Hsa_circ_0004776 regulates the retina neovascularization in progression of diabetic retinopathy via hsa-miR-382-5p/BDNF axis.

The aim of this work was to identify the regulatory function of hsa_circ_0004776 in the progression of diabetic retinopathy (DR). The direct interactions between hsa_circ_0004776 and hsa-miR-382-5p and between hsa-miR-382-5p and BDNF, were confirmed via dual-luciferase reporter assays. Quantitative Real-Time PCR analysis indicated that hsa_circ_0004776 was highly expressed in aqueous humour samples of DR patients and human retinal microvascular epithelial cells (hRECs) under a high-glucose environment, whereas hsa-miR-382-5p showed the opposite trend. Overexpressed hsa_circ_0004776 significantly enhanced DNA synthesis, proliferation, migration, and tube formation in hRECs in hyperglycaemia, while hsa-miR-382-5p mimics reversed these changes. Additionally, in a streptozotocin-induced Sprague-Dawley rat model of DR, vitreous microinjection of rno-miR-382-5p agomir reversed the pathologic features in the progression of DR, including retinal vascular leakage, capillary decellularization, loss of pericytes, fibrosis, and gliosis. Our results indicated that under hyperglycaemic conditions, hsa_circ_0004776 influences the progression of DR via hsa-miR-382-5p and thus represents a potential therapeutic target.

Hsa_circ_0010882 facilitates hepatocellular carcinoma progression by modulating M1/M2 macrophage polarization.

Hepatocellular carcinoma (HCC) is one common malignant tumour with a high immunosuppressive tumour microenvironment and poor outcomes. This study investigated the influence of hsa_circ_0010882 on M1/M2 macrophage polarization in the progression of HCC. A total of 125 paired tissue specimens from HCC patients who underwent hepatectomy were collected. M1 and M2 phenotypes macrophages were induced using THP-1. After co-cultured with macrophages and transfected HCC cells, the viability, migration and invasion of HCC cells were detected by cellular experiments. Bioinformatic databases and dual-luciferase reporter assays were used to predict and validate the interaction between circ_0010882 and miR-382. Expression of circ_0010882 was increased in HCC tissues and associated with shorter overall survival outcomes. The mRNA expression of M2 macrophage markers Arg-1, CD163 and CD206 were elevated in HCC tissues. Interfering with circ_0010882 increased M1-type macrophage markers (TNF-α and iNOS) while decreasing M2-type macrophage markers (Arg-1 and CD206). Silencing of circ_0010882 strengthened the capacity of M1 macrophages to suppress HCC cell viability, migration capacities and invasion potential while reducing the ability of M2 macrophages to promote above cellular abilities. MiR-382 was a direct target miRNA of circ_0010882. The circ_0010882 expression was increased in HCC tissues and associated with poor prognosis of HCC patients. Silencing of circ_0010882 inhibits macrophage M2 polarization in HCC progression by regulating miR-382 expression. Circ_0010882 may serve as a biomarker to provide novel strategies for the treatment of HCC and patient rehabilitation, thereby improving the prognosis of patients.

Circ_0006640 transferred by bone marrow-mesenchymal stem cell-exosomes suppresses lipopolysaccharide-induced apoptotic, inflammatory and oxidative injury in spinal cord injury.

BACKGROUND: Emerging proofs have shown that differentially expressed circular RNAs (circRNAs) are closely associated with the pathophysiological process of spinal cord injury (SCI). Mesenchymal stem cell (MSC)-exosomes have been demonstrated to possess favorable therapeutic effects in diseases. Herein, this work aimed to investigate the action of circ_0006640 transferred by MSC-exosomes functional recovery after SCI. METHODS: SCI animal models were established by spinal cord contusion surgery in mice and lipopolysaccharide (LPS)-stimulated mouse microglial cell line BV2. Levels of genes and proteins were detected by qRT-PCR and Western blot. Properties of BV2 cells were characterized using CCK-8 assay, flow cytometry and ELISA analysis. The oxidative stress was evaluated. Dual-luciferase reporter assay was used for verifying the binding between miR-382-5p and circ_0006640 or IGF-1 (Insulin-like Growth Factor 1). Exosome separation was conducted by using the commercial kit. RESULTS: Circ_0006640 expression was lower in SCI mice and LPS-induced microglial cells. Circ_0006640 overexpression protected microglial cells from LPS-induced apoptotic, inflammatory and oxidative injury. Mechanistically, circ_0006640 directly sponged miR-382-5p, which targeted IGF-1. MiR-382-5p was increased, while IGF-1 was decreased in SCI mice and LPS-induced microglial cells. Knockdown of miR-382-5p suppressed apoptosis, inflammation and oxidative stress in LPS-induced microglial cells, which were reversed by IGF-1 deficiency. Moreover, miR-382-5p up-regulation abolished the protective functions of circ_0006640 in LPS-induced microglial cells. Additionally, circ_0006640 was packaged into MSC-exosomes and could be transferred by exosomes. Exosomal circ_0006640 also had protective effects on microglial cells via miR-382-5p/IGF-1 axis. CONCLUSION: Circ_0006640 transferred by BMSC-exosomes suppressed LPS-induced apoptotic, inflammatory and oxidative injury via miR-382-5p/IGF-1 axis, indicating a new insight into the clinical application of exosomal circRNA-based therapeutic in the function recovery after SCI.

