Circ-USP9X accelerates deep vein thrombosis after fracture by acting as a miR-148b-3p sponge and upregulates SRC kinase signaling inhibitor 1.

OBJECTIVES: This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities. METHODS: An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) were developed. The expression levels of circUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments. RESULTS: The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified. CONCLUSION: circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.

Altered expression of miR-17 and miR-148b in pediatric familial mediterranean fever patients.

Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder with wide phenotypic variation that has been observed among individuals who have the same genotype. Modifying genes, epigenetic factors, or environmental factors might all have an impact on genotype-phenotype correlation in FMF. The current research aims to determine the expression levels of microRNAs (miR-148b and miR-17) in Egyptian FMF participants. We also aimed to investigate Caspase -1 gene expression to make a correlation with disease severity. The study comprised 25 clinically diagnosed FMF cases and 25 healthy subjects matched for age and sex. The molecular diagnosis of FMF cases was assessed using real-time SNP genotyping assay. MiR-148b and miR-17 expression were profiled using TaqMan assay technology. The expression level of Caspase -1 gene was also verified using qRT-PCR. MiR-17 in the studied cases was significantly upregulated compared to healthy individuals (P = 0.006), whereas miR-148b was significantly downregulated in the examined patients (P = 0.030). Moreover, statistically significant upregulation of Caspase-1 expression was also elucidated in relation to normal subjects (P = 0.033). The results obtained indicated that miR-17 and miR-148b might be potential regulatory biomarkers in FMF cases. We further hypothesized that the upregulation of Caspase-1 could hint at its significance as a future therapeutic target to alleviate the inflammatory process in these patients. Key Points • The role of miRNAs in FMF and various mechanisms involved in FMF pathogenesis has received increasing attention. • Studying the expression profiles of miR-17 and miR-148b in FMF patients revealed their potential role as regulatory biomarkers in these patients. • Significant upregulation of Caspase-1 expression in FMF cases could hint at its significance as a future therapeutic target. • Future studies on larger cohorts are warranted to clarify and better understand the role of miRNAs in the pathogenesis and severity of FMF.

Long non-coding RNA LINC01554 overexpression suppresses viability, migration, and invasion of liver cancer cells through regulating miR-148b-3p/EIF4E3.

Background: Long non-coding RNAs (lncRNAs) can be severed as competing endogenous RNAs (ceRNAs) to regulate target genes or mRNAs via sponging microRNAs (miRNAs). This study explored the effect of LINC01554 on liver cancer cells through the ceRNA mechanism. Methods: Five significantly down-regulated lncRNAs were selected for further verification, and then through bioinformatics, interactive miRNAs and mRNAs of lncRNAs were identified. The relationship between LINC01554, miR-148b-3p and EIF4E3 was detected by the dual luciferase reporter gene assay. Afterwards, HCCLM3 cells were transfected with pCDH-LINC01554, miR-148b-3p inhibitor and miR-148b-3p mimics. Cell viability, apoptosis, migration and invasion were measured by Cell Counting Kit-8, flow cytometer, and Transwell assays. Real-time quantitative PCR (RT-qPCR) and Western blot were used to measure the expressions of related genes and proteins. Results: LINC01554 was significantly down-regulated in the liver cancer cell lines, and was expressed in the cytoplasm of HCCLM3 cells. LINC01554 overexpression inhibited proliferation, migration, and invasion of HCCLM3 cells, and promote their apoptosis (P < 0.05). Besides, LINC01554 overexpression also significantly increased the levels of BAX, BCL2/BAX, P53, cleaved-Caspase3, TIMP3, E-cadherin and EIF4E3 (P < 0.05). Through bioinformatics and dual-luciferase reporter gene assay, LINC01554, miR-148b-3p and EIF4E3 were proved to interact with each other. Furthermore, the effects of miR-148b-3p knockdown on HCCLM3 cells were similar with those of LINC01554 overexpression, and miR-148b-3p mimics could reverse the changes of cell viability, apoptosis, migration, and invasion induced by LINC01554 overexpression. Conclusions: LINC01554 overexpression could suppress the growth and metastasis of HCCLM3 cells via miR-148b-3p/EIF4E3.

MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway.

BACKGROUND: Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-ß pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. METHODS: Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-ß pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. RESULTS: We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. CONCLUSIONS: Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-ß signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

BMSC-derived exosomal miR-148b-3p attenuates OGD/R-induced HMC3 cell activation by targeting DLL4 and Notch1.

