrna cells were significantly less invasive (*P < 0.05) than the FTC-238 cells and FTC-238-control shrna cells. D. A decrease of mean tumor weight in mice group with FTC-238-ST6GALNAC4 shrna tumors was observed, as compared to the control group (*P < 0.05). Data are the means 6 SD of triplicate determinants (*P < 0.05). injected subcutaneously into the right flank of each nude mouse, respectively. Once bearing palpable tumors (about 4 weeks after tumor cell inoculation), mice were sacrificed and their tumors were isolated, weighed, and photographed. Statistical Analysis The data were presented as mean 6 standard deviation (SD) from the triple tests of each group. SPSS17.0 software was used for statistical analysis and Student s t-test was selected to determine the significance of differences among the examined groups. P < 0.05 was considered to be statistically significant. Results Differential Expression of ST6GALNAC Gene Family in Three Kinds of Thyroid Cell Lines and Tissue Samples Real-time PCR analysis showed that the expression levels of ST6GALNAC gene family were differed among the three types of thyroid cell lines and tissue samples. As shown in Fig. 1, increased expression levels of ST6GALNAC2, ST6GALNAC4, ST6GALNAC5 were observed in FTC-238 cells in comparison with FTC-133 cells and Nthyor3-1 cells. Remarkably, ST6GAL- NAC4 showed a rather high expression levels (*P < 0.05). Only slight differences were observed in the expression levels of ST6GALNAC6.No statistically significant differences were found in the expression of ST6GALNAC1, ST6GALNAC3. Furthermore, as shown in Fig. 1, the expression level of ST6GAL- NAC4 was significantly increased in FTC tissues compared to it in the transitional tissues. Altered Expression of ST6GALNAC4 Gene Influences the Invasive Ability of FTC-238 Cells Both In Vitro and In Vivo Owing to the significant increase of ST6GALNAC4 mrna expression in FTC-238 cells, we silenced ST6GALNAC4 with ST6GALNAC4 shrna, to verify its direct effect on the invasive ability of FTC-238 cells. As shown in Figs. 2A and 2B, the expression level of ST6GALNAC4 was significantly reduced in MIAO ET AL. 139

5 IUBMB LIFE FIG 3 Upregulation of ST6GALNAC4 gene enhances the invasive ability of FTC-133 cells both in vitro and in vivo. A and B. After fulllength sequences transfection, ST6GALNAC4 mrna and protein were increased notably in FTC-133 cells by real time PCR and Western blot. C. The invasivity of cells was analyzed. FTC-133/ST6GALNAC4 cells were significantly more invasive (*P < 0.05) than the FTC-133 and FTC-133/mock cells. D. Tumor weight were increased obviously in nude mice bearing FTC-133/ST6GAL- NAC4 compared to the control group (*P < 0.05). Data are the means 6 SD of triplicate determinants (*P < 0.05). FTC-238-shRNA transfectants compared to that in the control transfectants (*P < 0.05). Transwell assay demonstrated that knockdown of ST6GALNAC4 gene significantly decreased invasive activity in FTC-238-ST6GALNAC4 shrna cells, suggesting that cell invasion capability was inhibited by treated with ST6GALNAC4 shrna (Fig. 2C). In xenograft tumor model, nude mice bearing FTC-238, FTC-238-control shrna, FTC-238-ST6GALNAC4 shrna were used to analyze the differences of tumor weight. Figure 2D showed that a significant reduction of tumor weight of FTC-238- ST6GALNAC4 shrna tumor compared to the control shrna group. The above results indicate that downregulation of ST6GALNAC4 gene suppress the invasive ability of FTC-238 cells. Altered Expression of ST6GALNAC4 Gene Effects on the Invasive Ability of FTC-238 Cells Both In Vitro and In Vivo After verifying that silence of ST6GALNAC4 gene suppress tumor cell invasive ability, we transfected FTC-133 cells with ST6GALNAC4 expression vector to determine the effect of overexpression of the gene on invasive ability of FTC-133 cells. The higher expression levels of mrna and protein of ST6GAL- NAC4 were detected in FTC-133 transfectants. Transwell invasion assay revealed that the invasion capability of FTC-133 cells transfected with ST6GALNAC4 expression vector was obviously increased in comparison with that of the FTC-133/mock cells (Fig. 3C). Nude mice were inoculated with tumor cells FTC-133, FTC- 133/mock, FTC-133/ST6GALNAC4.Tumor weight were increased obviously in the nude mice bearing FTC-133/ST6GALNAC4, as compared to it in the FTC-133/mock group (Fig. 3D). Therefore, the upregulation of the ST6GALNAC4 gene is shown to raise the invasion ability of follicular thyroid carcinoma cells. mir-4299 as a Negative Regulator of ST6GALNAC4 qrt PCR analysis, a quantitative reverse transcription PCR, was utilized to evaluate the change in mir-4299 expression in follicular thyroid cell lines and thyroid cancer and transitional tissues. As shown in Fig. 4A, the expression of mir-4299 was dramatically downregulated in FTC-238 cell lines. The expression of mir-4299 in thyroid cancer was also lower than that in transitional tissues group (Fig. 4A). 140 mir-4299 MEDIATES