LncRNA PSMB8-AS1 increases glioma malignancy via the miR-382-3p/BCAT1 axis.

BACKGROUND: This study aimed to investigate the specific roles of the long non-coding RNA (lncRNA) proteasome 20S subunit beta 8 (PSMB8)-antisense RNA 1 (AS1)/microRNA (miR)-382-3p/branched-chain amino acid transaminase 1 (BCAT1) interaction network in gliomas. METHODS: Western blotting and quantitative reverse transcription-polymerase chain reaction were performed to assess the expression levels of lncRNA PSMB8-AS1, BCAT1, and miR-382-3p. Moreover, the cell proliferation, migration, and apoptosis were assessed using the cell counting kit-8, Transwell, and caspase-3 activity assays, respectively. The biological role of lncRNA PSMB8-AS1 in glioma was investigated in vivo using a xenograft mouse model. Additionally, the associations among lncRNA PSMB8-AS1, miR-382-3p, and BCAT1 were analyzed using dual-luciferase and RNA immunoprecipitation assays and bioinformatics analyses. RESULTS: Glioma cell lines and tissues exhibited overexpression of lncRNA PSMB8-AS1 and BCAT1 and low expression of miR-382-3p. Knockdown of PSMB8-AS1 remarkably repressed the tumor growth in vivo and the migration and proliferation of glioma cells in vitro. In contrast, knockdown of lncRNA PSMB8-AS1 increased the cell apoptosis. Mechanistically, PSMB8-AS1 directly targeted miR-382-3p. By sponging miR-382-3p, lncRNA PSMB8-AS1 stimulated the migration and proliferation of glioma cells and suppressed their apoptosis. Additionally, miR-382-3p directly targeted BCAT1. Inhibition of miR-382-3p reversed the antitumor effects of BCAT1 silencing on glioma progression. CONCLUSION: Our study revealed that lncRNA PSMB8-AS1 aggravated glioma malignancy by enhancing BCAT1 expression after competitively binding to miR-382-3p. Therefore, lncRNA PSMB8-AS1 may be a potential biomarker and therapeutic target for glioma treatment.

The Role of circRNA-miRNA-mRNA Regulatory Network and its Potential Biomarker Function in Colorectal Cancer.

BACKGROUND: Revealing the process and mechanism of colorectal cancer will facilitate the discovery of new biomarkers and contribute to the development of targeted drugs. OBJECTIVES: This study aimed to explore the potentially functional circRNA-miRNA-mRNA network in colorectal cancer (CRC), and further explore its mechanism. METHODS: Bioinformatics analysis was used to identify the differentially expressed circRNAs and mRNAs. Gene set enrichment analysis and KEGG pathways analysis were used to screen out the differentially expressed genes and observe crucial pathways that might have a strong association with CRC. Then, a network targeting circRNA, miRNA, and mRNA has been built by using the Cytoscape software. In addition, the expression of circRNA_0001573, miR-382-5p, and FZD3 was detected by qRT-PCR in CRC tissues and cells (SW480, HCT116, and HT29). RESULTS: Abnormal expressions of circRNAs and mRNAs were obtained by bioinformatics analysis and visualized by Volcano plot and Heatmap. A series of highly correlated pathways were enriched by KEGG analysis. The interaction network of circRNA_0001573/miR-382-5p/FZD3 axis was predicted. The expressions of circRNA_0001573 and FZD3 were highly upregulated and the miR- 382-5p expression level was decreased in CRC tissues and cell lines (SW480, HCT116, and HT29). CONCLUSION: Our study suggests that circRNA_0001573 and circRNA_0001573/miR-382-5p/FZD3 regulatory networks might provide a potential diagnosis for colorectal cancer.

