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BACKGROUND & AIMS: Accumulating evidence has indicated the presence of mature microRNAs (miR) in the nucleus, but their effects on steatohepatitis remain elusive. We have previously demonstrated that the intranuclear miR-204-3p in macrophages protects against atherosclerosis, which shares multiple risk factors with metabolic dysfunction-associated steatotic liver disease (MASLD). Herein, we aimed to explore the functional significance of miR-204-3p in steatohepatitis. METHODS: miR-204-3p levels and subcellular localization were assessed in the livers and peripheral blood mononuclear cells of patients with MASLD. Wild-type mice fed high-fat or methionine- and choline-deficient diets were injected with an adeno-associated virus system containing miR-204-3p to determine the effect of miR-204-3p on steatohepatitis. Co-culture systems were applied to investigate the crosstalk between macrophages and hepatocytes or hepatic stellate cells (HSCs). Multiple high-throughput epigenomic sequencings were performed to explore miR-204-3p targets. RESULTS: miR-204-3p expression decreased in livers and macrophages in mice and patients with fatty liver. In patients with MASLD, miR-204-3p levels in peripheral blood mononuclear cells were inversely related to the severity of hepatic inflammation and damage. Macrophage-specific miR-204-3p overexpression reduced steatohepatitis in high-fat or methionine- and choline-deficient diet-fed mice. miR-204-3p-overexpressing macrophages inhibited TLR4/JNK signaling and pro-inflammatory cytokine release, thereby limiting fat deposition and inflammation in hepatocytes and fibrogenic activation in HSCs. Epigenomic profiling identified miR-204-3p as a specific regulator of ULK1 expression. ULK1 transcription and VPS34 complex activation by intranuclear miR-204-3p improved autophagic flux, promoting the anti-inflammatory effects of miR-204-3p in macrophages. CONCLUSIONS: miR-204-3p inhibits macrophage inflammation, coordinating macrophage actions on hepatocytes and HSCs to ameliorate steatohepatitis. Macrophage miR-204-3p may be a therapeutic target for MASLD. IMPACT AND IMPLICATIONS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic inflammatory disease ranging from simple steatosis to steatohepatitis. However, the molecular mechanisms underlying the progression of MASLD remain incompletely understood. Here, we demonstrate that miR-204-3p levels in circulating peripheral blood mononuclear cells are negatively correlated with disease severity in patients with MASLD. Nuclear miR-204-3p activates ULK1 transcription and improves autophagic flux, limiting macrophage activation and hepatic steatosis. Our study provides a novel understanding of the mechanism of macrophage autophagy and inflammation in steatohepatitis and suggests that miR-204-3p may act as a potential therapeutic target for MASLD.
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Fígado Gorduroso , MicroRNAs , Animais , Camundongos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/etiologia , Hepatócitos/metabolismo , Fígado/metabolismo , Fígado/patologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genéticaRESUMO
INTRODUCTION: Gap junctions can transmit signals between cells, including miRNAs, leading to the amplification of adjacent cell damage. No previous study has addressed gap junctions and miRNAs in sepsis because the internal mechanism of sepsis-induced intestinal injury is complex. Therefore, we studied the relationship between connexin43 (Cx43) and miR-181b and provided a research direction for further study of sepsis. METHODS: A mouse caecal ligation and puncture method was used to construct a mouse sepsis model. Firstly, damage to intestinal tissues at different time points was analysed. The levels of Cx43, miR-181b, Sirt1, and FOXO3a in intestinal tissues and the transcription and translation of the apoptosis-related genes Bim and puma, which are downstream of FOXO3a were analysed. Secondly, the effect of Cx43 levels on miR-181b and Sirt1/FOXO3a signalling pathway activity was explored by using the Cx43 inhibitor heptanol. Finally, luciferase assays were used to determine miR-181b binding to the predicted target sequence. RESULTS: The results show that during sepsis, intestinal injury becomes increasingly worse with time, and the expression of Cx43 and miR-181b increase. In addition, we found that heptanol could significantly reduce intestinal injury. This finding indicates that inhibiting Cx43 regulates the transfer of miR-181b between adjacent cells, thereby reducing the activity of the Sirt1/FOXO3a signalling pathway and reducing the degree of intestinal injury during sepsis. CONCLUSIONS: In sepsis, the enhancement of Cx43 gap junctions leads to an increase in miR-181b intercellular transfer, affects the downstream SIRT1/FOXO3a signalling pathway and causes cell and tissue damage.