6 FIG 4 mir-4299 Modulates the Invasive Ability of Human Thyroid Carcinoma Cells by Targeting ST6GALNAC4 In Vitro and In Vivo In order to investigate whether mir-4299 regulated the invasive ability of human thyroid carcinoma cells in vitro, FTC-238 cells were transfected with mir-4299 agomir, whereas FTCmiR-4299 as a negative regulator of ST6GALNAC4. A. The expression of mir-4299 was examined by qrt PCR in FTC-238 and FTC-133 cells and tissue samples (*P < 0.05). B. The expression of mir-4299 was studied by qrt PCR in cells transfected with the mimics or antagomir (*P < 0.05). C. ST6GALNAC4 were detected by qrt-pcr and Western blot in FTC-238 cells treated with mimics (*P < 0.05). D. The mrnas and proteins of ST6GALNAC4 were analyzed in FTC-133 cells treated with Antagomir (*P < 0.05). E. The nucleotides sequence of the target site of mir-4299 in ST6GALNAC4 3 -UTR (upper panel); Luciferase assay for the direct targeting of 3 -UTR of ST6GALNAC4 by mir-4299 (lower panel). The wide-type and mutant mir-4299 target sequences of ST6GALNAC4 were fused with luciferase reporter and transfected into FTC-238 cells, stably transfected with lentimir-4299 and its vector control. Each bar represents the relative luciferase activity (*P < 0.05). Using the bioinformatics algorithms TargetScan ( targetscan.org) and miranda ( we selected ST6GALNAC4 as potential mir-4299 target genes. Besides, luciferase activity assays were performed in order to test whether ST6GALNAC4 was direct targets of mir Transfection of FTC-238 cells with mir-4299 mimics (100 nm), we measured the changes in ST6GALNAC4 expression levels. The increased expression of ST6GALNAC4 was detected at both the mrna and protein levels after the transfection of mir-4299 mimics (Figs. 4B and 4C). Furthermore, the transfection of mir-4299 Antagomir in FTC-133 cells resulted in a low expression level of ST6GALNAC4 (Figs. 4B and 4C). To determine whether mir-4299 downregulate the expression of ST6GALNAC4,we then examined the target sequence of ST6GALNAC UTR (wt 3 0 -UTR), ST6GALNAC4 mutant sequence (mt 3 0 -UTR) into a luciferase reporter vector. Transfected FTC-238 cells with wt or mt 3 0 -UTR vector and mir-4299 mimics or mir-control. In FTC-238 cells, as shown in Fig. 4E, the effect of invasion suppression was reversed by the mutation of the target sequences in the UTRs of ST6GALNAC4. Taken together, our results demonstrate that mir-4299 targeting ST6GALNAC4 in follicular thyroid cells. MIAO ET AL. 141

7 IUBMB LIFE FIG 5 mir-4299 modulates the invasive ability of human thyroid carcinoma cells by targeting ST6GALNAC4 in vitro and in vivo. A. Knockdown of mir-4299 increased the invasive ability of FTC-238 cells in transwell invasion assay (*P < 0.05). B. Overexpression of mir-4299 decreased the invasive ability of FTC-133 cells in transwell invasion assay (*P < 0.05). C. The mean tumour weight were measured at the indicated time points (*P < 0.05). D. The expression of mir-4299 was detected by qrt-pcr in tumors (*P < 0.05). E. The expression levels of ST6GALNAC4 was analysed by qrt-pcr and western blotting (*P < 0.05). 133 cells were transfected with mir-4299 antagomir. Transwell invasion assay revealed that the invasion capability was obviously decreased of FTC-238 cells transfected with mir agomir (Fig. 5A). Meanwhile, invasion capability was observed increased after mir-4299 being inhibited by the antagomir in FTC-133 cells (Fig. 5B). Nude mice were inoculated with tumor cells FTC-238 that had been transfected with mir-4299 agomir or negative control (NC), to verify whether mir-4299 modulated the invasive ability of human thyroid carcinoma cells in vivo. The weight of the mir-4299 agomir transfected tumors were markedly increased (Fig. 5C). Meanwhile, the transfection of mir-4299 antagomir in FTC-133 cells led to the opposite consequence in tumor weight (Fig. 4D). The RNA and protein were extracted from the tumor tissues, which were isolated from the mice. Consistent with the findings in vitro, the qrt-pcr and western blotting showed that the mrna and protein levels of ST6GALNAC4 was decreased while the expression of mir-4299 was increased (Figs. 5E and 5G) and vice versa (Figs. 5F and 5H). 142 mir-4299 MEDIATES