Bone mesenchymal stem cell (BMSC)-derived exosome (BMSC-Exo) could be a treatment method for ischemic injury. In ischemic cerebrovascular disease (IC), microglia is pivotal in neuronal damage and remodeling. This study explores the mechanisms of BMSC-Exo miR-148b-3p in regulating oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human microglial clone 3 (HMC3) cell activation. Transmission electron microscopy (TEM) and qNano were used to assess BMSC-Exo features. The functions of BMSC-Exo miR-148 b-3p in OGD/R-induced HMC3 cell activation were explored via MTT assay, flow cytometry, scratch, transwell, and enzyme-linked immunosorbent assay (ELISA) assays. A dual-luciferase reporter assay was performed to determine the relationship between miR-148b-3p and Delta-like ligand 4(DDL4) or neurogenic locus notch homolog protein 1 (Notch1). OGD/R decreased miR-148b-3p expression in HMC3 cells. After BMSC-Exo treatment, miR-148b-3p expression was upregulated, cell viability and migration were inhibited, cell cycles remained in the G0/G1 phase, and proinflammatory cytokines were decreased in OGD/R-induced HMC3 cells. More importantly, BMSC-Exo miR-148b-3p could further strengthen BMSC-Exo effects. DDL4 and Notch1 are direct targets of miR-148b-3p, respectively. Moreover, the knockdown of DLL4 or Notch1 could inhibit OGD/R-induced HMC3 cell activation. BMSC-Exo miR-148b-3p inhibited OGD/R-induced HMC3 cell activation via inhibiting DLL4 and Notch1 expression, which provided a new strategy for treating cerebral ischemia.

miR-148b-3p suppresses the proliferation and migration of Schwann cells by targeting USP6 following sciatic nerve injury.

BACKGROUND: Peripheral nerve injury is a common disorder associated with damaged axons and distal myelin sheath degeneration, and Schwann cells play a paramount role in peripheral nerve regeneration. This study aims to explore the role of microRNA miR-148b-3p on Schwann cells after peripheral nerve injury. METHODS: Sciatic nerve transection was conducted in rat as the model of peripheral nerve injury. The expression level of miR-148b-3p and Ubiquitin Specific Peptidase 6 (USP6) was detected by qRT-PCR and Western blot at diverse time points after nerve transection. Cell migration and proliferation were determined in primary Schwann cells isolated from rat. The functional interaction of miR-148b-3p and USP6 mRNA was validated by dual-luciferase reporter assay. RESULTS: In the animal model of sciatic nerve injury, miR-148b-3p expression level in the proximal nerve stump showed downregulation after nerve transection procedure, while USP6 expression level was elevated. The overexpression of miR-148b-3p inhibited the proliferation and migration of primary Schwann cells, while suppressing miR-148b-3p showed the opposite effect. USP6 mRNA was identified as a target of miR-148b-3p, which was found to mediate the effect of miR-148b-3p. USP6 silencing suppressed the migration and proliferation in primary Schwann cells. CONCLUSION: Our data demonstrated the functional role of miR-148b-3p/USP6 axis in regulating the migration and proliferation of Schwann cells following peripheral nerve injury. miR-148b-3p showed downregulation and its target USP6 was upregulated after nerve transection procedure. Targeting miR-148b-3p/USP6 axis may provide a novel opportunity for peripheral nerve repair.

MiR-148b caused liver injury in rats with traumatic hemorrhagic shock by inhibiting SIRT6 expression.

BACKGROUND: The purpose of this study was to investigate the role of miR-148b in liver injury in rats with traumatic hemorrhagic shock (THS) and to elucidate its potential mechanism. METHODS: The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of rats were detected by enzyme-linked immune sorbent assay (ELISA), and the injury of rat liver was analyzed by hematoxylin-eosin (H&E) staining. Apoptosis of rat hepatocytes and normal rat liver cell line (BRL3A) was identified by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and flow cytometry, respectively. MiR-148b and sirtuin 6 (SIRT6) expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. Lactate dehydrogenase (LDH) content and cell viability were measured by commercial kits and cell counting kit-8 (CCK-8) assay, respectively. The binding sites of miR-148b and SIRT6 were predicted by the Starbase database and verified by dual luciferase reporter assay. RESULTS: MiR-148b expression in THS rats or ischemia-reperfusion (I/R)-treated cells was higher than in the control group. Overexpression of miR-148b further promoted the effects of I/R, which enhanced the levels of ALT, AST and LDH, cell apoptosis of liver tissue or BRL3A cells and decreased the expression of SITR6. Besides, miR-148b negatively correlated with SIRT6, and upregulated the expression of SIRT6 could partly reverse the effect of miR-148b. CONCLUSION: Hepatocyte injury induced by I/R was achieved by regulating miR-148b /SIRT6 axis.

