Studies have shown that the abnormal expression of circular RNA (circRNA) is inextricably linked to hepatocellular carcinoma (HCC). Recently, hsa_circ_0000518 (circ_0000518) was discovered in many cancer progressions. However, its function in HCC is still unclear. Through GEO database analysis combined with gene expression detection of HCC related clinical samples and cell lines, we identified that circ_0000518 was abnormally overexpressed in HCC. Cell and animal model experiments jointly indicated that circ_0000518 can stimulate HCC cell proliferation, migration, invasion and suppress apoptosis. Furthermore, we also found that knocking down the circ_0000518 could inhibit the Warburg effect in HCC cells. Mechanistically, circ_0000518 was found to be primarily localized in the cytoplasm, and sponge hsa-miR-326 (miR-326) promoted integrin alpha 5 (ITGA5) expression. In addition, circ_0000518 could enhance the stability of HuR-mediated ITGA5 mRNA, thereby activating the Warburg effect. In conclusion, this study elucidated that circ_0000518 was a cancer-promoting circRNA, which could enhance ITGA5 expression through competing endogenous RNAs (ceRNA) and RNA Binding Protein (RBP) mechanisms, thus facilitating the development of HCC. It provides a meaningful diagnostic and therapeutic target for HCC.

Circular RNAs (circRNAs) play critical roles in the recurrence and progression of non-small-cell lung cancer (NSCLC). This study aimed to investigate the function and underlying mechanism of a novel circRNA (circRPPH1) in NSCLC. Localization of circRPPH1 was determined via FISH assay, while cell proliferation was assessed via CCK8 and colony formation assay. Cell migration and invasion were studied using transwell assay, while binding sites between miR-326 and circRPPH1 or ERBB4 were verified by luciferase reporter, RIP, and RNA pull-down assays. Moreover, xenograft assay was performed to verify the in vivo roles of circRPPH1. Results indicated that circRPPH1 was highly expressed in NSCLC tissues and cells, where circRPPH1 levels were predictive of poor prognosis. The malignant behavior of NSCLC cells was exacerbated by overexpressing circRPPH1, while opposite effects were observed when it was knocked down. Direct interaction between miR-326 and circRPPH1 or ERBB4 was confirmed in NSCLC cells, while rescue experiment results showed that circRPPH1 exerted an oncogenic role via miR-326-ERBB4 signal axis. Moreover, in vitro, growth of NSCLC cells was significantly attenuated following circRPPH1 depletion. The study concluded that circRPPH1 was involved in promoting NSCLC progression via the miR-326/ERBB4 axis, which provided a novel potential target for the diagnosis or treatment of NSCLC.

Circular RNAs are highly stable regulatory RNAs that have been increasingly associated with tumorigenesis and progression. However, the role of many circRNAs in triple-negative breast cancer (TNBC) and the related mechanisms have not been elucidated.
In this study, we screened circRNAs with significant expression differences in the RNA sequencing datasets of TNBC and normal breast tissues and then detected the expression level of circRPPH1 by qRT‒PCR. The biological role of circRPPH1 in TNBC was then verified by in vivo and in vitro experiments. Mechanistically, we verified the regulatory effects between circRPPH1 and ZNF460 and between circRPPH1 and miR-326 by chromatin immunoprecipitation (ChIP), fluorescence in situ hybridization assay, dual luciferase reporter gene assay and RNA pull-down assay. In addition, to determine the expression of associated proteins, we performed immunohistochemistry, immunofluorescence, and western blotting.
The upregulation of circRPPH1 in TNBC was positively linked with a poor prognosis. Additionally, both in vivo and in vitro, circRPPH1 promoted the biologically malignant behavior of TNBC cells. Additionally, circRPPH1 may function as a molecular sponge for miR-326 to control integrin subunit alpha 5 (ITGA5) expression and activate the focal adhesion kinase (FAK)/PI3K/AKT pathway.
Our research showed that ZNF460 could promote circRPPH1 expression and that the circRPPH1/miR-326/ITGA5 axis could activate the FAK/PI3K/AKT pathway to promote the progression of TNBC. Therefore, circRPPH1 can be used as a therapeutic or diagnostic target for TNBC.

