TET1 is a Tumor Suppressor That Inhibits Papillary Thyroid Carcinoma Cell Migration and Invasion

Background. Ten-eleven translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) promoting demethylation in cells. However, the expression pattern and biologic significance of TET in papillary thyroid carcinoma (PTC) remain unclear. This study aimed to elucidate the biological functions of TET1 and the miRNA and mRNA expression levels in PTC cells with downregulated TET1. Methods. The expression of the TET family in 49 PTC tissues and corresponding tumor-adjacent tissues, as well as PTC cell lines (BCPAP, K1, and TPC-1) and the normal thyroid epithelial cell line (Nthy-ori 3-1), were detected using quantitative real-time polymerase chain reaction. The 5hmC level was detected in PTC tissues and cell lines using immunohistochemistry and dot blot assay, respectively. After silencing the TET1 gene with siRNAs in BCPAP and TPC-1 cells, cell proliferation was detected using EdU assay. Transwell assay was used to investigate cell migration and invasion. miRNA and mRNA expression arrays were conducted in TET1-depleted BCPAP cells. Results. The expression level of TET1 decreased in PTC tissues and cell lines and was consistent with the reduction in the 5hmC level. The knockdown of the TET1 gene promoted cell migration and invasion in BCPAP cells. The expression of miR-7, miR-15/16 cluster, and let-7 family was downregulated, while the expression of let-7e was upregulated after siRNA-TET1 treatment in BCPAP cells. The expression of WNT4, FZD4, CDK6, MCF2L, and EDN1 was upregulated as potential target genes of dysregulated miRNAs. Conclusion. The study showed that TET1 dysfunction inhibited the migration and invasion of BCPAP cells and might have a potential role in the pathogenesis of PTC.

5hmC Level Predicts Biochemical Failure Following Radical Prostatectomy in Prostate Cancer Patients with ERG Negative Tumors

This study aimed to validate whether 5-hydroxymethylcytosine (5hmC) level in combination with ERG expression is a predictive biomarker for biochemical failure (BF) in men undergoing radical prostatectomy (RP) for prostate cancer (PCa). The study included 592 PCa patients from two consecutive Danish RP cohorts. 5hmC level and ERG expression were analyzed using immunohistochemistry in RP specimens. 5hmC was scored as low or high and ERG was scored as negative or positive. Risk of BF was analyzed using stratified cumulative incidences and multiple cause-specific Cox regression using competing risk assessment. Median follow-up was 10 years (95% CI: 9.5–10.2). In total, 246 patients (41.6%) had low and 346 patients (58.4%) had high 5hmC level. No significant association was found between 5hmC level or ERG expression and time to BF (p = 0.2 and p = 1.0, respectively). However, for men with ERG negative tumors, high 5hmC level was associated with increased risk of BF following RP (p = 0.01). In multiple cause-specific Cox regression analyses of ERG negative patients, high 5hmC expression was associated with time to BF (HR: 1.8; 95% CI: 1.2–2.7; p = 0.003). In conclusion, high 5hmC level was correlated with time to BF in men with ERG negative PCa, which is in accordance with previous results.

44 Misregulation of ten-eleven translocation 3 CXXC domain leads to abnormal formation of 5-hydroxymethylcytosine and expression of pluripotency genes in pig embryos

Ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play an important role in regulation of DNA methylation patterns by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET3 protein, a member of TET family, is enriched in mature oocytes and early stage embryos and contributes to DNA demethylation of the paternal genome in zygotes. N-terminal CXXC domain of TET3 is thought to be important in catalysing 5mC oxidation through its DNA binding potential. However, it is not clear whether specific DNA binding of CXXC domain is required for 5hmC conversion in mammalian embryos. Here, we investigated the role of TET3 CXXC domain in controlling 5hmC formation in fertilized pig embryos by injecting TET3 CXXC domain into mature pig oocytes as a dominant negative to inhibit the direct binding of TET3 to the genome through the CXXC domain. The CXXC domain of pig TET3 was identified through bioinformatics comparison of TET3 sequences among different species and cloned from mature pig oocyte-derived cDNA. To construct the green fluorescent protein (GFP)-CXXC fusion protein, CXXC sequence was subcloned into N-terminal GFP fusion vector, and then mRNA was synthesised by in vitro transcription. The GFP-CXXC mRNA (100 ng/µL) was injected into oocytes matured in vitro for 36 to 37h. Then, the oocytes were fertilized at 42h post-maturation. Water-injected oocytes served as a control. At 17h post-fertilization, zygotes were collected to analyse 5hmC level by immunocytochemistry. The level of 5hmC was analysed using ImageJ (https://imagej.nih.gov/ij/). Expression of pluripotency-related genes at Day 7 blastocysts was examined through quantitative RT-PCR; ΔΔCt method was used to analyse the quantitative RT-PCR data and Student’s t-test was used for statistical analysis. All experiments were conducted at least three times and P-values of less than 0.05 were considered significant. The GFP-CXXC was exclusively localised in pronuclei, indicating that the CXXC domain may lead to nuclear localization of TET3. The level of 5hmC in zygotes was not altered by the overexpression of GFP-CXXC. Interestingly, in 2-cell stage embryos, the 5hmC level was reduced in GFP-CXXC injected embryos compared with the control group, suggesting that CXXC domain is important for 5hmC formation post-DNA replication. There was an increase in transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes compared with control blastocysts (P<0.05). There was no difference in the expression of POU5F1 and SOX2. In this study, we found that CXXC domain of TET3 is critical in maintaining the level of 5hmC formation at 2-cell stage and proper level of pluripotency-related genes (NANOG and ESRRB) in blastocysts. Future studies will focus on elucidating mechanisms behind the changes after overexpression of GFP-CXXC in pig embryos.