YTHDC1 modulates the malignant phenotype of retinoblastoma via SQSTM1-mediated autophagy

Abstract Retinoblastoma (RB) is the most common pediatric intraocular malignancy, yet the role of N6-methyladenosine (m6A) regulators in RB progression remains unclear. This study investigated the function and mechanism of the m6A reader YTHDC1 in RB invasion. The RNA-sequencing dataset GSE97508 from Gene Expression Omnibus was analyzed to identify differentially expressed m6A regulators between invasive and non-invasive RB. Their expression was next validated by RT-qPCR, Western blot, and immunohistochemistry. Functional assays including CCK-8, EdU, and Transwell assays were performed to evaluate the effects of YTHDC1 knockdown or overexpression on RB cells in vitro and in vivo. SQSTM1 was predicted to be a downstream target by bioinformatics and experimental validation. RNA stability and RIP-qPCR assays were performed to examine the regulation of SQSTM1 mRNA by YTHDC1. Autophagic flux was evaluated by LC3B Western blotting and mCherry-GFP-LC3B fluorescence imaging. YTHDC1 was significantly downregulated in invasive RB. YTHDC1 knockdown enhanced, whereas its overexpression suppressed, RB cell proliferation and invasion. YTHDC1 promoted SQSTM1 expression by stabilizing its mRNA. Knockdown of SQSTM1 in RB cells promoted cell proliferation, migration, and invasion, and partially counteracted the inhibition of cell function caused by YTHDC1 overexpression. Knockdown of YTHDC1 or SQSTM1 enhanced autophagic flux, and knockdown of SQSTM1 led to a substantial reduction of the mTOR pathway activity. This study highlights the critical role of YTHDC1 and SQSTM1 in the invasive progression of RB. Their downregulation promoted autophagy in RB cells, providing preliminary evidence of their involvement in RB progression.