Circ_0005015 upregulates BACH1 to promote aggressive behaviors in glioblastoma by sponging microRNA-382-5p.

To investigate the potential role and molecular mechanism of circ_0005015 in GBM progression. Circ_0005015, microRNA-382-5p (miR-382-5p), and BTB domain and CNC homolog 1 (BACH1) levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was determined by MTT, colony formation, and EdU assays. Cell apoptosis was analyzed using flow cytometry. Cell migration and invasion were assessed using wound healing and transwell assays. Glucose accumulation and lactate levels were examined by the corresponding kit. RNA pull-down and dual-luciferase reporter assays were performed to confirm the interaction between miR-382-5p and circ_0005015 or BACH1. Protein levels of MMP9, PCNA, and BACH1 were examined using western blot assay. Role of circ_0005015 on tumor growth in vivo was analyzed using a xenograft tumor model. Circ_0005015 content was up-regulated in GBM patients and cells, its knockdown restrained GBM cell proliferation, migration, invasion, glycolysis, and triggered apoptosis. Mechanistically, we found that circ_0005015 could directly interact with miR-382-5p and serve as a miRNA sponge to regulate BACH1 expression. In addition, circ_0005015 knockdown might repress tumor growth in vivo. Circ_0005015 boosted GBM progression via binding to miR-382-5p to up-regulate BACH1, which may offer new effective targets for GBM treatment.

Aerobic exercise improves cognitive impairment in mice with type 2 diabetes by regulating the MALAT1/miR-382-3p/BDNF signaling pathway in serum-exosomes.

BACKGROUND: It has been documented that aerobic exercise (AE) has a positive effect on improving cognitive function in type 2 diabetes (T2DM) patients. Here, we tried to explore how AE regulates the expression of long non-coding RNA in serum-exosomes (Exos), thereby affecting cognitive impairment in T2DM mice as well as its potential molecular mechanism. METHODS: T2DM mouse models were constructed, and serum-Exos were isolated for whole transcriptome sequencing to screen differentially expressed lncRNA and mRNA, followed by prediction of downstream target genes. The binding ability of miR-382-3p with a long non-coding RNA MALAT1 and brain-derived neurotrophic factor (BDNF) was explored. Then, primary mouse hippocampal neurons were collected for in vitro mechanism verification, as evidenced by the detection of hippocampal neurons' vitality, proliferation, and apoptosis capabilities, and insulin resistance. Finally, in vivo mechanism verification was performed to assess the effect of AE on insulin resistance and cognitive disorder. RESULTS: Transcriptome sequencing analysis showed that MALAT1 was lowly expressed and miR-382-3p was highly expressed in serum-Exos samples of T2DM mice. There were targeted binding sites between MALAT1 and miR-382-3p and between miR-382-3p and BDNF. In vitro experiments showed that MALAT1 upregulated BDNF expression by inhibiting miR-382-3p. Silencing MALAT1 or overexpressing miR-382-3p could reduce the expression of INSR, IRS-1, IRS-2, PI3K/AKT, and Ras/MAPK, inhibit neuronal proliferation, and promote apoptosis. In vivo experiments further confirmed that AE could increase the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thereby improving cognitive impairment in T2DM mice. CONCLUSION: AE may upregulate the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thus improving cognitive impairment in T2DM mice.

Overexpressed microRNA (miR)-382-3p promoted vascular remodeling via suppressing autophagy-related protein 7 (ATG7) in chronic thromboembolic pulmonary hypertension.