Tumor-derived exosomal miR-148b-3p mediates M2 macrophage polarization via TSC2/mTORC1 to promote breast cancer migration and invasion.

BACKGROUND: Emerging evidence has revealed that tumor-associated macrophages (TAMs) and exosomes play a crucial role in the microenvironment for tumor growth. However, the mechanisms through which exosomal miRNAs modulate TAMs and tumor development in breast cancer are not fully understood. METHODS: We constructed a macrophage model and an indirect coculture system consist of breast cancer cells and macrophages. Exosomes were isolated from BC cells culture supernatant and identified by transmission electron microscopy, Western blot and Nanosight LM10 system. The expression of miR-148b-3p in exosomes was determined by qRT-PCR and the effect of exosomal miR-148b-3p on macrophage polarization was measured using qRT-PCR and ELISA. The proliferation, migration and invasion of BC cells were estimated by EdU, wound healing assay and transwell assay. We employed bioinformatics, luciferase reporter assay and Western blot to identify the target gene of miR-148b-3p. Western blot was used to clarify the mechanism of exosomal miR-148b-3p mediated the crosstalk between BC cells and M2 macrophages. RESULTS: Cancer-derived exosomes could induce M2 polarization of macrophages, which promoted the migration and invasion of breast cancer cells. We found that exosomal miR-148b-3p was overexpressed in breast cancer cell-derived exosomes and correlated with lymph node metastasis, late tumor stage and worse prognosis. Upregulated miR-148b-3p expression in exosomes modulated macrophage polarization by targeting TSC2, which promoted the proliferation and might affect migration and invasion of breast cancer cells. Interestingly, we found that exosomal miR-148b-3p could induce M2 macrophage polarization via the TSC2/mTORC1 signaling pathway in breast cancer. CONCLUSION: Overall, our study elucidated that miR-148b-3p could be transported by exosomes from breast cancer cells to surrounding macrophages and induced M2 polarization by targeting TSC2, providing novel insights for breast cancer therapy.

LncRNA H19 promotes tumor angiogenesis in smokers by targeting anti-angiogenic miRNAs.

A key concept in drug discovery is the identification of candidate therapeutic targets such as long noncoding RNAs (lncRNAs) because of their extensive involvement in neoplasms, and impressionability by smoking. Induced by exposure to cigarette smoke, lncRNA H19 targets and inactivates miR-29, miR-30a, miR-107, miR-140, miR-148b, miR-199a and miR-200, which control the rate of angiogenesis by inhibiting BiP, DLL4, FGF7, HIF1A, HIF1B, HIF2A, PDGFB, PDGFRA, VEGFA, VEGFB, VEGFC, VEGFR1, VEGFR2 and VEGFR3. Nevertheless, these miRNAs are often dysregulated in bladder cancer, breast cancer, colorectal cancer, glioma, gastric adenocarcinoma, hepatocellular carcinoma, meningioma, non-small-cell lung carcinoma, oral squamous cell carcinoma, ovarian cancer, prostate adenocarcinoma and renal cell carcinoma. As such, the present perspective article seeks to establish an evidence-based hypothetical model of how a smoking-related lncRNA known as H19 might aggravate angiogenesis by interfering with miRNAs that would otherwise regulate angiogenesis in a nonsmoking individual.

A primary goal in the treatment of cancer is preventing the formation of new blood vessels, or angiogenesis, within the tumor, because these newly formed capillaries serve to supply tumor cells with oxygen, letting them live for longer periods of time and develop several other unfavorable traits that would complicate the entire treatment process. Although certain molecules are responsible for regulating angiogenesis, others such as lncRNA H19, cause significant deregulation in the level of these antiangiogenic molecules, enhancing tumor vascularization. Because H19 is induced in response to cigarette smoke, individuals who smoke might be at higher risk of treatment failure as a result of accelerated angiogenesis.

Infant consumption of microRNA miR-375 in human milk lipids is associated with protection from atopy.

