MicroRNA-124 conducts neuroprotective effect via inhibiting AK4/ATF3 after subarachnoid hemorrhage.

Spontaneous subarachnoid hemorrhage (SAH) accounts for approximately 5% of all cases of stroke. SAH is correlated with elevated rates of mortality and disability. Despite significant advancements in comprehending the pathogenesis and surgical management, efficacious clinical interventions remain restricted, and the prognosis is yet to be enhanced. MicroRNAs play a crucial role in various pathological processes in organisms. Revealing these regulatory processes is conducive to the development of new treatment methods. MicroRNA-124 is highly expressed in the nervous system and has significant research value for SAH. This study aims to explore the role of miR-124 in the early post-SAH period on neural function and verify whether it is involved in the pathological and physiological processes of SAH. In this study, we used methods such as comparing the expression levels of miR-124 in cerebrospinal fluid, establishing a rat SAH model, and a mouse embryonic primary neuron hemoglobin stimulation model to verify the downstream proteins of miR-124 in SAH. Through transfection techniques, we adjusted the expression of this small RNA in Vitro and in Vivo models using miR-124 inhibitor and mimic in the primary neuron hemoglobin stimulation model and rat SAH model, and observed the phenotype. Finally, by consulting the literature and verifying in Vivo and in Vitro methods, AK4 and downstream molecule ATF3 were identified as downstream targets of miR-124.

Promoter methylation levels of microRNA-124 in non-neoplastic rectal mucosa as a potential biomarker for ulcerative colitis-associated colorectal cancer in pediatric-onset patients.

PURPOSE: To determine the methylation level of the miR-124 promoter in non-neoplastic rectal mucosa of patients with pediatric-onset ulcerative colitis (UC) to predict UC-associated colorectal cancer (UC-CRC). METHODS: Between 2005 and 2017, non-neoplastic rectal tissue specimens were collected from 86 patients with UC, including 13 patients with UC-CRC; cancer tissues were obtained from the latter group. The methylation status of the miR-124 promoter was quantified using bisulfite pyrosequencing and compared between pediatric- and adult-onset UC patients. RESULTS: Patients with pediatric-onset UC experienced a significantly shorter disease duration than those with adult-onset UC. The levels of miR-124 promoter methylation in non-neoplastic rectal mucosa were positively correlated with the age at the diagnosis and duration of UC. The rate of increase in miR-124 methylation was accelerated in patients with pediatric-onset UC compared to those with adult-onset UC. Furthermore, the miR-124 methylation levels in non-neoplastic rectal mucosa were significantly higher in patients with UC-CRC than in those with UC alone (P = 0.02). A receiver operating characteristic analysis revealed that miR-124 methylation in non-neoplastic tissue discriminated between patients with pediatric-onset UC with or without CRC. CONCLUSION: miR-124 methylation in non-neoplastic rectal mucosa may be a useful biomarker for identifying patients with pediatric-onset UC who face the highest risk of developing UC-CRC.

Multiple knockout mouse and embryonic stem cell models reveal the role of miR-124a in neuronal maturation.

MicroRNA-124a (miR-124a) is one of the most abundantly expressed microRNAs in the central nervous system and is encoded in mammals by the three genomic loci miR-124a-1/2/3; however, its in vivo roles in neuronal development and function remain ambiguous. In the present study, we investigated the effect of miR-124a loss on neuronal differentiation in mice and in embryonic stem (ES) cells. Since miR-124a-3 exhibits only background expression levels in the brain and we were unable to obtain miR-124a-1/2/3 triple knockout (TKO) mice by mating, we generated and analyzed miR-124a-1/2 double knockout (DKO) mice. We found that these DKO mice exhibit perinatal lethality. RNA-seq analysis demonstrated that the expression levels of proneural and neuronal marker genes were almost unchanged between the control and miR-124a-1/2 DKO brains; however, genes related to neuronal synaptic formation and function were enriched among downregulated genes in the miR-124a-1/2 DKO brain. In addition, we found the transcription regulator Tardbp/TDP-43, loss of which leads to defects in neuronal maturation and function, was inactivated in the miR-124a-1/2 DKO brain. Furthermore, Tardbp knockdown suppressed neurite extension in cultured neuronal cells. We also generated miR-124a-1/2/3 TKO ES cells using CRISPR-Cas9 as an alternative to TKO mice. Phase-contrast microscopic, immunocytochemical, and gene expression analyses showed that miR-124a-1/2/3 TKO ES cell lines were able to differentiate into neurons. Collectively, these results suggest that miR-124a plays a role in neuronal maturation rather than neurogenesis in vivo and advance our understanding of the functional roles of microRNAs in central nervous system development.