BACKGROUND: This research has investigated the role of miR-382-3p in chronic thromboembolic pulmonary hypertension (CTEPH). METHODS: Human pulmonary artery smooth muscle cells (hPASMCs) were treated with PDGF-BB to induce proliferation, and then transfected with miR-382-3p mimic, miR-382-3p inhibitor, ATG7 overexpression plasmid, and siATG7. MiR-382-3p, ATG7, VEGF, PCNA, p62, and LC3-Ⅱ/LC3-I levels were detected by qRT-PCR and Western blotting. Cell viability and migration were tested through CCK-8 and wound healing assays, respectively. Target genes of miR-382-3p were predicted by Targetscan and starBase, and pathway analysis was implemented through WebGestalt. The binding relationship between miR-382-3p and ATG7 was analyzed by the dual-luciferase reporter and RIP assays. A CTEPH model was constructed in rats with the treatment of miR-382-3p antagomir or agomir, and mean pulmonary artery pressure (mPAP) was measured. Lung tissue was observed through the HE staining assay. RESULTS: MiR-382-3p level in hPASMCs was obviously upregulated with the increasing dose of PDGF-BB. MiR-382-3p mimic promoted yet miR-382-3p inhibitor suppressed hPASMC proliferation. MiR-382-3p directly targeted ATG7. ATG7 overexpression repressed hPASMC proliferation and migration, whereas siATG7 exerted the opposite effects. ATG7 overexpression partly neutralized the effects of miR-382-3p mimic on proliferation, migration, and autophagy-related proteins (ATG7, p62, and LC3-Ⅱ/LC3-I) in hPASMCs, whereas siATG7 partly offset the impacts of miR-382-3p inhibitor. MiR-382-3p antagomir reversed CTEPH-induced mPAP elevation, miR-382-3p upregulation, thickening of the pulmonary artery wall, and increased expressions of VEGF, PCNA, and autophagy-related proteins in rats, while miR-382-3p agomir potentiated these effects induced by CTEPH. CONCLUSION: Overexpressed miR-382-3p promotes vascular remodeling via ATG7 inhibition in CTEPH.

microRNA-382 as a tumor suppressor? Roles in tumorigenesis and clinical significance.

MicroRNAs (miRNAs) are small single-stranded RNAs belonging to a class of non-coding RNAs with an average length of 18-22 nucleotides. Although not able to encode any protein, miRNAs are vastly studied and found to play role in various human physiologic as well as pathological conditions. A huge number of miRNAs have been identified in human cells whose expression is straightly regulated with crucial biological functions, while this number is constantly increasing. miRNAs are particularly studied in cancers, where they either can act with oncogenic function (oncomiRs) or tumor-suppressors role (referred as tumor-suppressor/oncorepressor miRNAs). miR-382 is a well-studied miRNA, which is revealed to play regulatory roles in physiological processes like osteogenic differentiation, hematopoietic stem cell differentiation and normal hematopoiesis, and liver progenitor cell differentiation. Notably, miR-382 deregulation is reported in pathologic conditions, such as renal fibrosis, muscular dystrophies, Rett syndrome, epidural fibrosis, atrial fibrillation, amelogenesis imperfecta, oxidative stress, human immunodeficiency virus (HIV) replication, and various types of cancers. The majority of oncogenesis studies have claimed miR-382 downregulation in cancers and suppressor impact on malignant phenotype of cancer cells in vitro and in vivo, while a few studies suggest opposite findings. Given the putative role of this miRNA in regulation of oncogenesis, assessment of miR-382 expression is suggested in a several clinical investigations as a prognostic/diagnostic biomarker for cancer patients. In this review, we have an overview to recent studies evaluated the role of miR-382 in oncogenesis as well as its clinical potential.

Bone marrow mesenchymal stem cell-derived exosomal microRNA-382 promotes osteogenesis in osteoblast via regulation of SLIT2.

BACKGROUND: Osteoporosis (OP) is a systemic skeletal disorder with increased bone fragility. Human bone marrow mesenchymal stem cells (hBMSCs) have multi-lineage differentiation ability, which may play important roles in osteoporosis. In this study, we aim to investigate the role of hBMSC-derived miR-382 in osteogenic differentiation. METHODS: The miRNA and mRNA expressions in peripheral blood monocytes between persons with high or low bone mineral density (BMD) were compared. Then we collected the hBMSC-secreted sEV and examined the dominant components. The over-expression of the miR-382 in MG63 cell and its progression of osteogenic differentiation were investigated by qRT-PCR, western blot and alizarin red staining. The interaction between miR-382 and SLIT2 was confirmed by dual-luciferase assay. The role of SLIT2 was also confirmed through up-regulation in MG63 cell, and the osteogenic differentiation-associated gene and protein were tested. RESULTS: According to bioinformatic analysis, a series of differential expressed genes between persons with high or low BMD were compared. After internalization of hBMSC-sEV in MG63 cells, we observed that the ability of osteogenic differentiation was significantly enhanced. Similarly, after up-regulation of miR-382 in MG63 cells, osteogenic differentiation was also promoted. According to the dual-luciferase assay, the targeting function of miR-382 in SLIT2 was demonstrated. Moreover, the benefits of hBMSC-sEV in osteogenesis were abrogated through up-regulation of SLIT2. CONCLUSION: Our study provided evidence that miR-382-contained hBMSC-sEV held great promise in osteogenic differentiation in MG63 cells after internalization by targeting SLIT2, which can be served as molecular targets to develop effective therapy.

