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Lysosomes function as critical signaling hubs that govern essential enzyme complexes. LGALS proteins (LGALS3, LGALS8, and LGALS9) are integral to the endomembrane damage response. If ESCRT fails to rectify damage, LGALS-mediated ubiquitination occurs, recruiting autophagy receptors (CALCOCO2, TRIM16, and SQSTM1) and VCP/p97 complex containing UBXN6, PLAA, and YOD1, initiating selective autophagy. Lysosome replenishment through biogenesis is regulated by TFEB. LGALS3 interacts with TFRC and TRIM16, aiding ESCRT-mediated repair and autophagy-mediated removal of damaged lysosomes. LGALS8 inhibits MTOR and activates TFEB for ATG and lysosomal gene transcription. LGALS9 inhibits USP9X, activates PRKAA2, MAP3K7, ubiquitination, and autophagy. Conjugation of ATG8 to single membranes (CASM) initiates damage repair mediated by ATP6V1A, ATG16L1, ATG12, ATG5, ATG3, and TECPR1. ATG8ylation or CASM activates the MERIT system (ESCRT-mediated repair, autophagy-mediated clearance, MCOLN1 activation, Ca2+ release, RRAG-GTPase regulation, MTOR modulation, TFEB activation, and activation of GTPase IRGM). Annexins ANAX1 and ANAX2 aid damage repair. Stress granules stabilize damaged membranes, recruiting FLCN-FNIP1/2, G3BP1, and NUFIP1 to inhibit MTOR and activate TFEB. Lysosomes coordinate the synergistic response to endomembrane damage and are vital for innate and adaptive immunity. Future research should unveil the collaborative actions of ATG proteins, LGALSs, TRIMs, autophagy receptors, and lysosomal proteins in lysosomal damage response.

Cisplatin (DDP) is a commonly used first-line chemotherapy drug for non-small cell lung cancer (NSCLC), but the emergence of DDP resistance greatly reduces its therapeutic effect. Arrestin domain-containing 3 (ARRDC3) has been found to play an anti-cancer role in NSCLC, but whether ARRDC3 regulates DDP resistance in NSCLC is still unclear. The levels of ARRDC3, myeloid ecotropic insertion site 1 (MEIS1), protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2), and autophagy-related proteins were detected by quantitative real-time PCR (qRT-PCR) or western blot. Cell counting kit 8 (CCK8) assay, 3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry, and transwell assay were used to measure cell DDP resistance, proliferation, apoptosis and invasion. The interaction between ARRDC3 and MEIS1 or PRKAA2 was confirmed by dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay and Co-immunoprecipitation (Co-IP) assay. Xenograft tumor models were constructed to test the effect of ARRDC3 on NSCLC tumorigenesis and DDP resistance in vivo. ARRDC3 expression was downregulated in DDP-resistant NSCLC tissues and cells. Functional experiments showed that ARRDC3 overexpression restrained DDP resistance, growth, invasion and autophagy in NSCLC cells. Moreover, MEIS1 promoted ARRDC3 transcription by binding to its promoter region, and the inhibitory effects of MEIS1 overexpression on cell DDP resistance, growth, invasion and autophagy were reversed by ARRDC3 knockdown. Besides, ARRDC3 could interact with PRKAA2, and PRKAA2 overexpression also eliminated the regulation of ARRDC3 on cell DDP resistance, growth, invasion and autophagy. Furthermore, MEIS1 decreased PRKAA2 expression by activating ARRDC3 transcription. Additionally, upregulation of ARRDC3 reduced NSCLC tumorigenesis and DDP resistance in vivo. MEIS1-activated ARRDC3 represses NSCLC cell DDP resistance, growth, invasion and autophagy by reducing PRKAA2 expression, confirming the crucial role of MEIS1/ARRDC3/PRKAA2 axis in the chemotherapy resistance of NSCLC.

Objectives Increasing evidence have demonstrated the N6-methyladenosine (m 6 A) plays critical roles in osteoarthritis (OA) progression, but the role of m 6 A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m 6 A demethylase fat mass and obesity-associated protein ( FTO ) in OA progression. Materials and methods The FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2 . Results FTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO -mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p , which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2 , thereby alleviating OA cartilage damage. Conclusions Our results attested that FTO alleviated the OA cartilage damage by mediating FTO / miR-3591-5p / PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.