MicroRNA-124a regulates the differentiation of bone marrow mesenchymal stem cells into neurons.

OBJECTIVE: This study investigated the effects of microRNA-124a on the differentiation of bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanism. METHODS: Flow cytometry was used for isolation and identification of BMSCs. Real-time polymerase chain reaction (RT-PCR) was used to detect gene mRNA expression. Apoptosis was detected using Annexin V-FITC/PI Apoptosis Detection Kit. Cell proliferation ability was tested using Cell Counting Kit-8 (CCK-8). The differentiation of BMSCs into neuron inducers ß-thiol ethanol or baicalin formed the basis of the study. RESULTS: ß-thiol ethanol markedly suppressed the microRNA-124a expression of BMSCs, baicalin markedly induced the microRNA-124a expression of BMSCs and ß-thiol ethanol or baicalin promoted apoptosis and reduced the growth of BMSCs. Only the microRNA-124a inhibitor did not affect apoptosis or the differentiation of BMSCs, and it increased the effects of ß-thiol ethanol or baicalin on the apoptosis of BMSCs. CONCLUSION: ß-thiol ethanol and baicalin treatment could affect microRNA-124a expression in BMSCs. We demonstrated that microRNA-124a promoted the differentiation of BMSCs into neurons.

Downregulation of miR-124-3p suppresses the development of the deep retinal blood vessels by enhancing the Stat1/Ripk1 pathway in mouse retinal microglia.

The study aimed to investigate the role of microRNA (miR)-124-3p in retinal angiogenesis in a mouse model. An intravitreal injection of miR-124-3p antagomir was used to knockdown the expression of miR-124-3p in the mouse retina at postnatal day (P)3. Immunofluorescent staining of both retinal frozen sections and whole retina were used to observe retinal vascular development in the P6, P9 and P12 mice, as well as the changes in retinal ganglion cells, astrocytes, Müller cells and microglia. Whole retinal RNA extracted from P9 mice was used for transcriptome sequencing. Following gene set enrichment analysis, the enriched genes caused by miR-124-3p inhibition were analyzed by immunofluorescent staining and western blot. Results indicated that deep vascular development was significantly inhibited by the activation of M1 phenotype microglia. Moreover, there were no notable effects on superficial retinal vascular development, the retinal ganglion cells, astrocytes, and Müller cells. The expression of the Stat1/Irf9/Eif2ak2/Ripk1 axis in the miR-124-3p knockdown group was significantly increased. The microglia penetrated deep into the retina and the activation of Ripk1(+) microglia significantly increased, which was accompanied by an increased level of apoptosis to inhibit the deep vascular sprout. Downregulation of miR-124-3p during the early retinal development can suppress the development of the deep retinal blood vessels by enhancing the expression level of the Stat1/Irf9/Eif2ak2/Ripk1 axis and inducing the cell apoptosis of the activation of Ripk1(+) microglia.

MicroRNA-124-3p alleviates cerebral ischaemia-induced neuroaxonal damage by enhancing Nrep expression.