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Apoptose , MicroRNAs , Sepse , Animais , Camundongos , Apoptose/genética , Conexina 43/genética , Conexina 43/farmacologia , Modelos Animais de Doenças , Heptanol/farmacologia , MicroRNAs/genética , Sepse/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuína 1/farmacologiaRESUMO
Intestinal injury has long been considered to play a crucial role in the pathophysiology of sepsis and has even been characterized as the "motor" of it. Thus, we explored the effects of connexin43 (Cx43) on sepsis-induced intestinal injury in order to provide potential therapeutic strategies. Rat cecal ligation and puncture (CLP) models in vivo and cell models (IEC-6 cells) pretreated with LPS in vitro were used in the current study. Firstly, different methods, such as Cx43 inhibitors (18-α-GA and oleamide) or siRNA targeting Cx43 and N-acetyl cysteine (NAC) (a kind of ROS scavenger), were used to observe the effects of Cx43 channels mediating ROS transfer on intestinal injury. Secondly, the influence of ROS content on the activity of the JNK1/Sirt1/FoxO3a signaling pathway was explored through the application of NAC, sp600125 (a JNK1 inhibitor), and nicotinamide (a Sirt1 inhibitor). Finally, luciferase assays and ChIP were used to determine the direct regulation of FoxO3a on proapoptotic proteins, Bim and Puma. The results showed that sepsis-induced intestinal injury presented a dynamic change, coincident with the alternation of Cx43 expression. The inhibition of Cx43 attenuated CLP-induced intestinal injury in vivo and LPS-induced IEC-6 injury in vitro. The changes of Cx43 channel function regulated ROS transfer between the neighboring cells, which mediated the activation of the JNK1/Sirt1/FoxO3a signaling pathway. FoxO3a directly affected its downstream target genes, Bim and Puma, which are responsible for cell or tissue apoptosis. In summary, our results suggest that Cx43 inhibition suppresses ROS transfer and inactivates the JNK1/Sirt1/FoxO3a signaling pathway to protect against sepsis-induced intestinal injury.
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Conexina 43/metabolismo , Proteína Forkhead Box O3/metabolismo , Intestinos/lesões , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sepse/complicações , Sirtuína 1/metabolismo , Animais , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Masculino , Ácidos Oleicos/farmacologia , Ratos , Ratos Sprague-Dawley , Sepse/tratamento farmacológicoRESUMO
BACKGROUND/AIM: Recent studies have demonstrated the crucial regulatory roles of circular RNAs (circRNAs) in cancer initiation and progression. The sponge mechanism of circRNAs has been shown to be widely active in various types of tumors. However, many circRNAs still have not been verified to function through this mechanism. This study aimed to investigate the regulatory mechanism of hsa_circ_0079557 in colorectal cancer (CRC) and its role in CRC progression. MATERIALS AND METHODS: Raw gene expression profile datasets were downloaded from Gene Expression Omnibus (GEO) and combined to form a new dataset. Hsa_circ_0079557 was found to be highly expressed in CRC. Its role was evaluated in vitro and in vivo through a series of experiments, including quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, colony formation, cell counting kit-8 (CCK-8), transwell assays, scratch wound healing assays, nude mice experiments, and immunohistochemistry (IHC). The association between hsa_circ_0079557 and the identified target microRNAs (miRNA) was confirmed through fluorescence in situ hybridization (FISH) and dual-luciferase reporter assays. The downstream target proteins were predicted using the web-based tool "TargetScan," and their expressions were determined using Western blot (WB). RESULTS: Hsa_circ_0079557 was found to be relatively up-regulated in CRC tissues and cell lines. Suppression of hsa_circ_0079557 expression inhibited cell proliferation in vitro and in vivo. Additionally, hsa_circ_0079557 acted as a "molecular sponge" for miR-502-5p, up-regulating the expression of Cyclin D1 (CCND1). CONCLUSION: In this study, we identify a highly expressed circRNA in CRC and propose a novel pathway of hsa_circ_0079557/miR-502-5p/CCND1 in CRC.
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Neoplasias Colorretais , MicroRNAs , Humanos , Animais , Camundongos , Ciclina D1 , Hibridização in Situ Fluorescente , Camundongos Nus , RNA Circular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , MicroRNAs/genéticaRESUMO
Background: Tumor dormancy is an important way to develop drug resistance. This study aimed to identify the characteristics of colorectal cancer (CRC) cell dormancy. Methods: Based on the CRC cohorts, a total of 1,044 CRC patients were included in this study, and divided into a dormant subgroup and proliferous subgroup. Non-negative matrix factorization (NMF) was used to distinguish the dormant subgroup of CRC via transcriptome data of cancer tissues. Gene Set Enrichment Analysis (GSEA) was used to explore the characteristics of dormant CRC. The characteristics were verified in the cell model, which was used to predict key factors driving CRC dormancy. Potential treatments for CRC dormancy were also examined. Results: The dormant subgroup had a poor prognosis and was more likely to relapse. GSEA analysis showed two defining characteristics of the dormant subgroup, a difference in energy metabolism and synergistic effects of cancer-associated fibroblasts (CAFs), which were verified in a dormant cell model. Transcriptome and clinical data identified LMOD1, MAB21L2, and ASPN as important factors associated with cell dormancy and verified that erlotinib, and CB-839 were potential treatment options. Conclusion: Dormant CRC is associated with high glutamine metabolism and synergizes with CAFs in 5-FU resistance, and the key effectors are LMOD1, MAB21L2, and ASPN. Austocystin D, erlotinib, and CB-839 may be useful for dormant CRC.