PurposeAutoimmune thyroiditis (AIT) is one of the most common autoimmune endocrine diseases. The currently recognized causes are genetic susceptibility, environmental factors and immune disorders. It is important to clarify the pathogenesis for the prevention, diagnosis, treatment of AIT and scientific iodine supplementation. This study analyzed the DNA methylation levels of PRKAA2, ITGA6, PRL and THEM4 genes related to PI3K-AKT signaling pathway, compared the DNA methylation levels between cases and controls from different water iodine levels in Shandong Province of China, and evaluated the contribution of PI3K-AKT signaling pathway-related genes in AIT.MethodsA total of 176 adult AIT patients were included from three different water iodine areas, and 176 healthy controls were included according to gender, age and BMI. According to the results of the Illumina Methylation 850 K BeadChip in our previous research, the significant methylation differences of genes on the PI3K-AKT signaling pathway related to AIT were determined. The MethylTarget™ assay was used to detect the methylation levels of the target genes, and real-time PCR experiments were used to verify the mRNA expression levels.ResultsCompared with the control group, PRKAA2_3 and 15 CpG sites were hyper-methylated. ITGA6 gene and 2 CpG sites were hypo-methylated in AIT cases. The mRNA expression of ITGA6 gene was negatively correlated with the DNA methylation levels of ITGA6 gene and 2 CpG sites. Compared with cases and controls in areas with different water iodine levels, methylation differences were mainly in PRKAA2 and ITGA6 genes. The methylation levels of PRKAA2_1 and PRKAA2_3 were positively correlated with age. The methylation levels of PRL and THEM4 genes were negatively correlated with age. The methylation level of PRKAA2_3 was positively correlated with FT4.ConclusionIn summary, we identified aberrant DNA methylation levels of PRKAA2 and ITGA6 genes related to PI3K-AKT signaling pathway in the blood of AIT patients. Both iodine supplementation after long-term iodine deficiency and iodine excess can affect the DNA methylation levels of PRKAA2 and ITGA6 genes, and the former affects more obviously. In ITGA6 gene, this aberrant epigenetic modification is associated with the increased mRNA expression.

Background Lung adenocarcinoma (LUAD) is a common type of lung cancer and its pathogenic mechanism is complicated. A profound research for the molecular mechanism in LUAD is indispensable. Methods Gene levels were detected via real-time quantitative polymerase chain reaction and western blot. Proliferation, migration and apoptosis were assessed using colony formation assay, wound healing assay, and flow cytometry. Ferroptosis was evaluated through oxidative stress and iron level. Relations between genes were analyzed using Immunoprecipitation (IP) assay and ubiquitination assay, as well as ChIP assay and dual-luciferase reporter assay. USP18 function in vivo was explored using xenograft model. Results Ubiquitin-specific protease 18 (USP18) was overexpressed in LUAD tissues and cells. LUAD cell proliferation and migration were suppressed but apoptosis and ferroptosis were enhanced after USP18 knockdown. Pou domain, class 4, transcription factor 1 (POU4F1) protein expression was stabilized through USP18-mediated deubiquitination. Function of USP18 silence was reversed by POU4F1 overexpression in LUAD cells. POU4F1 promoted transcription of AMPK-α2 (PRKAA2) and USP18 modulated PRKAA2 protein level via affecting POU4F1. POU4F1 regulated LUAD cell behaviors by upregulating PRKAA2. USP18 enhanced tumor growth in vivo via mediating POU4F1 and PRKAA2. Conclusion All data demonstrated that USP18 acted as an oncogene in LUAD via interacting with POU4F1/PRKAA2 axis. Highlights Silence of USP18 promotes lung adenocarcinoma cell apoptosis and ferroptosis. USP18 affects lung adenocarcinoma cell progression by stabilizing POU4F1 protein. USP18 regulates PRKAA2 via enhancing POU4F1.