OBJECTIVE: Ischaemic stroke has a high death rate and frequently results in long-term and severe brain damage in survivors. miRNA-124-3p (miR-124-3p) treatment has been suggested to reduce ischaemia and play a vital function in avoiding neuron death. An investigation of the role of miR-124-3p, in the ischaemia damage repair or protection in the middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation/reperfusion (OGD/R) model, was the purpose of this research. METHODS: The expression of miRNA and mRNA in the MCAO model was predicted using bioinformatics analysis. The OGD/R neuronal model was developed. We examined the influence of a number of compounds on the OGD/R model in vitro using gain- and loss-of-function approaches. RESULTS: For starters, miR-124-3p and Nrep level in the MCAO model were found to be lower in the model predicted by bioinformatics than in the sham-operated group. And then in the OGD/R model, miR-124-3p treatment reduced OGD/R neuronal damage, increased neuronal survival, and reduced apoptosis in cell lines. Moreover, we further looked at the impact of miR-124-3p on downstream Rnf38 and Nrep using the OGD/R model. Western blot analysis and dual-luciferase reporter assays indicated that miR-124-3p binds and inhibits Rnf38. Finally, although Nrep expression was reduced in the OGD/R model neuronal model, it was shown that miR-124-3p administration reduced apoptosis and increased neuronal activity, particularly with regard to axon regeneration-related proteins. CONCLUSION: Our studies have shown that miR-124-3p may reduce neuronal injury by preventing Rnf38-mediated effects on the Nrep axis.

[Research Progress of microRNA-7/124/155 in Parkinson's Disease].

Parkinson's disease(PD)is the second most common neurodegenerative disease after Alzheimer's disease,with high morbidity and high disability rate.Since the early symptoms of PD are not typical and often similar to those of normal aging or other diseases.It is easy to missed diagnosis and misdiagnosis,which seriously affects the diagnosis and treatment of this disease and aggravetes the burden on the patients' life.MicroRNAs(miRNA)are a class of endogenous non-coding RNAs that are involved in post-transcriptional regulation by binding to target messenger RNAs(mRNA).They are highly conserved,short,easy to obtain,and can stably exist in peripheral body fluids.They have been used as biomarkers for a variety of diseases.Recent studies have demonstrated that miRNA play an important role in the development of PD.This paper reviews the recent research progress of miR-7/124/155,three mature miRNA in PD,aiming to provide reference for clarifying the pathogenesis and guiding the diagnosis and treatment of PD.

MicroRNA-124 ameliorates autophagic dysregulation in glaucoma via regulation of P2X7-mediated Akt/mTOR signaling.

Glaucoma is a neurodegenerative disease that leads to irrevocable blindness. In glaucoma, even though axonal damage and function deficit culminates in retinal ganglion cell (RGC) degeneration, our knowledge on the autophagic mechanisms and the role of specific microRNAs is still limited. In this study, we investigated the role of microRNA-124 (MiR-124) in surgically induced glaucomatous neurodegeneration using a mouse model. Animals were segregated into four cohorts of 10 each: (i) sham-operated (n = 10); (ii) surgically induced glaucoma (SIG; n = 10); (iii) SIG + miR-124 mimic; (iv) SIG + miR-NC. Chronic elevation of intraocular pressure (IOP) is a critical risk factor for glaucoma. In our study, chronically elevated IOP caused anterograde axonal transport (AAT) defect, increased the autophagic activity (manifested by significantly (p < 0.05) increased LC3-II/LC3-I ratio, beclin-1 and Atg7 protein expressions) and also downmodulated the protein expression of p-Akt and p-mTOR, mediated by the purinergic P2 receptor subtype 7 (P2X7) upmodulation-leading to retinal degeneration. However, administration of miR-124 mimic improved the retinal integrity and function, as indicated by the improved AAT function, normalized the autophagic dysfunction, modulated the protein expression of P2X7-mediated p-Akt and p-mTOR. Hence, we propose that development of miR-124-based advanced therapies might be a potential avenue in the treatment of glaucomatous neurodegeneration.

Effect of miR-124a on collagen-induced arthritis in mice and the underlying mechanisms. / MiR-124aå¯¹èƒ¶åŽŸè¯±å¯¼æ€§å ³èŠ‚ç‚Žå°é¼ çš„å½±å“åŠå ¶æœºåˆ¶.

