Long Non-coding RNA YTHDF3-DT Suppresses The Autophagy-dependent Ferroptosis Of Lung Adenocarcinoma Via Stabilizing INHBA

Objective: This study aimed to verify the expression of YTHDF3-DT in lung cancer and analyze its biological function;screen and verify the downstream target genes and pathways of YTHDF3-DT;clarify the molecular mechanism of YTHDF3-DT regulating downstream target genes and pathways;to explore the regulatory mechanism of YTHDF3-DT on RSL-3-induced ferroptosis and autophagy;to analyze the relationship between autophagy and ferroptosis in LUAD cells regulated by YTHDF3-DT.This study will provide a theoretical basis for a new approach to cancer therapy based on the induction of LUAD ferroptosis.Methods: RT-q PCR was used to detect the expression levels of lnc RNA and m RNA in clinical tissues and cell lines,and it could also be used to detect the stability of m RNA,and the subcellular localization of lnc RNA;online database TCGA,KM-plotter were used to analyze the expression of YTHDF3-DT in large samples and its correlation with prognosis;Western Blot was used to detect the intracellular protein expression level;Lentiviral transfection was used to construct stable expression cell models,and transient transfection was used to acquire temporary gene expression;MTS,clone formation,transwell invasion and migration assays were used to verify the effect of YTHDF3-DT and downstream genes on the biological function of LUAD cells;MTS was also used to detect cell viability;Trypan blue assay was used to detect the proportion of dead cells;Iron assay was used to detect the level of total iron and ferrous ions in cells;flow cytometry was used to detect cell apoptosis,cell cycle and lipid ROS levels;electron microscope was used to observe subcellular structure and mitochondrial morphological changes;RSL-3 and Ferrostatin-1 were used to induce or inhibit ferroptosis;RNA-seq was used to screen out downstream target genes and analyze downstream signaling pathways;Act D was used to inhibit RNA synthesis and detect m RNA stability;dual luciferase reporter was used to detected the interaction between mi RNA and m RNA or lnc RNA;subcutaneous xenotransplanted tumor model was used to verify the effect of YTHDF3-DT on LUAD tumor formation in vivo.Results: YTHDF3-DT is located in both the nucleus and cytoplasm,and it is highly expressed in clinical samples and cell lines of lung adenocarcinoma,patients with high expression of YTHDF3-DT have a poor prognosis;It can significantly increase the proliferation ability,clone formation ability,invasion and metastasis of LUAD cells,and also increase the tumorigenic ability of LUAD cells in vivo;YTHDF3-DT significantly increased the cell activity but reduced cell death,reduced total iron and ferrous iron of LUAD cell after the treatment of RSL-3.YTHDF3-DT also reduced lipid ROS levels and eliminate the ferroptic changes of mitochondrial of LUAD;the ferroptic and autophagy-related markers were detected by WB,and the results showed that YTHDF3-DT can inhibit RSL-3-induced ferroptosis and autophagy;a total of 246 differential expressed genes were screened by RNA-seq,among which INHBA was filtered as the target gene,and the GSEA analyses found that genes regulated by YTHDF3-DT were mainly enriched in m TORC1,PI3K/Akt/m TOR and TGFβ pathways;YTHDF3-DT acted as ce RNA competitively bind with mi RNA-301a-3p and significantly increase the stability of INHBA m RNA.YTHDF3-DT can activate the PI3K/Akt/m TOR pathway through INHBA,then inhibit the autophagy and autophagy-dependent ferroptosis in LUAD cells.Conclusion: Lnc RNA YTHDF3-DT is distributed in both the cytoplasm and nucleus of LUAD cells,highly expressed in LUAD tissues and cell lines,and its expression is negatively correlated with the prognosis of patients;overexpression of YTHDF3-DT promotes the proliferation and clone formation of LUAD cells,migration and invasion and tumor formation speed in vivo;YTHDF3-DT can block the inhibitory effect of RSL-3 on LUAD cell activity,and inhibit RSL-3-induced ferroptosis and autophagy in LUAD cells;YTHDF3-DT can significantly upregulate the expression of INHBA and YTHDF3-DT can regulate LUAD autophagy-dependent ferroptosis by activating PI3K/Akt/m TOR through the Activin A/SMAD pathway.