The m6A methyltransferase METTL3 drives thyroid cancer progression and lymph node metastasis by targeting LINC00894.

BACKGROUND: Long noncoding RNAs (lncRNAs) are significant contributors to various human malignancies. The aberrant expression of lncRNA LINC00894 has been reported in various human malignancies. We aimed to illustrate the role of LINC00894 and its underlying mechanism in the development of papillary thyroid carcinoma (PTC). METHODS: We performed bioinformatics analysis of differentially expressed RNAs from TCGA and GEO datasets and selected the target lncRNA LINC00894. SRAMP analysis revealed abundant M6A modification sites in LINC00894. Further analysis of StarBase, GEPIA, and TCGA datasets was performed to identify the related differentially expressed genes METTL3. Colony formation and CCK-8 assays confirmed the relationship between LINC00894, METTL3, and the proliferative capacity of PTC cells. The analysis of AnnoLnc2, Starbase datasets, and meRIP-PCR and qRT‒PCR experiments confirmed the influence of METTL3-mediated m6A modification on LINC00894. The study employed KEGG enrichment analysis as well as Western blotting to investigate the impact of LINC00894 on the expression of proteins related to the Hippo signalling pathway. RESULTS: LINC00894 downregulation was detected in PTC tissues and cells and was even further downregulated in PTC with lymphatic metastasis. LINC00894 inhibits the lymphangiogenesis of vascular endothelial cells and the proliferation of cancer cells. METTL3 enhances PTC progression by upregulating LINC00894 by enhancing LINC00894 mRNA stability through the m6A-YTHDC2-dependent pathway. LINC00894 may inhibit PTC malignant phenotypes through the Hippo signalling pathway. CONCLUSION: The METTL3-YTHDC2 axis stabilizes LINC00894 mRNA in an m6A-dependent manner and subsequently inhibits tumour malignancy through the Hippo signalling pathway.

LINC00894 Regulates Cerebral Ischemia/Reperfusion Injury by Stabilizing EIF5 and Facilitating ATF4-Mediated Induction of FGF21 and ACOD1 Expression.

The non-coding RNA LINC00894 modulates tumor proliferation and drug resistance. However, its role in brain is still unclear. Using RNA-pull down combined with mass spectrometry and RNA binding protein immunoprecipitation, EIF5 was identified to interact with LINC00894. Furthermore, LINC00894 knockdown decreased EIF5 protein expression, whereas LINC00894 overexpression increased EIF5 protein expression in SH-SY5Y and BE(2)-M17 (M17) neuroblastoma cells. Additionally, LINC00894 affected the ubiquitination modification of EIF5. Adeno-associated virus (AAV) mediated LINC00894 overexpression in the brain inhibited the expression of activated Caspase-3, while increased EIF5 protein level in rats and mice subjected to transient middle cerebral artery occlusion reperfusion (MCAO/R). Meanwhile, LINC00894 knockdown increased the number of apoptotic cells and expression of activated Caspase-3, and its overexpression decreased them in the oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro models. Further, LINC00894 was revealed to regulated ATF4 protein expression in condition of OGD/R and normoxia. LINC00894 knockdown also decreased the expression of glutamate-cysteine ligase catalytic subunit (GCLC) and ATF4, downregulated glutathione (GSH), and the ratio of GSH to oxidized GSH (GSH: GSSG) in vitro. By using RNA-seq combined with qRT-PCR and immunoblot, we identified that fibroblast growth factor 21 (FGF21) and aconitate decarboxylase 1 (ACOD1), as the ATF4 target genes were regulated by LINC00894 in the MCAO/R model. Finally, we revealed that ATF4 transcriptionally regulated FGF21 and ACOD1 expression; ectopic overexpression of FGF21 or ACOD1 in LINC00894 knockdown cells decreased activated Caspase-3 expression in the OGD/R model. Our results demonstrated that LINC00894 regulated cerebral ischemia injury by stabilizing EIF5 and facilitating EIF5-ATF4-dependent induction of FGF21 and ACOD1.

LINC00894 inhibited neuron cellular apoptosis and regulated activating transcription factor 3 expression.

LINC00894 may be associated with synaptic function, but its biology function in neural cells is still unknown. In this study, LINC00894 knockdown decreased the EdU incorporated into newly synthesized DNA and cell viability in MTT or CCK-8 assay in HEK-293T and BE(2)-M17 (M17) neuroblastoma cells. And LINC00894 knockdown increased cellular apoptosis in Annexin V-FITC staining, the expression of activated Caspase3 and the level of reactive oxygen species (ROS) both in HEK-293T and M17 cells. Moreover, LINC00894 also protected cells from hydrogen peroxide induced apoptosis in in vitro models. Utilizing RNA sequencing (RNA-seq) integrated with quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunoblot, we identified that LINC00894 affected activating transcription factor 3 (ATF3) expression in HEK-293T, M17, and SH-SY5Y neuroblastoma cells. Finally, we found that ectopic expression of ATF3 restored cell proliferation and inhibited cell apoptosis in LINC00894 downregulated M17 cells. While knockdown of ATF3 also significantly increased the cell viability inhibition and apoptosis promotion induced by LINC00894 knockdown in M17 cells. Our results from in vitro models revealed that LINC00894 could promote neuronal cell proliferation and inhibit cellular apoptosis by affecting ATF3 expression.

LINC00894 targets Annexin A2 to regulate oxaliplatin resistance in hepatocellular carcinoma: ANXA2 protein function.

To investigate the role of LINC00894 in oxaliplatin chemoresistance of hepatocellular carcinoma (HCC) and its mechanisms. The oxaliplatin-resistant HCC cell lines were established. IC50 of oxaliplatin was calculated by CCK-8 assay. Cell viability was detected using clonal formation experiment, while cell apoptosis was accessed by flow cytometry. RNA binding protein immunoprecipitation and RNA pull-down were performed to explore the interaction of LINC00894 and ANXA2. The expressions of RNA and protein were tested by qRT-PCR and western blot respectively. Tumor xenograft was performed to detect the effect of LINC00894 in vivo. The expression of ki67 was evaluated by immunohistochemistry staining. LINC00894 was overexpressed in HCC cells resistant to oxaliplatin. Elevated LINC00894 promoted HCC cells resistance to oxaliplatin, whereas silence of LINC00894 improved HCC sensitivity to oxaliplatin. LINC00894 could bind to the ANXA2 protein, enhanced the stability of the ANXA2 protein and reduced its ubiquitination. Furthermore, LINC00894 modulated HCC resistance to oxaliplatin both in vitro and in vivo by targeting the ANXA2 protein.LINC00894 enhanced the stability of ANXA2 protein and attenuated its ubiquitination by interacting with it, thereby promoting oxaliplatin resistance in HCC. Our findings contributed to understanding the role of these mechanisms in the process of oxaliplatin resistance in HCC.