OBJECTIVES: Rheumatoid arthritis (RA) is a chronic autoimmune disease. MicroRNA has been shown to play an important role in RA. MicroRNA-124a (miR-124a) has anti-proliferative and anti-inflammatory effects in RA fibroblast synovial cells. This study aims to explore the effects of miR-124a overexpression on arthritis in collagen-induced arthritis (CIA) mice and the underlying mechanisms. METHODS: Bovine type II collagen and complete Ferris adjuvant were used to induce CIA model from DBA/1 mice. Twenty-eight days after initial immunization (D28), CIA mice were randomly divided into a model group, a miR-124a treatment group, and a negative control (NC) group. Physiological saline, miR-124a agomir, and miR-124a agomir NC were injected into the skin at the tail root of mice every 3 days for 4 times, respectively. The degree of joint swelling and arthritis index of mice were recorded accordingly. Sixty-three days after initial immunization (D63), the mice were sacrificed to obtain the synovial tissue of ankle joint. HE staining was used to observe the proliferation of synovial cell, infiltration of inflammatory cell, pannus, and bone erosion of synovial tissues; TUNEL staining was used to detect cell apoptosis; qRT-PCR was used to detect the mRNA expression of miR-124a, phosphatidylinositol-3-kinase catalytic subunit alpha (PIK3CA) and its downstream genes Bcl-2 and Bax. Immunohistochemistry was used to detect the protein expression of PIK3CA, Bcl-2, and Bax protein in synovial tissues of each group. RESULTS: Different degrees of swelling presented in the paws of DBA/1 mice at D28, which indicated the CIA model was constructed successfully. Forty-eight days after initial immunization (D48), the paws of mice in the miR-124a treatment group were only slightly red and swollen, while the paws of mice in the model group and the NC group were obviously red and swollen. The arthritis index of mice in the miR-124a treatment group were decreased significantly compared to the NC group at D51, D53, D59, and D62 (51, 53, 59, 62 days after initial immunization) (all P<0.05). Sixty-three days after initial immunization (D63), HE staining indicated that the scores of synovial cell proliferation, inflammatory cell infiltration, synovial pannus, and bone erosion were significantly reduced in the miR-124a treatment group (P<0.05 or P<0.01), while cell apoptosis was increased in the miR-124a treatment group compared with the model group and NC group (P<0.01 or P<0.001). Besides, the expression of miR-124a and Bax in the synovial tissue in miR-124a treatment group was significantly higher than those in the model group and NC group (P<0.01 or P<0.001), while the expressions of PIK3CA and Bcl-2 were decreased (P<0.05 or P<0.01 or P<0.001), and the ratio of Bcl-2 to Bax was significantly decreased (P<0.01 or P<0.001). CONCLUSIONS: Overexpression of miR-124a can reduce arthritis in CIA mice bacause it could promote synovial cell apoptosis and inhibit synovial cell proliferation via targeting PIK3CA and regulating its downstream pathways.

Long noncoding RNA MAPKAPK5-AS1 promoted lipopolysaccharide-induced inflammatory damage in the myocardium by sponging microRNA-124-3p/E2F3.

BACKGROUND: Myocardial dysfunction caused by sepsis (SIMD) leads to high mortality in critically ill patients. We investigated the function and mechanism of long non-coding RNA MAPKAPK5-AS1 (lncRNA MAPKAPK-AS1) on lipopolysaccharide (LPS)-induced inflammation response in vivo and in vitro. METHOD: Male SD rats were utilized for in vivo experiments. Rat cardiomyocytes (H9C2) were employed for in vitro experiments. Western blotting was employed to measure protein expression, and RT-PCR was performed to measure mRNA expression of inflammation factors. TUNEL and flow cytometry were carried out to evulate cell apoptosis. RESULT: The results showed that the expression of MAPKAPK5-AS1 was increased, while the expression of miR-124-3p was decreased in the inflammatory damage induced by LPS in vivo and in vitro. Knockdown of MAPKAPK5-AS1 reduced LPS-induced cell apoptosis and inflammation response, while overexpression of miR-124-3p weakened the effects of MAPKAPK5-AS1 knockdown on LPS-induced cell apoptosis and inflammation response. Moreover, miR-124-3p was identified as a downstream miRNA of MAPKAPK5-AS1, and E2F3 was a target of miR-214-3p. MAPKAPK5-AS1 knockdown increased the expression of miR-124-3p, while miR-124-3p overexpression reduced the expression of MAPKAPK5-AS1. In addition, miR-124-3p was found to downregulate E2F3 expression in H9C2 cells. CONCLUSION: MAPKAPK5-AS1/miR-124-3p/E2F3 axis regulates LPS-related H9C2 cell apoptosis and inflammatory response.