Amyotrophic lateral sclerosis (ALS) is the most prevalent and lethal class of severe motor neuron diseases (MND) with no efficacious treatment. The pathogenic mechanisms underlying ALS remain unclear. Nearly 90% of patients exhibit sporadic onset (sALS). Therefore, elucidating the pathophysiology of ALS is imperative. Long non-coding RNA (lncRNA) is a large class of non-coding RNAs that regulate transcription, translation, and post-translational processes. LncRNAs contribute to the pathogenesis of diverse neurodegenerative disorders and hold promise as targets for interference in the realm of neurodegeneration. However, the mechanisms of which lncRNAs are involved in ALS have not been thoroughly investigated. We identified and validated a downregulated lncRNA, lnc-HIBADH-4, in ALS which correlated with disease severity and overall survival. Lnc-HIBADH-4 acted as a \"molecular sponge\" regulating lysosomal function through the lnc-HIBADH-4/miR-326/CTSD pathway, thereby impacting autophagy-lysosome dynamics and the levels of cell proliferation and apoptosis. Therefore, this study discovered and revealed the role of lnc-HIBADH-4 in the pathogenesis of ALS. With further research, lnc-HIBADH-4 is expected to provide a new biomarker in the diagnosis and treatment of ALS.

Adhesion G protein-coupled receptor G2 (ADGRG2) is an orphan adhesion G protein-coupled receptor (GPCR), which performs a tumor-promoting role in certain cancers; however, it has not been systematically investigated in hepatocellular carcinoma (HCC). In the current study, we utilized multiple databases to analyze the expression and diagnostic and prognostic value of ADGRG2 in HCC and its correlation with immune infiltration and inflammatory factors. The function and upstream regulatory miRNA of ADGRG2 were validated through qPCR, Western blot, CCK8, wound healing, and dual luciferase assays. It turned out that ADGRG2 was significantly higher in HCC and had a poor survival rate, especially in AFP ≤ 400 ng/mL subgroups. Functional enrichment analysis suggested that ADGRG2 may be involved in cancer pathways and immune-related pathways. In vitro, siRNA-mediated ADGRG2 silencing could inhibit the proliferation and migration of Huh7 and HepG2 cells. There was a highly significant positive correlation between ADGRG2 and neutrophils. Moreover, NET-related genes were filtered and confirmed, such as ENO1 and S100A9. Meanwhile, the high expression of ADGRG2 was also accompanied by the highest number of inflammatory cytokines, chemokines, and chemokine receptors and good immunotherapy efficacy. Finally, AGDGR2 may be sensitive to two drugs (PIK-93 and NPK76-II-72-1) and can be targeted by miR-326. In conclusion, ADGRG2 may serve as a novel biomarker and drug target for HCC diagnosis, immunotherapy, and prognosis and was related to neutrophils and the inflammatory process of liver cancer development.

Pancreatic ductal adenocarcinoma (PDAC) is a notoriously aggressive malignancy with a survival rate of merely 9%. The prognosis in patients with PDAC is relatively poor, particularly in patients with advanced distant metastases. However, the mechanisms of PDAC progression remain elusive. Circular RNAs (circRNAs) have been implicated in the development of various malignancies, including PDAC. Therefore, this study aimed to investigate how a novel circRNA, circATP13A1, regulates PDAC progression. We used the GEO database to determine circATP13A1 expression levels in cancer and adjacent cells and employed the limma package of R software to identify differentially expressed circRNAs. We detected the expression of circATP13A1, miR-186, and miR-326 using qRT-PCR and investigated the effect of circATP13A1 on cell proliferation, migration, invasion, and apoptosis in vitro using the Cell Counting Kit-8 (CCK-8), the transwell migration assay, and the flow cytometry assay. We then performed RNA pull-down assay, RNA immunoprecipitation (RIP), and Western blot to verify the interaction between circATP13A1, miR-186, miR-326, and HMGA2. Moreover, we used a naked mice model to determine how circATP13A1 affects tumor growth and progression in vivo. Loss and gain of function analyses revealed that circATP13A1 upregulation promotes cell proliferation, migration, invasion and tumor growth both in vitro and in vivo, which results in PDAC progression and poor prognosis in patients. CircATP13A1 knockdown significantly impaired cell proliferation and migration of PDAC cell lines. Additionally, circATP13A1 knockdown significantly increased the expression of miR-186 and miR-326, while reducing the expression of HMGA2 (P < 0.05), indicating that miR-186 and miR-326 are downstream targets of circATP13A1. Rescue experiments support the interactions between circATP13A1, miR-186, miR-326, and HMGA2. In conclusion, we demonstrated that circATP13A1 sponges the miR-186/miR-326/HMGA2/axis, acting as an oncogene to promote PDAC development.

