miR-552-3p modulates transcriptional activities of FXR and LXR to ameliorate hepatic glycolipid metabolism disorder.

BACKGROUND & AIMS: The nuclear location of miRNAs has been known for more than a decade, but the exact function of miRNAs in the nucleus has not been fully elucidated. We previously discovered that intranuclear miR-552-3p has an inhibitory role on gene transcription and contains a particular AGGTCA-like sequence, the cis-elements of the NR1 subfamily of nuclear receptors. Here, we aim to explore the potential effect of miR-552-3p and its AGGTCA-like sequence on NR1s and its possible application in improving hepatic glycolipid metabolism. METHODS: RNA-seq, mass spectrometry, and bioinformatics analysis were used to reveal the possible pathways influenced by miR-552-3p. High fat-high fructose diet-fed mice and db/db mice transfected with AAV2/8-miR-552-3p were established to investigate the in vivo effects of miR-552-3p on hepatic glycolipid metabolism. Fluorescence resonance energy transfer, pull-down, electrophoretic mobility shift, and chromatin immunoprecipitation assays were performed to explore the mechanism by which miR-552-3p regulates NR1s. RT-PCR was conducted to analyse miR-552-3p levels in liver biopsies from patients with NAFLD and normal controls. RESULTS: MiR-552-3p could inhibit metabolic gene expression in vitro and displayed beneficial effects on glycolipid metabolism in vivo. Intranuclear miR-552-3p primarily regulated the LXRα and FXR pathways; this was achieved by its binding to the complementary sequence of AGGTCA to modulate the transcriptional activities of LXRα and FXR. Moreover, LXRα and FXR ligands could restore the effects of miR-552-3p on gene expression and glycolipid metabolism. Additionally, the hepatic miR-552-3p level was significantly decreased in liver samples from patients with NAFLD compared to normal controls. CONCLUSIONS: The mechanism by which miR-552-3p modulates LXRα and FXR has revealed a new method of miRNA-mediated gene regulation. In addition, the beneficial effects in vivo and clinical relevance of miR-552-3p suggest that it might be a potential therapeutic target for the treatment of glycolipid metabolic disease. LAY SUMMARY: Glycolipid metabolic diseases, which have become a major public health concern worldwide, are triggered by abnormalities in lipid and glucose metabolism. Herein, we show that miR-552-3p has the ability to ameliorate hepatic glycolipid metabolic diseases by modulating the transcriptional activities of LXRα and FXR in the nucleus. These findings provide evidence that miR-552-3p may serve as a potential therapeutic target.

New PCSK9 inhibitor miR-552-3p reduces LDL-C via enhancing LDLR in high fat diet-fed mice.

PCSK9 has emerged as a promising new therapeutic target for hyperlipidemia. The efficacy of PCSK9 siRNA in clinic trials clues the feasibility of exploring more PCSK9 inhibitors based on genetic inhibition in the treatment of hyperlipidemia. MicroRNAs (miRNAs) as a class of endogenous non-coding small RNAs can regulate genes at transcriptional and/or translational level. Here, we screened miRNAs from the prediction of TargetScan database with possible inhibitory activities in PCSK9 protein level via AlphaLISA and Western blotting, in which miR-552-3p was selected out for its strongest inhibitory effect. MiR-552-3p could bind to the 3' untranslated region (3'-UTR) of PCSK9 to inhibit translation and interact with the promoter of PCSK9 to suppress transcription. Further in vitro and in vivo experiments proved the effects of miR-552-3p on PCSK9 and downstream effectors: it could increase LDLR protein level, promote LDL-C uptake in HepG2 cells and lower serum LDL-C in high fat diet (HFD)-fed mice. In conclusion, our findings firstly identified miR-552-3p as a new PCSK9 inhibitor with the dual-inhibition mechanism, which suggested the possible application of miR-552-3p in the treatment of hyperlipidemia.

Exosomal miR-552-3p isolated from BALF of patients with silicosis induces fibroblast activation.

BACKGROUND: Silica particles can cause silicosis, a disease characterized by diffuse fibrosis of the lungs. Various signaling pathways composed of different types of cells and cytokines are involved in the development of silicosis. Exosomes have become a research hotspot recently. However, the role of exosomal microRNA (miRNA) in silicosis remains unclear. METHODS: In this study, we generated exosomal miRNA sequences from exosomes isolated from bronchoalveolar lavage fluid (BALF) of silicosis patients and the control group by high-throughput sequencing. Functional annotation and analysis of miRNA identified key target miRNAs. Levels of target miRNAs were analyzed in patient and animal samples and cells. Effects of increased miRNA were assessed through protein levels in target signaling pathways in cells treated with silica, miRNA mimics, and inhibitors. RESULTS: Our study identified 40 up-regulated and 70 down-regulated miRNAs, with miR-552-3p and its putative target gene Caveolin 1 (CAV1) as targets for further research. We found that the levels of exosomal miR-552-3p increased in silicosis patients' BALF samples, silicosis model mice, and A549 cells exposed to silica. Inhibition of miR-552-3p suppressed the expression of fibrosis markers. The increased miR-552-3p leads to the up-regulation of fibronectin and α-smooth muscle actin (α-SMA) and the suppression of caveolin 1 in fibroblast cells. Mitogen-activated protein kinase (MAPK) signaling pathways are activated in cells treated with silica and miR-552-3p mimics. CONCLUSIONS: These results help to understand exosomal miRNA-mediated intercellular communication and its key role in fibroblast activation and silicosis.