A paclitaxel and microRNA-124 coloaded stepped cleavable nanosystem against triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is one of the most biologically aggressive breast cancers and lacks effective treatment options, resulting in a poor prognosis. Therefore, studies aiming to explore new therapeutic strategies for advanced TNBC are urgently needed. According to recent studies, microRNA-124 (miR124) not only inhibits tumour growth but also increases the sensitivity of TNBC to paclitaxel (PTX), suggesting that a platform combining PTX and miR124 may be an advanced solution for TNBC. RESULTS: Herein, we constructed a stepped cleavable calcium phosphate composite lipid nanosystem (CaP/LNS) to codeliver PTX and miR124 (PTX/miR124-NP). PTX/miR124-NP exhibited superior tumor microenvironment responsive ability, in which the surface PEG layer was shed in the mildly acidic environment of tumor tissues and exposed oligomeric hyaluronic acid (o-HA) facilitated the cellular uptake of CaP/LNS by targeting the CD44 receptor on the surface of tumor cells. Inside tumour cells, o-HA detached from CaP/LNS due to the reduction of disulfide bonds by glutathione (GSH) and inhibited tumour metastasis. Then, PTX and miR124 were sequentially released from CaP/LNS and exerted synergistic antitumour effects by reversing the Epithelial-Mesenchymal Transition (EMT) process in MDA-MB-231 cells. Moreover, PTX/miR124-NP showed significant antitumour efficiency and excellent safety in mice bearing MDA-MB-231 tumours. CONCLUSION: Based on these results, the codelivery of PTX and miR124 by the CaP/LNS nanosystem might be a promising therapeutic strategy for TNBC.

MicroRNA-124 regulates lens epithelial cell apoptosis by affecting Fas alternative splicing by targeting polypyrimidine tract-binding protein in age-related cataract.

BACKGROUND: Age-related cataract (ARC) is a primary cause of visual blindness worldwide. Lens epithelial cell (LEC) apoptosis, in which Fas plays an essential role, is a vital cytological basis for cataractogenesis. However, the regulatory mechanism of Fas-dependent LEC apoptosis is not well understood. This study aimed to investigate whether MicroRNA (miRNA)-124 can regulate LEC apoptosis by targeting polypyrimidine tract-binding protein (PTB) and thereby affecting Fas alternative splicing in ARC. METHODS: Lens capsule samples from patients with ARC and cornea donors with a transparent lens were collected. HLE-B3 cells were cultured and treated with hydrogen peroxide (H2 O2 ) to establish an apoptosis model in LECs. The expression of miRNA-124, PTB, Fas, and Fas isoforms in tissues and cell lines was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, polyacrylamide gel electrophoresis, and flow cytometry. The miRNA-124 mimic and inhibitor were transfected into HLE-B3 cells, and the effects of the miRNA-124/PTB/Fas pathway in LECs were assessed by analysis of their related targets. RESULTS: High expression of miRNA-124 and membrane Fas (mFas) mRNA and decreased PTB expression were observed in the lens capsule samples. In cells undergoing H2 O2 -induced apoptosis, mFas expression was increased, accompanied by decreased PTB and increased miRNA-124 expression. Subsequently, miRNA-124 upregulation suppressed PTB expression, elevated the mFas level without affecting total Fas expression and promoted apoptosis; miRNA-124 downregulation exerted the opposite effects. CONCLUSION: This study revealed that miRNA-124 promotes LEC apoptosis in ARC by upregulating mFas through targeted inhibition of PTB. Moreover, the identification of the miRNA-124/PTB/Fas pathway provides novel insight into Fas-dependent LEC apoptosis.

Tumor Suppressive Effects of miR-124 and Its Function in Neuronal Development.

