Methyltransferase-like 14 promotes ferroptosis in sepsis-induced acute kidney injury via increasing the m6A methylation modification of LPCAT3.

Acute kidney injury (AKI) is one of the most serious and common complications in the course of sepsis, known for its poor prognosis and high mortality rate. Recently, ferroptosis, as a newly discovered regulatory cell death, might be closely associated with the progression of AKI. METTL14 is a writer of RNA m6A, an abundant epigenetic modification in transcriptome with broad function. Hence, the purpose of our study is to explore the potential function and mechanism of METTL14 on the ferroptosis in sepsis-induced AKI. In this paper, TCMK-1 cells and mice treated with LPS were used to constructe AKI model in vitro and in vivo. Pathological changes of renal tissue were observed by HE staining. The fluorescent probe C11-BODIPY and 4HNE kits were used to measure the lipid peroxidation. The ferroptosis index was evaluated by MDA, GSH and Fe2+ kits. The total m6A levels were analyzed by EpiQuik M6A RNA methylation kit, and the m6A levels of LPCAT3 were examined by Me-RIP assay. Finally, the interaction between LPCAT3 and METTL14 was clarified using RIP and dual-luciferase reporter gene assays. Our works revealed that the m6A level and ferroptosis was markedly ascended in LPS-induced TCMK-1 cells. The silence of METTL14 lowered the cell viability, the levels of MDA, Fe2+ and lipid peroxidation in the LPS-stimulated AKI model in vitro and in vivo, but increase GSH levels. Moreover, the up-regulation of ferroptosis-related proteins by LPS was notably inhibited by the knockdown of METTL14. In addition, silencing METTL14 reduced the m6A and mRNA levels of LPCAT3. Furthermore, the efficacy of METTL14 downregulation on the ferroptosis in the LPS-induced TCMK-1 cells were antagonized by LPCAT3 overexpression. Taken together, our findings revealed that METTL14 knockdown resisted ferroptosis in sepsis-induced AKI through lessening the level of LPCAT3 mediated by m6A modification.

Xu L ，Wang QJ ，Nie MX ，Chen ZF ... -  《-》

NDUFS3 alleviates oxidative stress and ferroptosis in sepsis induced acute kidney injury through AMPK pathway.

In recent years, ferroptosis has been found to play an important role in various acute kidney injury (AKI). However, relatively little research has been conducted on sepsis-induced acute kidney injury (SI-AKI). As an important trigger of ferroptosis, how mitochondrial damage plays a regulatory role in SI-AKI is still unclear. To explore the potential relationship between mitochondria and ferroptosis, we established a SI-AKI rat model by intraperitoneal injection of lipopolysaccharide (LPS). Transcriptome sequencing was used to detect changes in gene transcription levels in the control group, LPS 3 h group, LPS 6 h group and LPS 12 h group. The severity of kidney injury was determined based on serum creatinine (CRE), blood urea nitrogen (BUN), tissue HE staining, TUNEL staining and inflammatory factor levels. Cytoscape software was utilized to screen several mitochondria-related HUB genes, and NADH dehydrogenase [ubiquinone] ferrithionein 3 (NDUFS3) was selected for subsequent validation due to its novelty and feasibility. qRT-PCR, Western blot was employed to evaluate the expression of NDUFS3 in kidney tissues. GO enrichment analysis revealed that up-regulated genes in the LPS 12 h group were enriched in several cell death terms while down-regulated genes were enriched in lipid metabolic process and oxidation-reduction progress terms. Furthermore, Western blot, IHC, MDA, GSH and iron content levels were used to assess ferroptosis in the kidney tissue of the SI-AKI rats, dihydroethidium (DHE) assay and ATP kit were used to assess mitochondrial ROS levels and mitochondrial function. To further validate the function of NDUFS3, we constructed overexpression rats using hydrodynamic method by tail vein injection of pc DNA3.1-NDUFS3 overexpression plasmid. we utilized LPS to stimulate HK-2 cells and establish an in vitro model. We then overexpressed NDUFS3 using pcDNA 3.1. The overexpression of NDUFS3 was found to inhibit LPS-induced ferroptosis and mitochondrial damage in HK-2 cells, as evidenced by Western blot, MDA, GSH, divalent iron, ROS levels, Mitosox red, ATP content and transmission electron microscopy. Finally, the use of Compound C to inhibit AMPK in HK-2 cells demonstrated that NDUFS3 plays a protective role through the AMPK pathway. Therefore, our study supports the emerging role of NDUFS3 in SI-AKI, providing new potential mitochondria-related targets for the treatment of SI-AKI.

Wang Y ，Lv W ，Ma X ，Diao R ，Luo X ，Shen Q ，Xu M ，Yin M ，Jin Y ... -  《-》

METTL14 Promotes Lipopolysaccharide-Induced Myocardial Damage via m6A-Dependent Stabilization of TRPM7 mRNA.

Wu X ，Huang P ，Xiao Y ，Zha L ，Ma J ，Xiao H ... -  《-》

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

《Jove-Journal of Visualized Experiments》

Exosomal miR-130a-3p confers cisplatin resistance in esophageal cancer by regulating ferroptosis via the suppression of METTL14-mediated m6A RNA methylation of FSP1.

Exosomal microRNA (miRNA)s have been proven to affect recipient cell chemoresistance in several cancers. This research attempted to uncover the role of exosomal miRNA and the regulatory mechanism in cisplatin resistance of esophageal cancer (EC). Cisplatin-resistant EC cells (KYSE-150-CisR and TE-1-CisR) were established by the parental cells (KYSE-150 and TE-1) treated with a gradual increase of cisplatin concentration. Exosomes from both cisplatin-resistant EC cells and the parental one were obtained with ultracentrifugation (CisR-exo and CisS-exo), and identified by transmission electron microscopy and nanoparticle tracking analysis. The effect of CisR-exo on the cisplatin resistance of EC was assessed by in vitro (and in vivo functional experiments.Intracellular reactive oxygen species and iron were determined by the corresponding kits. The m6A dot blot assay and methylated RIP-qPCR was conducted to analyze the m6A modification level. Dual-luciferase reporter assay was performed to confirm the intermolecular interaction. Increased levels of miR-130a-3p were observed in both KYSE-150CisR and TE-1CisR cells, as well as their derived CisR-exos when compared with the parental cells and CisS-exos. Exosomal miR-130a-3p from cisplatin-resistant EC cells conferred cisplatin resistance to EC in vitro and in vivo, which might be mediated by the suppression of ferroptosis. Mechanically, KYSE-150CisR derived exosomal miR-130a-3p interacted with METTL14 to inhibit FSP1 (a ferroptosis suppressor) m6A modification of recipient cells. Overexpressing METTL14 restrained the cisplatin resistance disseminated by CisR-exos in KYSE-150 cells. Cisplatin-resistant EC cell-isolated exosomal miR-130a-3p suppressed the m6A modification of FSP1 to modulate ferroptosis, enhancing cisplatin resistance.

Han H ，Li Y ，Lin Z ，Ma X ，Huang W ，Lu C ，Ma R ，Han R ... -  《-》