Exosomes (EXOs) secreted by cancer-associated fibroblasts (CAFs) can induce malignant phenotypes of tumor cells. The long non-coding RNA LINC00930 is known for promoting cancer cell glycolysis and proliferation. However, its role and mechanism in colorectal cancer (CRC) are unclear. This study aimed to determine whether CAF-derived exosomal LINC00930 is involved in CRC progression. In our study, LINC00930 was significantly downregulated in CRC samples and cell lines, as well as in CAFs and CAF-derived EXOs. Functionally, CAF-derived exosomal LINC00930 inhibited CRC cell proliferation, migration, invasion, and glycolysis in vitro and impaired tumor growth in patient-derived xenograft models. LINC00930 improved PRKAA2 stability by recruiting MBNL1. Furthermore, PRKAA2 silencing partly reversed CAF-derived exosomal LINC00930-mediated effects on CRC cell proliferation, migration, invasion, and glycolysis. In conclusion, this study identifies LINC00930 as a tumor-suppressive lncRNA delivered by CAF-derived EXOs that inhibits CRC progression through the MBNL1-PRKAA2 axis, suggesting that LINC00930 could be a viable therapeutic target for CRC.

Background Chemotherapy can significantly improve the disease‐free survival and overall survival of patients with advanced gastric cancer (GC). 5‐fluorouracil (5‐FU) is frequently applied in the clinic, acting as a first‐line chemotherapy drug of advanced GC, which could be used alone or combining platinum drugs. However, its efficacy is significantly attenuated by chemoresistance, which is associated with patients’ poor survival. Recently, there is evidence suggesting that dysregulation of autophagy may contribute to drug resistance in cancer, and circular RNAs (circRNAs) also take part in chemoresistance. However, whether circRNAs participate in 5‐FU chemoresistance through autophagy remains largely unknown. Methods RNA sequencing technologies and bioinformatics analysis were performed in GC. Sanger sequencing, Actinomycin D assay and RNase R assay confirmed the circular structure of circular CPM (circCPM). Various cell line models and animal models were used to explore related functions in vitro and in vivo. Quantitative Real‐time PCR (qRT‐PCR), fluorescence in situ hybridization, ribonucleic acid; (RNA) pulldown assays, RNA binding protein immunoprecipitation assays and Luciferase reporter assays were applied to explore involved pathways. Results circCPM was up‐regulated in 5‐FU resistant GC cell lines and tissue. Moreover, high circCPM expression is positively associated with poor survival. Silencing circCPM greatly improved chemosensitivity in vitro and in vivo. Mechanistically, it directly binds to miR‐21‐3p in the cytoplasm and therefore increases the expression of PRKAA2, contributing to the activation of autophagy and chemoresistance. Conclusion Our results reveal that circCPM has a crucial role in regulating GC autophagy and 5‐FU resistance by targeting PRKAA2. It may function as a new theory basis for assessing the curative effect of GC and reversing 5‐FU chemoresistance. CircCPM is up‐regulated in 5‐FU resistant gastric cancer cells and tissues. CircCPM modulates autophagy by working as a sponge of miR‐21‐3p, thereby up‐regulating PRKAA2 expression. CircCPM regulates gastric cancer 5‐FU chemoresistance through the miR‐21‐3p/ PRKAA2 axis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a rapidly growing global health concern. However, the molecular mechanisms underlying its onset and progression remain incompletely understood. This study aimed to investigate the molecular mechanism by which the transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates hepatic lipid accumulation through the miR-335-5p–mediated PRKAA2/AMPK signaling pathway. MASLD models were established both in high-fat diet (HFD)-fed mice and in free fatty acid (FFA)-treated hepatocytes. Gene expression, lipid accumulation, and related signaling pathways were analyzed using quantitative RT–PCR, Western blotting, Oil Red O staining, dual-luciferase reporter assays, and rescue experiments. We identified a novel regulatory axis in which C/EBPα transcriptionally activates miR-335-5p, leading to suppression of PRKAA2, the catalytic subunit of AMPKα2, and subsequent inhibition of AMPK signaling. This cascade promotes the expression of lipogenic genes and enhances hepatic lipid deposition. Inhibition of either C/EBPα or miR-335-5p restored PRKAA2/AMPK activity, reduced intracellular triglyceride and cholesterol levels, and alleviated hepatic steatosis in both cellular and murine models. Conversely, PRKAA2 silencing reproduced the lipogenic phenotype, while its overexpression rescued miR-335-5p-induced lipid accumulation. Our findings reveal a previously unrecognized C/EBPα/miR-335-5p/PRKAA2 axis that drives hepatic lipogenesis and steatosis in MASLD. Targeting this signaling pathway—by inhibiting C/EBPα or miR-335-5p, or activating PRKAA2/AMPK—may provide a promising therapeutic strategy for MASLD.