MicroRNA-124 (miR-124) is strongly expressed in neurons, and its expression increases as neurons mature. Through DNA methylation in the miR-124 promoter region and adsorption of miR-124 by non-coding RNAs, miR-124 expression is known to be reduced in many cancer cells, especially with high malignancy. Recently, numerous studies have focused on miR-124 due to its promising tumor-suppressive effects; however, the overview of their results is unclear. We surveyed the tumor-suppressive effect of miR-124 in glial cell lineage cancers, which are the most frequently reported cancer types involving miR-124, and in lung, colon, liver, stomach, and breast cancers, which are the top five causes of cancer death. Reportedly, miR-124 not only inhibits proliferation and accelerates apoptosis, but also comprehensively suppresses tumor malignant transformation. Moreover, we found that miR-124 exerts its anti-tumor effects by regulating a wide range of target genes, most notably STAT3 and EZH2. In addition, when compared to the original role of miR-124 in neuronal development, we found that the miR-124 target genes that contribute to neuronal maturation share similarities with genes that cause cancer cell metastasis and epithelial-mesenchymal transition. We believe that the two apparently unrelated fields, cancer and neuronal development, can bring new discoveries to each other through the study of miR-124.

microRNA-124 inhibits stem-like properties and enhances radiosensitivity in nasopharyngeal carcinoma cells via direct repression of expression of JAMA.

Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA-124 (miR-124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR-124 in NPC CSCs. qRT-PCR was performed to measure miR-124 expression in NPC tissues and cell lines and the effects of miR-124 on stem-like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR-124 with the 3'UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR-124 in an animal model and clinical samples. Down-regulation of miR-124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR-124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up-regulation of miR-124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR-124 can inhibit stem-like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.

CX3CL1/CX3CR1 axis attenuates early brain injury via promoting the delivery of exosomal microRNA-124 from neuron to microglia after subarachnoid hemorrhage.

BACKGROUND: Microglial activation-mediated neuroinflammation is a major contributor to early brain injury (EBI) after subarachnoid hemorrhage (SAH). MicroRNA-124 (miR-124) is the most abundant miRNAs in the central nervous system (CNS) and plays a vital role in microglial activation by targeting protein CCAAT-enhancer-binding protein α (C/EBPα). It has been reported that the CX3CL1/CX3CR1 axis is involved in the delivery of miR-124 from neurons to microglia. METHODS: An experimental rat SAH model was established by injecting autologous arterial blood into the prechiasmatic cistern, and cultured primary neurons and microglia were exposed to oxyhemoglobin to mimic SAH in vitro. We additionally exploited specific expression plasmids encoding CX3CL1 and CX3CR1. RESULTS: We observed significant decreases in CX3CL1 and CX3CR1 in the brain tissues of SAH patients. We also observed decreases in the levels of CX3CL1 in neurons and CX3CR1 in microglia after SAH in rats. Moreover, microglia exhibited an activated phenotype with macrophage-like morphology and high levels of CD45 and major histocompatibility complex (MHC) class II after SAH. After overexpression of CX3CL1/CX3CR1, the level of CD45 and MHC class II and the release of inflammatory factors tumor necrosis factor α, interleukin 1α and complement 1q were significantly decreased. There was also increased neuronal degeneration and behavior dysfunction after SAH, both of which were inhibited by CX3CL1/CX3CR1 overexpression. Additionally, we found that the delivery of exosomal miR-124 from neurons to microglia was significantly reduced after SAH, accompanied by an increase in C/EBPα expression, and was inhibited by CX3CL1/CX3CR1 overexpression. In conclusion, the CX3CL1/CX3CR1 axis may play protective roles after SAH by promoting the delivery of exosomal miR-124 to microglia and attenuate microglial activation and neuroinflammation. CONCLUSIONS: CX3CL1/CX3CR1 axis may be a potential intervention target for the inhibition of SAH-induced EBI by promoting exosome transport of miR-124 to microglia.

Midazolam inhibits proliferation and accelerates apoptosis of hepatocellular carcinoma cells by elevating microRNA-124-3p and suppressing PIM-1.