OBJECTIVE: MicroRNA-326 is abnormally expressed in autoimmune diseases, but its roles in autoimmune hepatitis (AIH) are unknown. In this study, we aimed to investigate the effect of miR-326 on AIH and the underlying mechanism.
METHODS: Concanavalin A was administrated to induce AIH in mice and the expression levels of miR-326 and TET2 was evaluated by qRT-PCR and western blot, respectively. The percentages of Th17 and Treg cells were evaluated by flow cytometry and their marker proteins were determined by western blot and ELISA. The mitochondrial membrane potential (MMP) and ROS level were tested with the JC-1 kit and DCFH-DA assay. The binding relationships between miR-326 and TET2 were verified by dual-luciferase reporter assay. The liver tissues were stained by the HE staining. In vitro, AML12 cells were cocultured with mouse CD4+T cells. The expression levels of pyroptosis-related proteins were assessed by western blot.
RESULTS: Concanavalin A triggered AIH and enhanced the expression level of miR-326 in mice. It increased both Th17/Treg ratio and the levels of their marker proteins. The expression of TET2 was decreased in AIH mice. Knockdown of miR-326 could decrease the levels of pyroptosis-related proteins, the ROS level and increase MMP. In mouse CD4+T cells, miR-326 sponged TET2 to release IL-17A. Coculture of AML12 cells with isolated CD4+T cells from miR-326 knockdown AIH mice could relieve pyroptosis.
CONCLUSIONS: Knockdown of miR-326 exerted anti-pyroptosis effects via suppressing TET2 and downstream NF-κB signaling to dampen AIH. We highlighted a therapeutic target in AIH.

Idiopathic Pulmonary Fibrosis (IPF) is a progressively fatal and incurable disease characterized by the loss of alveolar structures, increased epithelial-mesenchymal transition (EMT), and aberrant tissue repair. In this study, we investigated the role of Nuclear Factor I-B (NFIB), a transcription factor critical for lung development and maturation, in IPF. Using both human lung tissue samples from patients with IPF, and a mouse model of lung fibrosis induced by bleomycin, we showed that there was a significant reduction of NFIB both in the lungs of patients and mice with IPF. Furthermore, our in vitro experiments using cultured human lung cells demonstrated that the loss of NFIB was associated with the induction of EMT by transforming growth factor beta (TGF-β). Knockdown of NFIB promoted EMT, while overexpression of NFIB suppressed EMT and attenuated the severity of bleomycin-induced lung fibrosis in mice. Mechanistically, we identified post-translational regulation of NFIB by miR-326, a miRNA with anti-fibrotic effects that is diminished in IPF. Specifically, we showed that miR-326 stabilized and increased the expression of NFIB through its 3\'UTR target sites for Human antigen R (HuR). Moreover, treatment of mice with either NFIB plasmid or miR-326 reversed airway collagen deposition and fibrosis. In conclusion, our study emphasizes the critical role of NFIB in lung development and maturation, and its reduction in IPF leading to EMT and loss of alveolar structures. Our study highlights the potential of miR-326 as a therapeutic intervention for IPF. The schema shows the role of NFIB in maintaining the normal epithelial cell characteristics in the lungs and how its reduction leads to a shift towards mesenchymal cell-like features and pulmonary fibrosis. A In normal lungs, NFIB is expressed abundantly in the epithelial cells, which helps in maintaining their shape, cell polarity and adhesion molecules. However, when the lungs are exposed to factors that induce pulmonary fibrosis, such as bleomycin, or TGF-β, the epithelial cells undergo epithelial to mesenchymal transition (EMT), which leads to a decrease in NFIB. B The mesenchymal cells that arise from EMT appear as spindle-shaped with loss of cell junctions, increased cell migration, loss of polarity and expression of markers associated with mesenchymal cells/fibroblasts. C We designed a therapeutic approach that involves exogenous administration of NFIB in the form of overexpression plasmid or microRNA-326. This therapeutic approach decreases the mesenchymal cell phenotype and restores the epithelial cell phenotype, thus preventing the development or progression of pulmonary fibrosis.

