Fig 2: METTL3 reduces TACSTD2 mRNA stability via an m6A-YTHDC2-dependent signaling. A: Prediction of m6A sites of TACSTD2 by SRAMP database. B: Analyzing m6A modification sites in the TACSTD2 coding sequence region through database. C: METTL3 expression level of THCA in GEPIA2 databases. D: RT-qPCR was used to evaluate the mRNA level adjusted to GAPDH of methyltransferases and demethylases in THCA and paired tissues. The expression levels of TACSTD2 in METTL3-overexpressing and METTL3 knockdown PTC cells were detected by WB (E) and qRT-PCR (F). G: MeRIP-qPCR analysis was employed to demonstrate METTL3-mediated TACSTD2 m6A modifications. The m6A modification of TACSTD2 was increased on upregulation of METTL3, while it was depleted on METTL3 silencing. H-I: TACSTD2 mRNA level in METTL3-overexpressing PTC cells treated with actinomycin D at the indicated time points were detected by qRT-PCR. METTL3 overexpression reduced the half-life of TACSTD2 transcript. J: YTHDC2 expression level of THCA in GEPIA2 databases. K: RT-qPCR was used to evaluate the mRNA level adjusted to GAPDH of N6-methyladenosine readers in THCA and paired tissues. L: The expression levels of TACSTD2 in YTHDC2-overexpressing PTC cells were detected by qRT-PCR. M: The expression levels of TACSTD2 in YTHDC2-overexpressing and YTHDC2 knockdown PTC cells were detected by WB. N: RIP-qPCR evaluated enrichment level of TACSTD2 in PTC cells after YTHDF2 knockdown. O: TACSTD2 mRNA level in YTHDC2 overexpression PTC cells treated with actinomycin D at the indicated time points were detected by qRT-PCR. YTHDC2 overexpression reduced the half-life of TACSTD2 transcript. P: WB was used to assess TACSTD2 level in PTC cells transfected with NC or OE-METTL3 with sh-NC or sh-YTHDC2.

Fig 3: TACSTD2 gain-of-function promotes PTC cell proliferation and migration. A-B: RT-qPCR and WB assess the activation effect on TACSTD2 following FTC-133 and TPC-1 cell transfection with pcDNA3.1-TACSTD2 or pcDNA3.1-NC. C–E: Determination of the viability of FTC-133 and TPC-1 cells after TACSTD2 overexpression through CCK-8, colony formation, and EdU assays. F–H: Evaluation of the migration ability of FTC-133 and TPC-1 cells after TACSTD2 overexpression by wound healing and transwell migration/invasion assays.

Fig 4: Knockdown of TACSTD2 promotes apoptosis by activating the TNF signaling pathway and blocking EMT to inhibit PTC progression. A: Difference volcano plot of KTC-1 cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#2, n = 3, |log2(FC)|> 1 and P < 0.05. B: KEGG analysis: TNF signaling pathway in PTC with TACSTD2-low expression was the top pathway based on mRNA-seq analysis. C–D: WB was used to measure TNF-α, TNF-R1, TNF-R2 and TRADD expression in PTC cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#1/2. E: WB was used to measure Bax, Bcl-2, Caspase-3, cl-Caspase-3, Caspase-9 and cl-Caspase-9 expression in PTC cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#1/2. F: WB was used to measure E-cadherin, N-cadherin, β-catenin, Snail, and Vimentin expression in PTC cells transfected with Lv-sh-NC and Lv-sh-TACSTD2#1/2.

Fig 5: Knockdown of TACSTD2 inhibits PTC xenograft growth in vivo. A: In vivo tumor metastasis assay: Subcutaneous injection of KTC-1 cells into the mice flanks, followed by multipoint intratumor injection with Lv-sh-TACSTD2 or Lv-sh-NC. B: Representative images of tumors in nude mice. Xenograft tumor weight (C) and growth curve (D) at 28 days after injection. E: IHC analysis of TACSTD2, Ki-67, and PCNA expression in xenograft sections.