The aberrant downregulation of circCDC6 in colorectal cancer (CRC) was previously identified by circRNA microarray analysis. However, the detailed role of circCDC6 in CRC is still lacking. We thus investigated the function of circCDC6 in CRC. The expression of circCDC6, miR-3187-3p and PRKAA2 mRNA was checked by real-time quantitative PCR (RT-qPCR). Cell growth was evaluated by MTT, EdU and colony formation assays. Cell apoptosis was evaluated by flow cytometry. Glycolysis was evaluated by glycolysis stress test and lactic acid level. The expression of PRKAA2, HK2 and LDHA proteins was checked by western blotting. The potential binding between miR-3187-3p and circCDC6 or PRKAA2 was confirmed by dual-luciferase reporter assay, RIP assay and pull-down assay. Xenograft model was established in nude mice. CircCDC6 showed poor expression in CRC tumor samples and cells. CircCDC6 ectopic expression repressed CRC cell proliferation, survival and glycolysis energy metabolism. MiR-3187-3p was targeted by circCDC6, and miR-3187-3p depletion also repressed CRC cell growth and glycolysis. PRKAA2 was a downstream target of circCDC6/miR-3187-3p pathway, and circCDC6 upregulated PRKAA2 expression via targeting miR-3187-3p. PRKAA2 knockdown rescued the functional effects of circCDC6 ectopic expression. CircCDC6 overexpression in vivo impeded tumor development in animal models. CircCDC6, acting as a tumor inhibitor, repressed tumor growth and glycolysis metabolism in CRC via targeting the miR-3187-3p/PRKAA2 axis, which partly clarified the role of circCDC6 in CRC.

Background Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as the most prevalent type of chronic liver disease, posing a significant threat to human health. Protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) plays a pivotal role in regulating metabolic diseases. Nevertheless, the underlying molecular mechanisms by which PRKAA2 influences the pathogenesis of MASLD remain unclear. Method Bioinformatics analysis of public datasets identified the potential role of PRKAA2 in MASLD, verified its immune cells correlation, and constructed its competitive endogenous RNA (ceRNA) network. We assessed the mRNA and protein expression of PRKAA2, along with phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) phosphorylation levels after PRKAA2 knockdown. Pro-inflammatory cytokines were quantified by Enzyme-linked immunosorbent assay (ELISA), and lipid species were profiled using Liquid chromatography-mass spectrometry (LC-MS). In vivo, hepatic morphology and lipid deposition were evaluated by Hematoxylin-eosin (H&E) staining. Immunofluorescence measured PRKAA2, phospho-PI3K (p-PI3K) and phospho-AKT (p-AKT) expression. The regulatory interaction between PRKAA2 and its upstream miRNA was confirmed by dual-luciferase reporter assay. Results Our bioinformatics analysis identified PRKAA2 as a significantly upregulated gene in MASLD. Both in vitro and in vivo experiments consistently revealed markedly elevated PRKAA2 expression levels in MASLD models. Knockdown of PRKAA2 significantly reduced lipid accumulation, suppressed production of pro-inflammatory cytokines and attenuated the phosphorylation ratios of PI3K and AKT. Further mechanistic investigations confirmed that hsa-let-7b-5p directly targets PRKAA2 by binding to its wild-type (WT) 3’UTR, establishing this miRNA as a key upstream regulator of PRKAA2 in MASLD pathogenesis. Conclusions Our findings collectively demonstrated that PRKAA2 serves as a crucial mediator in MASLD pathogenesis, functioning through a novel regulatory axis involving the upstream hsa-let-7b-5p and the downstream activation of the PI3K/AKT pathway.