The diagnostic significance of circulating miRNAs and metabolite profiling in early prediction of breast cancer in Egyptian women.

OBJECTIVE: Breast cancer (BC) is one of the most commonly diagnosed solid malignancies in women worldwide. PURPOSE: Finding new non-invasive circulating diagnostic biomarkers will facilitate the early prediction of BC and provide valuable insight into disease progression and response to therapy using a safe and more accessible approach available every inspection time. Therefore, our present study aimed to investigate expression patterns of potentially circulating biomarkers that can differentiate well between benign, malignant, and healthy subjects. METHODS: To achieve our target, quantitative analyses were performed for some circulating biomarkers which have a role in the proliferation and tumor growth, as well as, glutamic acid, and human epidermal growth receptor 2 (HER2) in blood samples of BC patients in comparison to healthy controls using qRT-PCR, liquid chromatography/mass spectrometry (LC/MS/MS), and ELISA. RESULTS: Our findings showed that the two miRNAs (miRNA-145, miRNA-382) were expressed at lower levels in BC sera than healthy control group, while miRNA-21 was expressed at higher levels in BC patients than control subjects. Area under ROC curves of BC samples revealed that AUC of miRNA-145, miRNA-382, miRNA-21, and glutamic acid was evaluated to equal 0.99, 1.00, 1.00 and 1.00, respectively. Besides, there was a significantly positive correlation between miRNA-145 and miRNA-382 (r = 0.737), and a highly significant positive correlation between miRNA-21 and glutamic acid (r = 0.385). CONCLUSION: Based on our results, we conclude that the detection of serum miRNA-145, -382 and -21 as a panel along with glutamic acid, and circulating HER2 concentrations could be useful as a non-invasive diagnostic profiling for early prediction of breast cancer in Egyptian patients. It can provide an insight into disease progression, discriminate between malignancy and healthy control, and overcome the use limitations (low sensitivity and specificity, repeated risky exposure, and high cost) of other detecting tools, including mammography, magnetic resonance imaging, and ultrasound.

miRNA-382-5p Carried by Extracellular Vesicles in Osteoarthritis Reduces Cell Viability and Proliferation, and Promotes Cell Apoptosis by Targeting PTEN.

The objective of the study was to identify extracellular vesicle (EV) microRNAs (miRNAs) that play important roles in knee osteoarthritis (OA). Models of knee OA were surgically induced in nine male Sprague-Dawley rats. Tissue samples were collected at 0 weeks (Control), 6 weeks (6 weeks), and 12 weeks (12 weeks). The EVs were isolated and analyzed for size. Various biomarkers, including recombinant tetraspanin 30 cluster of differentiation (CD)63 and CD9 were detected. An Agilent array was used to screen for differentially expressed (DE) miRNAs. The levels of DE miRNAs and their target mRNAs were evaluated by quantitative reverse transcription-polymerase chain reaction and western blotting. The viability, proliferation, and apoptosis of lipopolysaccharide (LPS)-induced human synovial cells (HSCs) were examined by using Cell Counting Kit-8, EdU (5-ethynyl-2'-deoxyuridine), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays, respectively. The OA model rats had significantly increased levels of inflammatory activity, damaged cells, and rough articular cartilage when compared with rats in the control group. The EVs from the model rats appeared as round vesicle-like structures with a mean diameter of ∼145 nm. Five miRNAs that showed gradual increases in the model rats were selected for further analysis; those miRNAs included miR-127-3p, miR-132-3p, miR-141-3p, miR-345-5p, and miR-382-5p. miR-382-5p was found to reduce the viability and proliferation and promote the apoptosis of LPS-induced HSCs. Moreover, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was negatively regulated by miR-382-5p. Our findings revealed that EVs produced by the OA rats contained miR-382-5p, which might reduce cell viability and proliferation, and promote cell apoptosis by targeting PTEN.

Therapeutic effect of fastigial nucleus stimulation is mediated by the microRNA-182 & microRNA-382/BDNF signaling pathways in the treatment of post-stroke depression.