OBJECTIVE: Recently, the impact of microRNAs (miRNAs) has been identified in hepatocellular carcinoma (HCC), this study was designed to assess the effects of miR-124-3p and midazolam (MDZ) in HCC with the involvement of PIM-1. METHODS: HepG2 human HCC cells were selected for our study, which were treated with different concentrations of MDZ. The gain- and loss-of-function experiments were performed to elucidate the migration, invasion, proliferation, colony formation ability, cell cycle, and apoptosis of HepG2 cells upon treatment of MDZ, miR-124-3p mimics, or miR-124-3p inhibitor. The expression levels of miR-124-3p, PIM-1, Bax, Bcl-2, P21, and Ki-67 in HepG2 cells were assessed by reverse transcription quantitative polymerase chain reaction and western blot analysis. Moreover, HepG2 cell growth in vivo was measured by subcutaneous tumorigenesis in nude mice, and the target relation between miR-124-3p and PIM-1 was evaluated using dual luciferase reporter gene assay. RESULTS: We have found that after treated with overexpression of miR-124-3p and MDZ, there exhibited elevated miR-124-3p, declined expression of PIM-1, attenuated migration, invasion, proliferation and colony formation ability, and promoted apoptosis of HepG2 cells. Additionally, it could be observed that the tumor volume and weight were all reduced upon treatment of overexpression of miR-124-3p and MDZ. Meanwhile, the results in the HepG2 cells that treated with down-regulated miR-124-3p were the opposite. Furthermore, PIM-1 was found to be a target gene of miR-124-3p. CONCLUSION: Our study found that MDZ could inhibit proliferation and accelerate apoptosis of HCC cells by elevation of miR-124-3p and suppressing PIM-1, which may be an effective method in the treatment of HCC.

Downregulation of microRNA-124 prevents the development of acute liver failure through the upregulation of PIM-3.

NEW FINDINGS: • What is the central question of this study? Does miR-124 affect cell proliferation and apoptosis in acute liver failure (ALF) mice? • What is the main finding and its importance? Inhibiting miR-124 targets PIM-3 and thus upregulates its expression, consequently inhibiting liver cell apoptosis and promoting cell proliferation, ultimately preventing the progression of ALF. This highlights a promising competitive new target for ALF treatment. ABSTRACT: Acute liver failure (ALF) is a complicated syndrome frequently leading to dysfunction and failure of various organs. MicroRNAs (miRNAs) have played crucial roles in the development and progression of human diseases, including ALF. However, the potential role of miR-124 in ALF still remains elusive. Thus, we investigated the underlying mechanism by which miR-124 influences ALF in a mouse model of ALF. Initially, ALF mouse models were established using d-galactosamine and lipopolysaccharide. Then we detected the serum biochemical parameters of liver, and pathological characteristics and ultrastructure of liver tissues. Next, we determined miR-124 and PIM-3 expression in liver tissues and cells using RT-qPCR and western blot analysis. The interaction between miR-124 and PIM-3 was identified using the dual luciferase reporter gene assay. Subsequently, expression of miR-124 and PIM-3 in liver cells was altered to explore their effects on primary liver cell proliferation, the cell cycle and apoptosis. The results obtained showed that ALF mice exhibited a decreased cholinesterase level with increased levels of alanine aminotransferase, aspartate transaminase and total bilirubin as well as abundant liver cell apoptosis and necrosis. miR-124 was upregulated while PIM-3 was downregulated in ALF tissues and cells. Besides, the PIM-3 gene was a target of miR-124 and was inhibited by miR-124. Overexpression of miR-124 or silencing of PIM-3 reduced Bcl-2 expression but elevated tumour necrosis factor α expression, and resulted in a reduction in liver cell proliferation but an increase in cell apoptosis in ALF mice. Altogether, miR-124 functions as a disease-promoting miRNA with potential in stimulating ALF by targeting PIM-3.

MicroRNA-124a contributes to glucocorticoid resistance in acute-on-chronic liver failure by negatively regulating glucocorticoid receptor alpha.