LINC01705 derived from adipocyte exosomes regulates hepatocyte lipid accumulation via an miR-552-3p/LXR axis.

AIMS/INTRODUCTION: High glucose increases the accumulation of lipid droplets in hepatocytes, which eventually results in nonalcoholic fatty liver disease in patients with diabetes. However, the specific mechanism or communication between adipocyte and hepatocyte lipid metabolism is still ambiguous. MATERIALS AND METHODS: In this study, exosomes released from human adipocytes were isolated and identified by their morphology, size, and marker proteins by using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting (WB). Gene expression was detected by qRT-PCR and WB. Lipid accumulation was determined by oil red O staining and analyses of total cholesterol (TC) and triglyceride (TG) content. RESULTS: Our results showed that co-culture of HepG2 cells with adipocytes under high glucose conditions stimulated lipid deposition and LINC01705 expression in the HepG2 cells. Exosomes extracted from adipocytes cultured under high glucose conditions had higher levels of LINC01705 than exosomes extracted from adipocytes cultured under normal glucose conditions. Moreover, LINC01705 expression was also elevated in exosomes extracted from diabetes patients when compared with exosomes isolated from normal volunteers, and exosomes from patients who had diabetes complicated with fatty liver (DCFL) had the highest levels of LINC01705 expression. Treatment of HepG2 cells with exosomes extracted from high glucose-stimulated adipocytes promoted lipid deposition and LINC01705 expression in HepG2 cells. Further experiments showed that overexpression of LINC01705 promoted HepG2 lipid metabolism, while inhibition of LINC01705 had the opposite effect. Mechanistically, LINC01705 competitively bound to miR-552-3p, and treatment with miR-552-3p inhibitor reversed the effects induced by LINC01705 knockdown. Moreover, miR-552-3p was found to regulate the transcription activity of LXRα and thereby modulate lipid metabolism-related gene expression. CONCLUSIONS: When taken together, our findings showed that high glucose increased the LINC01705 levels in adipocyte exosomes, and thereby improved HepG2 lipid accumulation via an miR-552-3p/LXR axis.

MiR-552-3p Regulates Multiple Fibrotic and Inflammatory genes Concurrently in Hepatic Stellate Cells Improving NASH-associated Phenotypes.

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic steatosis, inflammation, and progressive fibrosis. Our previous study demonstrated that microRNA-552-3p (miR-552-3p) was down-regulated in the livers of patients with NASH and alleviated hepatic glycolipid metabolic disorders. However, whether miR-552-3p affects NASH progression remains unclear. In this current study, we found that hepatic miR-552-3p expression was negatively correlated with the degree of liver fibrosis and inflammation of NASH patients. Interestingly, the level of miR-552-3p was decreased during hepatic stellate cell (HSC) activation in vitro. Overexpression of miR-552-3p could not only inhibit the expression of fibrotic and inflammatory genes, but also restrain the activation of TGF-ß1/Smad3 signaling pathway by down-regulating the expression of TGFBR2 and SMAD3 in HSCs, finally suppressing HSC activation. More importantly, overexpression of miR-552-3p ameliorated liver fibrosis and inflammation in two murine models: high fat/high fructose/high cholesterol diet-induced NASH model and carbon tetrachloride (CCl4)-treated liver fibrosis model. In conclusion, miR-552-3p plays a crucial role in the pathogenesis of NASH by limiting multiple fibrotic and inflammatory pathways in HSCs, which may shed light on its therapeutic potential in NASH.

Circular RNA PIP5K1A Promotes Glucose and Lipid Metabolism Disorders and Inflammation in Type 2 Diabetes Mellitus.

Disorders of glucose and lipid metabolism are an important cause of type 2 diabetes mellitus (T2DM). Identifying the molecular mechanism of metabolic disorders is key to the treatment of T2DM. The study was to investigate the effect of circRNA PIP5K1A (circPIP5K1A) on glucose and lipid metabolism and inflammation in T2DM rats. A T2DM rat model was established, and then the T2DM rats were injected with lentiviral vectors that interfere with circPIP5K1A, miR-552-3p, or ENO1 expression. Fasting blood glucose (FBG) and fasting insulin (FINS) levels of rats were detected by an automatic analyzer and insulin detection kit, and HOMA-IR was calculated. Lipid metabolism was assessed by measuring serum levels of TG, TC, LDL-C, leptin, and resistin. Serum levels of inflammatory factors (TNF-α and IL-6) were detected by ELISA. The pathological conditions of pancreatic tissue were observed by HE staining. circPIP5K1A, miR-552-3p and ENO1 levels were recorded. The experimental results showed that circPIP5K1A and ENO1 were up-regulated, and miR-552-3p was down-regulated in T2DM rats. Down-regulating circPIP5K1A or up-regulating miR-552-3p reduced blood glucose and lipid levels, inhibited inflammation, and improved pancreatic histopathological changes in T2DM rats. In addition, up-regulating ENO1 rescued the ameliorating effects of down-regulated circPIP5K1A on T2DM rats. In general, downregulating circPIP5K1A improves insulin resistance and lipid metabolism disorders and inhibits inflammation by targeting miR-552-3p to mediate ENO1 expression.