The deregulation of Brain-Derived Neurotrophic Factor (BDNF) was reported to be responsible for the development of post-stroke depression (PSD), while the stimulation of the fastigial nucleus (FN) can be used to treat PDS by down-regulating the expression of miR-182 and miR-382. Therefore, we aim to test the hypothesis that the therapeutic effect of FN stimulation obtained in the treatment of PSD is mediated by the miR-382&miR-182/BDNF mRNA signaling pathways. Rat models of PSD were established and divided into sham, stroke, PSD and PSD + FNS groups to receive different treatments. Post-stroke depression-like behaviors were observed after the initiation of the treatments. TUNEL assay, Western Blot, IHC assay, real-time PCR, bioinformatics tools and luciferase assays were performed to examine the effect of FN stimulation on the expression of miR-182, miR-382 and BDNF mRNA/protein, as well as to further clarify the role of miR-382&miR-182/BDNF mRNA signaling pathways in FN stimulation. Post-stroke depression-like behaviors were significantly reduced in PSD rats. In contrary, the treatment by FN stimulation alleviated the symptoms of PSD and reduced the apoptosis index in the PSD group. Furthermore, in the PSD group, BDNF mRNA/protein levels were suppressed while the miR-382/miR-182 levels were both significantly up-regulated. After the treatment of FN stimulation, BDNF mRNA/protein levels were partly recovered, while miR-382/miR-182 levels was decreased. Furthermore, BDNF was identified as a virtual target of miR-382 and miR-182. In conclusion, FN stimulation increases the expression of BDNF via down-regulating the expression of miR-382/miR-182, thus exhibiting a positive effect in the management of PSD.

MiR-382 Functions on the Regulation of Melanogenesis via Targeting dct in Rainbow Trout (Oncorhynchus mykiss).

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that are involved in a diverse collection of biological processes as important post-transcriptional regulators. However, little is known about the molecular regulatory mechanism of miRNAs in fish skin pigmentation. In this study, we first confirmed that dopachrome tautomerase (dct), a key gene of melanogenesis, is a target of miR-382 in rainbow trout (Oncorhynchus mykiss) using luciferase reporter assay. The analysis of different developmental stages and tissue expression patterns between wild-type and yellow mutant rainbow trout suggested that miR-382 is a potential regulator during the process of skin pigmentation. In vitro, miR-382 mimics in rainbow trout primary liver cells significantly downregulated dct expression and resulted in decreased expression of key melanogenic genes including tyrosine-related protein 1 (tyrp1) and premelanosome protein (pmel), whereas the expression level of dct was markedly increased after transfected with miR-382 inhibitor. In vivo, overexpression of miR-382 by injection of miR-382 agomir significantly depressed the expression of dct in dorsal skin, tail fin, and liver and then reduced the expression levels of tyrp1 and pmel. Furthermore, transfection of miR-382 mimics inhibited cell proliferation and induced apoptosis. Taken together, our results identified a functional role of miR-382 in rainbow trout skin pigmentation through targeting dct, which facilitate understanding the regulatory mechanism of rainbow trout skin color at the post-transcriptional level and provide a theoretical basis for molecular breeding with skin color as the target trait.

miR-382-5p promotes cell invasion in hepatocellular carcinoma by targeting PTEN to activate PI3K/Akt signaling pathway.

PURPOSE: This study aimed at investigating miR-382-5p expression in tissues and cell lines with hepatocellular carcinoma (HCC), its effects on the invasion of HCC cells, and related mechanisms. METHODS: miR-382-5p expression in HCC tissues, adjacent tissues, cell lines of normal hepatic cells, and HCC cells were detected by qRT-PCR, indicating its upregulation or downregulation in HCC cell lines (Hep3B and HCCLM3). The effect of miR-382-5p on cell invasion was observed by the Transwell experiment. The targeting relationship of miR-382-5p and the phosphatase and tensin homolog (PTEN) was analyzed using bioinformatics tools and the luciferase reporter gene assay. The correlation between miR-382-5p and PTEN was analyzed with Spearman correlation analysis. PTEN expression was observed after upregulation and downregulation of miR-382-5p expression. The effect of miR-382-5p on the expression of key proteins in PI3K/Akt signaling pathway was determined by Western blot. RESULTS: miR-382-5p expression was upregulated in both HCC tissues and cell lines (both P<0.05). Upregulation or downregulation of miR-382-5p significantly promoted or inhibited the invasion of cell lines, Hep3B, and HCCLM3. The luciferase reporter gene assay confirmed that PTEN is a target of miR-382-5p. The expressions of miR-382-5p and PTEN were negatively correlated (r=-0.742, P<0.001). Upregulation of PTEN expression by plasmid transfection can reverse the invasive effect of miR-382-5p on HCC cells. Upregulation of miR-382-5p can activate PI3K/Akt signaling pathway, and downregulation of miR-382-5p can inhibit PI3K/Akt signaling pathway. CONCLUSIONS: miR-382-5p can activate the PI3K/Akt signaling pathway by targeting PTEN and promote HCC cell invasion.