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BACKGROUND: Actin cytoskeleton is connected with the processes of cell proliferation and migration in colorectal cancer (CRC). However, it is unknown how to accomplish these adjustments in CRC by actin cytoskeleton genes (ACGs) and here we investigated the role of hub prognosis-related ACGs-Diaphanous-related formin 3 (DIAPH3) in CRC, as a potential, novel target. METHODS: The ACGs gene set from the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to group CRC patients and select prognosis-related ACGs by univariate and multivariate Cox regression for constructing prognostic model. Next, we tested hub prognosis-related ACGs- DIAPH3 expression in CRC and clarified the role of DIAPH3 by shRNA constructs in KM12 and SW480. Activation of EGFR was analyzed by western blot and immunofluorescence. RESULTS: The results showed that actin cytoskeleton function is a significant prognostic factor for CRC patients and related to clinicopathological characteristics such as T stage and lymph node metastasis. A prognostic model constructed by four prognosis-related ACGs has a moderate intensity to 1-year Survival (AUC = 0.71). And hub prognosis-related ACGs DIAPH3 is downregulated in CRC. Knockdown of DIAPH3 could promote the proliferation and migration capacity of CRC. In addition, DIAPH3-silenced cells increase EGFR phosphorylation by inhibiting EGFR transportation to lysosome. CONCLUSIONS: ACGs play a significant role in tumor invasion and have the potential to predict the prognosis of CRC. Prognosis-related ACGs DIAPH3 might be a new prognostic biomarker and DIAPH3 could inhibit CRC progression through maintaining EGFR degradation.
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Neoplasias Colorretais , Humanos , Prognóstico , Neoplasias Colorretais/patologia , Forminas , Proliferação de Células , Metástase Linfática , Receptores ErbB/genéticaRESUMO
Colorectal cancer (CRC) is one of the most common primary malignancies worldwide. Numerous studies have demonstrated that long non-coding RNAs (lncRNAs) are considered as crucial regulators of tumor progression. In particular, upregulation of the lncRNA KCNQ1OT1 was reported in various types of malignancy as a promoter of tumor progression. However, the role and underlying mechanism of KCNQ1OT1 in CRC remain unclear. Thus, the present study aimed to investigate the role of KCNQ1OT1 in colorectal cancer through GEPIA, reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses, and cell assays. GEPIA analysis demonstrated that high expression levels of KCNQ1OT1 in CRC tissues predicted a poor prognosis for patients with CRC. KCNQ1OT1 was overexpressed in CRC tissues and cell lines via RT-qPCR analysis. Furthermore, the results from the cell viability assay, colony formation assay, wound healing assay, invasion assay and flow cytometric analysis demonstrated that KCNQ1OT1 knockdown significantly inhibited CRC cell proliferation, migration and invasiveness, and promoted CRC cell apoptosis, leading to cell cycle arrest. Western blot analysis demonstrated that KCNQ1OT1 knockdown inhibited the PI3K/AKT signaling pathway. These results suggest that KCNQ1OT1 may act as an oncogene through the PI3K/AKT signaling pathway in CRC.
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BACKGROUND: BMP3 gene is often found hypermethylated and hence inactivated in several types of cancers including colorectal cancer (CRC), indicating that it has a suppressor role in carcinogenesis. Though BMP3 is a reliable biomarker for screening CRC, the molecular mechanism of BMP3 in carcinogenesis remains largely unknown. METHODS: The expression level of BMP3 was examined by immunohistochemistry staining and western blot. Methylation-specific PCR (MSP) and real-time quantitative MSP were used to test the hypermethylation status of BMP3 gene. Analyses of BMP3 function in colon cancer cell proliferation, migration, invasion, and apoptosis were performed using HCT116 and KM12 cells. BMP3 was further knocked down or overexpressed in CRC cells, and the effects on cell growth of xenograft tumors in nude mice were assessed. Co-immunoprecipitation and immunofluorescence staining were used to analyze the association between BMP3 and BMPR2 or BMP3 and ActRIIB. Microarray analysis was performed to identify most differentially expressed genes and pathways regulated by BMP3. The BMP3-regulated SMAD2-dependent signaling pathway and TAK1/JNK signal axes were further investigated by quantitative PCR and western blot. RESULTS: BMP3 gene was hypermethylated and its expression was downregulated in both CRC tissues and cell lines. Expressing exogenous BMP3 in HCT116 inhibited cell growth, migration, and invasion and increased rate of apoptosis both in vitro and in vivo. However, shRNA-mediated attenuation of endogenous BMP3 in KM12 reversed such inhibitory and apoptotic effects. Furthermore, BMP3 could bind to ActRIIB, an activin type II receptor at the cellular membrane, thereby activating SMAD2-dependent pathway and TAK1/JNK signal axes to regulate downstream targets including caspase-7, p21, and SMAD4 that play crucial roles in cell cycle control and apoptosis. CONCLUSIONS: Our study reveals a previously unknown mechanism of BMP3 tumor suppression in CRC and provides a rationale for future investigation of BMP3 as a potential target for the development of novel therapeutic agents to fight CRC.