MiR-552-3p promotes malignant progression of gallbladder carcinoma by reactivating the Akt/ß-catenin signaling pathway due to inhibition of the tumor suppressor gene RGMA.

BACKGROUND: Gallbladder carcinoma (GBC) remains a highly lethal disease worldwide. MiR-552 family members promote the malignant progression of a variety of digestive system tumors, but the role of miR-552-3p in GBC has not been elucidated. miR-552-3p was predicted to target the 3'-untranslated region (3'UTR) of the mRNA for the tumor suppressor gene "repulsive guidance molecule BMP co-receptor a" (RGMA). The aim of the present study was to clarify the roles and mechanisms of miR-552-3p targeting RGMA in the malignant progression of GBC. METHODS: In vitro: expression of miR-552-3p was detected by real-time quantitative PCR (qRT-PCR) in tumor and non-tumor adjacent tissues (NATs). Lentivirus-miR-552-3p was employed to knockdown this miRNA in GBC cell lines. Stem cell-related transcription factors and markers were assessed by qRT-PCR. Cell Counting Kit-8 (CCK-8), sphere formation and transwell assays were used to determine the malignant phenotypes of GBC cells. Targeting the 3'UTR of RGMA by miR-552-3p was verified by integrated analysis including bioinformatics prediction, luciferase assays, measures of changes of gene expression and rescue experiments. In vivo: mouse models of subcutaneous tumors and lung metastases were established to observe the effect of miR-552-3p on tumorigenesis and organ metastasis, respectively. RESULTS: MiR-552-3p was abnormally highly expressed in GBC tissues and cancer stem cells. Interference with miR-552-3p in SGC-996 and GBC-SD cells significantly inhibited GBC stem cell expansion. Reciprocally, miR-552-3p promoted GBC cell proliferation, migration and invasion both in vitro and in vivo; hence, interference with this miRNA impeded the malignant progression of GBC. Furthermore, the important tumor suppressor gene RGMA was identified as a target of miR-552-3p. The effects of miR-552-3p on cell proliferation and metastasis were abrogated or enhanced by gain or loss of RGMA function, respectively. Mechanistically, miR-552-3p promoted GBC progression by reactivating the Akt/ß-catenin pathway and epithelial-mesenchymal transformation (EMT). Clinically, miR-552-3p correlated with multi-malignant characteristics of GBC and acted as a prognostic marker for GBC outcome. CONCLUSIONS: MiR-552-3p promotes the malignant progression of GBC by inhibiting the mRNA of the tumor suppressor gene RGMA, resulting in reactivation of the Akt/ß-catenin signaling pathway.

MiR-552-3p facilitated cell proliferation, migration and invasion by sponging Fibulin 5 in non-small cell lung cancer via activation of ERK/GSK3ß/ß-catenin signaling pathway.

Apart from the fact that miR-552-3p is known to promote cell progression among various cancers, its function on non-small cell lung cancer (NSCLC) is unknown which therefore emerges as the purpose of this research. TargetScan, Starbase, miRWalk, miRDB and the Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) were utilized to analyze the target genes of miR-552-3p. NSCLC cells were transfected with miR-552-3p mimic, miR-552-3p inhibitor, Fibulin 5 (FBLN5) overexpression plasmid, and small interfering FBLN5 (siFBLN5) and treated with extracellular regulated protein kinases (ERK) pathway inhibitor PD98059. MiR-552-3p, FBLN5, p-ERK, ERK, p-glycogen synthase kinase 3ß (GSK3ß) and ß-catenin levels were detected through quantitative reverse transcription-polymerase chain reaction and western blot. The binding sites between miR-552-3p and FBLN5 were predicted by TargetScan, which was tested through dual luciferase reporter analysis. Cell viability, migration and invasion were determined by cell counting kit-8 (CCK-8) assay, wound healing assay and transwell assay, respectively. MiR-552-3p expression was upregulated in NSCLC and FBLN5 functioned as its target. MiR-552-3p mimic promoted proliferation, migration, invasion, p-ERK, p-GSK3ß and ß-catenin expressions in NSCLC cells while miR-552-3p inhibitor did the opposite. Overexpressed FBLN5 suppressed proliferation, migration, invasion, p-ERK, p-GSK3ß and ß-catenin expressions in NSCLC cells whereas siFBLN5 exerted the effects opposite to overexpressed FBLN5. PD98059 enhanced the effect of overexpressed FBLN5 on NSCLC cell migration and invasion while reversing the effect of siFBLN5. MiR-552-3p facilitated cell proliferation, migration and invasion in NSCLC through sponging FBLN5 via activation of ERK/GSK3ß/ß-catenin pathway.