Acute myeloblastic leukemia (AML) is the most prevalent form of AML in adults. Despite the availability of various treatment options, including radiotherapy and chemotherapy, many patients fail to respond to treatment or relapse. Copper is a necessary cofactor for all organisms; however, it turns toxic when concentrations reach a certain threshold maintained by homeostatic systems that have been conserved through evolution. However, the mechanism through which excess copper triggers cell death remains unknown. In this study, data on long non-coding RNAs (lncRNAs) related to cuproptosis were retrieved from publicly available databases. LASSO and univariate and multivariate Cox regression analyses were performed to establish an lncRNA model associated with cuproptosis specific to AML. To investigate the risk model, the Kaplan-Meier curve, principal component analysis, functional enrichment analysis, and nomographs were employed. The underlying clinicopathological characteristics were determined, and drug sensitivity predictions against the model were identified. Six cuproptosis-related lncRNA-based risk models were identified as the independent prognostic factors. By regrouping patients using a model-based method, we were able to more accurately differentiate patients according to their responses to immunotherapy. In addition, prospective compounds targeting AML subtypes have been identified. Using qRT-PCR, we examined the expression levels of six cuproptosis-associated lncRNAs in 30 clinical specimens. The cuproptosis-associated lncRNA risk-scoring model developed herein has implications in monitoring AML prognosis and in the clinical prediction of the response to immunotherapy. Furthermore, we identified and verified the ceRNA of the cuproptosis-related lncRNA HAGLR/miR-326/CDKN2A regulatory axis using bioinformatic tools. HAGLR is highly expressed in AML and AML cell lines. HAGLR inhibition significantly reduced the proliferation of AML cells and promoted apoptosis. Elesclomol promotes the degradation of CDKN2A and inhibits the proliferation of AML cells. Elesclomol combined with si-HAGLR inhibited the AML progression of AML both in vitro and in vivo.

Zinc finger protein ZNF322A is an oncogenic transcription factor. Overexpression of ZNF322A activates pro-metastasis, cancer stemness, and neo-angiogenesis-related genes to enhance lung cancer progression. However, the upstream regulator of ZNF322A is not well defined. Dysregulation of microRNAs (miRNAs) can mediate cancer cell growth, migration, and invasion to promote tumorigenesis. Here, we uncover the mechanism of miRNA-mediated transcriptional regulation in ZNF322A-driven oncogenic events. ZNF322A harbors several putative miRNA-binding sites in the 3\'-untranslated region (UTR). We validated that miR-326 downregulated ZNF322A-3\'-UTR luciferase activity and mRNA expression. Furthermore, miR-326 suppressed the expression of ZNF322A-driven cancer-associated genes such as cyclin D1 and alpha-adducin. Reconstitution experiments by ectopic overexpression of ZNF322A abolished miR-326-suppressed cancer cell proliferation and cell migration capacity. Moreover, miR-326 attenuated ZNF322A-induced tumor growth and lung tumor metastasis in vivo. Clinically, the expression of miR-326 negatively correlated with ZNF322A mRNA expression in surgically resected tissues from 120 non-small cell lung cancer (NSCLC) patients. Multivariate Cox regression analysis demonstrated that NSCLC patients with low miR-326/high ZNF322A profile showed poor overall survival. Our results reveal that the deregulated expression of miR-326 leads to hyperactivation of ZNF322A-driven oncogenic signaling. Targeting the miR-326/ZNF322A axis would provide new therapeutic strategies for lung cancer patients.