MicroRNA-382 Promotes M2-Like Macrophage via the SIRP-α/STAT3 Signaling Pathway in Aristolochic Acid-Induced Renal Fibrosis.

Aristolochic acid nephropathy (AAN) is a type of drug-induced nephropathy and is correlated with a potentially progression of kidney fibrosis. However, whether miR-382 is implicated in macrophage activation in AA-induced kidney fibrosis remains elusive. Here, cell-sorting experiments defined a significant miR-382 enrichment in renal macrophage after AAN 14 days. Then, we found that treatment of AA induced a significant switch in the phenotype of macrophage both in vivo and in vitro. Furthermore, miR-382 knockout (KO) mice and miR-382-/- bone marrow-derived macrophage (BMDM) were subjected to AA induction. We found that both systemic KO and macrophage-specific miR-382 depletion notably suppressed M2-like macrophage activation as well as kidney interstitial fibrosis. Additionally, adoptive transfer of miR-382 overexpression BMDMs into mice promoted AA-induced kidney injury. Moreover, in cultured macrophage, upregulation of miR-382 promoted M2-related gene expression, accompanied by downregulation of signal regulatory protein α (SIRP-α) and activation of signal transducer and activator of transcription 3 (STAT3). The interaction between miR-382 and SIRP-α was evaluated via dual-luciferase assay. Knockdown of SIRP-α upregulated phosphorylated STAT3 at S727 and Y705. Pharmacological inhibition of STAT3 was performed both in vivo and in vitro. Inhibition of STAT3 attenuated AA-induced kidney fibrosis, in parallel to lesser macrophage M2 polarization. Coculture experiments further confirmed that overexpressed miR-382 in macrophage promoted injuries of tubular cells. Luminex bio-chip detection suggested that IL-4 and CCL-5 were critical in the cross talk between macrophages and tubular cells. Taken together, our data suggest that miR-382 is a critical mediator in M2-like macrophage polarization and can be a promising therapeutic target for kidney fibrosis.

LncRNA LINC00707 serves as a sponge of miR-382-5p to alleviate lipopolysaccharide (LPS)-induced WI-38 cell injury through upregulating NKAP in infantile pneumonia.

Infantile pneumonia (IP) is an acute lower respiratory infection that imposes a heavy burden on children's health. Increasing evidence has demonstrated that long non-coding RNA (lncRNA) LINC00707 participates in the regulation of the pneumonia process. Cell proliferative ability and apoptosis were measured using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Bcl-2 related X protein (Bax), NF-kB activating protein (NKAP), p-P65, P65, p-IκBα, and IκBα protein levels were detected using western blot assay. The binding between miR-382-5p and LINC00707 or NKAP was predicted by starBase v2.0 and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. LINC00707 and NKAP levels were increased, and miR-382-5p was decreased in LPS-stimulated WI-38 cells. Furthermore, the silencing of LINC00707 could abrogate LPS-engendered WI-38 cell proliferation, apoptosis, and oxidative stress. LINC00707 deficiency could relieve LPS-triggered WI-38 cell damage by regulating the miR-382-5p/NKAP axis, providing a new therapeutic strategy for IP treatment.

CircHIPK3 alleviates inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis in spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is one of the serious neurological diseases with high morbidity which may be treated with hematopoietic stem cell (HSC) transplants. Circular RNAs (circRNAs) play vital roles in SCI. The study aimed to reveal the function and mechanism of circRNA homeodomain interacting protein kinase 3 (HIPK3) in SCI. METHODS: SCI model in vitro was established by treating neuronal cells AGE1.HN with oxygen-glucose deprivation (OGD) and CoCl2. The levels of circHIPK3, miR-382-5p and dual specificity phosphatase 1 (DUSP1) were examined using quantitative real-time PCR (qRT-PCR) or western blot assay. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors (IL-6 and TNF-α). Cell proliferation and apoptosis were evaluated by 5'-ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry. Caspase-3 Colorimetric Assay Kit was used to detect aaspase-3 activity. The interactions among circHIPK3, miR-382-5p and DUSP1 were confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. RESULTS: CircHIPK3 and DUSP1 were down-regulated, while miR-382-5p was up-regulated in OGD-induced AGE1.HN cells. Overexpression of circHIPK3 suppressed inflammatory response and cell apoptosis and promoted proliferation in OGD-induced AGE1.HN cells by sponging miR-382-5p. CircHIPK3 regulated DUSP1 expression by targeting miR-382-5p. MiR-382-5p inhibition hindered inflammatory response of IL-6 and TNF-α and neuronal apoptosis and promoted apoptosis via targeting DUSP1. CONCLUSION: CircHIPK3 overexpression alleviated OGD-induced AGE1.HN cell inflammatory response and neuronal apoptosis via regulating miR-382-5p/DUSP1 axis, indicating that circHIPK3 might be a promising therapeutic target for SCI.