INTRODUCTION AND OBJECTIVES: Glucocorticoid resistance frequently associating with inflammation, may severely compromise the therapeutic effect of glucocorticoids. In this study, we aimed to investigate the regulation of glucocorticoid resistance by microRNA-124a (miR-124a) in patients with acute-on-chronic liver failure (ACLF). MATERIALS AND METHODS: The miR-124a levels and glucocorticoid receptor alpha (GRα) expressions in peripheral blood monocytes and liver tissues were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and western blot analyses in the following four groups: healthy controls (HC), moderate chronic hepatitis B (CHB) patients, hepatitis B virus-related ACLF (HBV-ACLF) patients, and alcohol-induced ACLF (A-ACLF) patients. In addition, the serum miR-124a levels and multiple biochemical indices were determined. The effects of miR-124a transfection on GRα expression were assayed by qRT-PCR and western blotting in U937 and HepG2 cells stimulated with lipopolysaccharide (LPS). RESULTS: Compared with the CHB patients and HC, the miR-124a levels in HBV-ACLF and A-ACLF patients increased, while GRα expressions decreased. No significant differences in miR-124a levels and GRα expressions were observed between the HBV-ACLF and A-ACLF patients. For the ACLF patients, miR-124a level was negatively related to GRα expression in monocytes and positively correlated with the inflammatory factors such as interleukin-1 beta (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). In U937 and HepG2 cells, LPS stimulated miR-124a levels but inhibited GRα expressions; meanwhile, increasing miR-124a levels reduced GRα expressions, and inhibiting miR-124a levels increased GRα expressions. CONCLUSIONS: This study provides evidence that GRα expression was negatively regulated by miR-124a, which primarily determines the extent of acquired glucocorticoid resistance in ACLF.

Evaluation of MicroRNA-124 Expression in Renal Cell Carcinoma.

The Wingless/INT (WNT) signaling network has roles in renal cancer development. It was shown that the tumor-suppressor microRNA-124 (miR-124) is associated with the Wnt pathway. Thus, we aimed to measure miR-124 expression levels to evaluate whether it is a prognostic marker or a potential treatment strategy. Thirty tumor and 30 surrounding healthy kidney tissues from the same subjects diagnosed with renal cell carcinoma (RCC), were included in the study. The expression levels of miR-124 were measured with real-time polymerase chain reaction (qPCR) and determined by the 2-ΔΔCT method. The Statistical Package for Social Science (SPSS) version 22 program was used for statistical analyses and a p value of 0.05 was considered to be statistically significant. The expression levels of miR-124 was found to be about 3-fold lower in tumors than in healthy tissues (p 0.001) and decreased expression levels correlated with tumor stage, tumor diameter, body mass index (BMI) and neutrophil values (p 0.05). Our results showed that miR-124 expression levels are associated with RCC. MicroRNA-124 may be assessed as a biomarker in prognosis and the restoration of miR-124 expression might be effective in the treatment of RCC.

microRNA-124 attenuates isoflurane-induced neurological deficits in neonatal rats via binding to EGR1.

Isoflurane anesthesia induces neuroapoptosis in the development of the brain. In this study, neonatal rats and hippocampal neurons were subjected to isoflurane exposure, in which the effect of miR-124 on the neurological deficits induced by isoflurane was evaluated. Isoflurane anesthesia models were induced in neonatal SD rats aged 7 days and then treated with miR-124 agomir, miR-124 antagomir, or LV-CMV-early growth response 1 (EGR1) plasmids. Then, the spatial learning and memory ability of rats were evaluated by Morris water maze. Furthermore, primary hippocampal neurons cultured 7 days were also exposed to isoflurane and transfected with miR-124 agomir, miR-124 antagomir, or LV-CMV-EGR1 plasmids. The targeting relationship of miR-124 and EGR1 was verified by the dual-luciferase reporter gene assay. To identify the effect of miR-124 on neuron activities, the viability and apoptosis of hippocampal neurons were assessed. In response to isoflurane exposure, miR-124 expression was reduced and EGR1 expression was increased in the hippocampal tissues and neurons. The isoflurane anesthesia damaged rats' spatial learning and memory ability, and reduced viability, and promoted apoptosis of hippocampal neurons. EGR1 was targeted and negatively regulated by miR-124. The treatment of miR-124 agomir improved rats' spatial learning and memory ability and notably increased hippocampal neuron viability and resistance to apoptosis, corresponding to an increased brain-derived neurotrophic factor (BDNF) expression, inhibited expression of proapoptotic factors (cleaved-Caspase-3 and Bax), and enhanced the expression of antiapoptotic factor (Bcl-2). Upregulated miR-124 inhibited the expression of EGR1, by which mechanism miR-124 reduced the neurological deficits induced by isoflurane in neonatal rats through inhibiting apoptosis of hippocampal neurons.