ALKBH5, an m6A demethylase, attenuates tumor growth and inhibits metastasis in papillary thyroid carcinoma.

The significance of ALKBH5 in erasing mRNA methylation in mRNA biogenesis, decay, and translation control has emerged as a prominent research focus. Additionally, ALKBH5 is associated with the development of numerous human cancers. However, it remains unclear whether ALKBH5 regulates the growth and metastasis of papillary thyroid carcinoma (PTC). Here, we compared cancer tissues and paracancerous tissues from PTC patients, along with cultured cells expressing ALKBH5 (overexpression, silent gene expression, normal stable expression). Our primary objective was to investigate the impact of ALKBH5 on PTC. Selected 30 cases of PTC tissues and their adjacent noncancerous tissues to compare the protein expression levels of ALKBH5 between the two groups using immunohistochemical analysis. qRT-PCR and western blot were used to detect the expression of ALKBH5 in normal thyroid follicular epithelial cells (Nthy-ori3-1) and 4 PTC cell lines (human PTC cell lines K1, BCPAP, IHH4, and TPC1). Appropriate cell lines were screened for subsequent experiments. Immunofluorescence staining was used to localize the high accumulation of ALKBH5 in cells. Construct the ALKBH5 knockdown vector and ALKBH5 overexpression vector separately, and construct the overexpression ALKBH5-mut vector with m6A domain mutation. The impact of different levels of ALKBH5 in the three cell lines on RNA m6A methylation levels was compared using qRT-PCR and western blot methods. Furthermore, cell viability was assessed using the CCK-8 assay, while the impact on cell proliferation was examined using plate colony formation assay. Cell invasion was evaluated using the Transwell assay. Immunohistochemical staining results showed that the expression of ALKBH5 protein in PTC cancer tissue was significantly lower than in adjacent non-cancerous tissue (P < 0.05). Lymph node metastasis in PTC patients may have been linked to ALKBH5 protein levels in their cancerous tissues (P = 0.034). The expression of ALKBH5 in PTC cell lines BCPAP, IHH4, and TPC1 was significantly lower than Nthy-ori3-1 (P < 0.05). IHH4 and TPC1 cell lines were selected for subsequent experiments. Immunofluorescence single staining results showed a high accumulation of ALKBH5 protein in the cell nucleus. Cell viability results suggested that compared to the overexpression-negative control group, cell proliferation, and invasion were significantly decreased in the ALKBH5 overexpression group (P < 0.05) and the mut-ALKBH5 overexpression group (P < 0.05). Additionally, compared to the ALKBH5 overexpression group, cell proliferation and invasion were significantly more decreased in the mut-ALKBH5 overexpression group (P < 0.05). However, compared to the interference-negative control group, cell proliferation and invasion were significantly increased in the ALKBH5 interference group (P < 0.05). The presented findings suggested that m6A demethylase ALKBH5 inhibits tumor growth and metastasis in PTC. Moreover, effective inhibition of m6A modification of ALKBH5 might constitute a potential treatment strategy for PTC.