Circ_0000190 sponges miR-382-5p to suppress cell proliferation and motility and promote cell death by targeting ZNRF3 in gastric cancer.

This study was to explore the role of circRNA_0000190 (circ_0000190) in gastric cancer (GC) progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were applied to measure RNA and protein expression. 5-Ethynyl-2'-deoxyuridine (EdU) assay and cell counting kit-8 (CCK8) assay were implemented to analyze cell proliferation ability. Transwell assays were conducted to analyze cell motility. Cell ferroptosis was assessed using commercial kit. The target relationship between microRNA-382-5p (miR-382-5p) and circ_0000190 or zinc and ring finger 3 (ZNRF3) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Murine xenograft model was used to analyze the function of circ_0000190 in GC progression in vivo. Circ_0000190 was down-regulated in GC tissues and cell lines. Low expression of circ_0000190 predicted dismal prognosis in GC patients. Circ_0000190 overexpression inhibited the proliferation, migration and invasion and promoted Erastin- or ras selective lethal 3 (RSL3)-mediated ferroptosis in GC cells. MiR-382-5p was a target of circ_0000190, and circ_0000190 suppressed GC progression partly via serving as miR-382-5p sponge. ZNRF3 was a target of miR-382-5p, and miR-382-5p accelerated the proliferation, motility and restrained the ferroptosis of GC cells partly via regulating ZNRF3. Circ_0000190 overexpression restrained xenograft tumor growth in vivo. Collectively, Circ_0000190 suppressed GC progression via miR-382-5p-dependent regulation of ZNRF3.

LncRNA-GAS5/miR-382-3p axis inhibits pulmonary artery remodeling and promotes autophagy in chronic thromboembolic pulmonary hypertension.

BACKGROUND: We have clarified the role of miR-382-3p in chronic thromboembolic pulmonary hypertension (CTEPH), but what is less clear lies in its upstream regulatory mechanism. OBJECTIVE: To explore the regulation mechanism of GAS5/miR-382-3p axis on CTEPH. METHODS: In vitro, we constructed cell models by treating Pulmonary Artery Smooth Muscle Cells (PASMCs) with platelet-derived growth factor-BB (PDGF-BB). The effects of different concentrations of PDGF-BB on the activity of PASMCs were tested by cell counting kit-8 (CCK-8). The upstream lncRNA of miR-382-3p was screened and confirmed through bioinformatics analysis, RNA pull-down, quantitative reverse transcription polymerase chain reaction (qRT-PCR), dual luciferase reporter gene and RNA immunoprecipitation assays. The effects of GAS5/miR-382-3p axis on the viability, migration, and expressions of autophagy- and angiogenesis-related proteins were confirmed by rescue experiments (CCK-8, wound healing and western blot). In vivo, animal models by perfusing autologous blood vessels, the effects of GAS5 overexpression or silencing on the expressions of miR-382-3p, angiogenesis- and autophagy-related genes, mean pulmonary arterial pressure (mPAP) and pulmonary artery wall were determined by biological signal acquisition system, hematoxylin-eosin staining, qRT-PCR and western blot. RESULTS: PDGF-BB dose-dependently promoted PASMCs viability. XIST and GAS5 expressions in PASMCs were affected by the concentration of PDGF-BB, but only GAS5 can be pulled down by miR-382-3p probe. GAS5 targeted miR-382-3p to inhibit the viability and migration of PAMSCs, mPAP in CTEPH rats, pulmonary artery wall thickening and angiogenesis, and promote autophagy. CONCLUSIONS: GAS5/miR-382-3p axis is involved in the regulation of pulmonary artery remodeling and autophagy in CTEPH.