8 Discussion Aberrant sialylation is observed in various types of cancer with invasive potential (24,25). The biosynthetic pathway of sialylated glycans highlights the importance of sialyltransferases in cancer properties. Altered mrna expression of STs has been detected in human breast cancer, cervical cancer, ovarian cancer, hepatocellular carcinoma (26 28). Using real-time PCR analysis, we found that the expression profiles of ST6GALNAC gene family were modified in three thyroid cell lines FTC-238, FTC-133, Nthyor3-1. Comparing to FTC-133 and Nthyor3-1 cells, FTC-238 cells showed overexpression levels of ST6GAL- NAC2, ST6GALNAC4, ST6GALNAC5 genes (Fig. 1A), and ST6GALNAC4 showed the highest level of expression. Furthermore, FTC-133 and Nthyor3-1 cells overexpressed of ST6GAL- NAC6 (Fig. 1A). With altered mrna expressions in carcinoma tissues, STs can be regarded as prognostic factors and potential targets for therapeutic approaches (29). In this study, real-time PCR analysis was used to probe the differential expression of ST6GALNAC family in FTC tissues and transitional tissues. As depicted in Fig. 1B, FTC tissues had a higher expression level of ST6GALNAC4 than it in the transitional tissues, whereas ST6GALNAC6 expression was at a high level in transitional tissues. The results from the clinical samples confirm that the altered level of ST6GALNAC4 is probably associated with invasion and metastasis in follicular thyroid cancer. Whether the ST6GALNAC gene family and its related proteins affect the tumor invasion is an interesting question. We targeted ST6GALNAC4 gene that was significantly differentially expressed in FTC-238 and FTC-133 cells. The altered level of ST6GALNAC4 was responsible for changed invasiveness, proliferative ability of the two cell lines both in vitro and in vivo (Figs. 2 and 3). The results from above give a hint of utilization of ST6GALNAC4 gene as a useful biomarker for clinical diagnosis and a functional regulator of tumor metastasis in follicular thyroid cancer. A few mirnas have been demonstrated to enhance the invasiveness of cancer. mir-20a/106a plays a vital role in GSC invasion and may serve as a targets for treatment of glioblastoma (30). mir-21 regulates cell proliferation and invasion in esophageal squamous cell carcinoma by targeting PDCD4 (31). Recently, changed expression of mirnas has been linked to epigenetic mechanisms (32,33). In this study, mir-4299 showed significantly higher expressional levels in FTC-133 cells and transitional tissues than those in FTC-238 cells and FTC tissues (Fig. 4A). Concurrent with the decreased expression of mir-4299 in FTC-133 cells, ST6GALNAC4 showed upregulating expression. It also showed down-regulating expression with the increased expression of mir-4299 in FTC-238 cells (Figs. 4B 4D). Furthermore, mir-4299 directly targeted sequences within the 3 0 -UTR of ST6GALNAC4 (Fig. 4E). Based on these data, we conclude that ST6GALNAC4 is a target of mir In order to validate this therapeutic concept, alternative approaches that change the expression of mirna were applied. The altered level of mir-4299 led to significantly changed ST6GALNAC4-mediated invasiveness, proliferative ability of the two cell lines both in vitro and in vivo (Fig. 5). These results further demonstrate that mir-4299 mediates the invasive properties of human follicular thyroid carcinoma by targeting ST6GALNAC4. In general, our work reveals differential expression of ST6GALNAC4 gene in two human follicular thyroid carcinoma cell lines and follicular thyroid cancer specimens. In this study, mir-4299 is observed to regulates the invasive properties of human follicular thyroid carcinoma via targeting ST6GAL- NAC4. Modification of mir-4299 primarily mediates invasion phenotypes. 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