BACKGROUND: Human milk is thought to reduce infant atopy risk. The biologic mechanism for this protective effect is not fully understood. OBJECTIVES: We tested the hypothesis that infant consumption of 4 microRNAs (miR-146b-5p, miR-148b-3p, miR-21-5p, and miR-375-3p) in human milk would be associated with reduced atopy risk. METHODS: The Breast Milk Influence of the Microtranscriptome Profile on Atopy in Children over Time (IMPACT) study involved a cohort of mother-infant dyads who planned to breastfeed beyond 4 mo. Infant consumption of the 4 human milk microRNAs (miRNAs) in the first 6 mo was calculated as the product of milk miRNA concentration and the number of human milk feeds, across 3 lactation stages: early milk (0-4 wk), transitional milk (4-16 wk), and mature milk (16-24 wk). The primary outcome was infant atopy in the first year, defined as atopic dermatitis (AD), food allergies, or wheezing. The final analysis included 432 human milk samples and 7824 wk of longitudinal health data from 163 dyads. RESULTS: Seventy-three infants developed atopy. Forty-one were diagnosed with AD (25%), 33 developed food allergy (20%), and 10 had wheezing (6%). Eleven developed >1 condition (7%). Infants who did not develop atopy consumed higher concentrations of miR-375-3p (d = 0.18, P = 0.022, adj P = 0.044) and miR-148b-3p (d = 0.23, P = 0.007, adj P = 0.028). The consumption of miR-375-3p (X2 = 5.7, P = 0.017, OR: 0.92, 95% CI: 0.86, 0.99) was associated with reduced atopy risk. Concentrations of miR-375-3p increased throughout lactation (r = 0.46, F = 132.3, P = 8.4 × 10-34) and were inversely associated with maternal body mass (r = -0.11, t = -2.1, P = 0.032). CONCLUSIONS: This study provides evidence that infant consumption of miR-375-3p may reduce atopy risk.

Circulating miR-148b-3p and miR-27a-3p can be potential biomarkers for diagnosis of pre-diabetes and type 2 diabetes: integrating experimental and in-silico approaches.

BACKGROUND: In view of the growing global prevalence of type 2 diabetes (T2D), detection of prediabetes and type 2 diabetes in the early stages is necessary to reduce the risk of developing diabetes, prevent the progression of the disease, and dysfunction of different organs. Since miRNAs are involved in the initiation and progression of numerous pathogenic processes, including diabetes, in the present study, we aimed to investigate the expression of miR-148b-3p and miR-27a-3p in prediabetic and T2D patients and to evaluate the diagnostic potential of these miRNAs. METHODS: We evaluated the expression of miR-148b-3p and miR-27a-3p in the plasma of three groups: 20 prediabetic patients, 20 T2D patients, and 20 healthy controls. The biochemical parameters were determined by the auto-analyzer. The possible target genes of these miRNAs were identified using an in-silico approach. RESULTS: Our results showed that, as compared to the healthy controls, there was a significant up regulation and down regulation in the expression of miR-148b-3p and miR-27a-3p in the T2D patients, respectively. The results of receiver operating characteristic curve analysis also suggested that miR-148b-3p acted successfully in discriminating the prediabetic and diabetic patients from the control group. According to in-silico analysis, miRs influence biological pathways involved in T2DM development, such as insulin signaling. CONCLUSIONS: The miR148b-3p and miR-27a-3p expression levels were deregulated in diabetes and pre-diabetes. Furthermore, miR-148b-3p showed significant ability in discriminating between diabetic and healthy individuals, suggesting a potential diagnostic use of miR-148b-3p in the detection of T2D.

LINC00665 affects the malignant biological behavior of ovarian cancer via the miR-148b-3p/KLF5.

This study investigated the expression and clinical significance of long intergenic noncoding RNA 00665 (LINC00665) in ovarian cancer (OC), as well as its effect on the malignant biological behavior of OC cells. The expression of LINC00665, miR-148b-3p, and Krüppel-like factor 5 (KLF5) in OC tissues and cells were determined by RT-qPCR. Western blot was used to detect the protein expression of KLF5. The expression patterns of LINC00665 in nuclear and cytoplasm fractions were undertaken using RT-qPCR. In addition, CCK-8 assay, clone formation assay, transwell, scratch test, and flow cytometry were respectively used to detect the cell activity, proliferation, invasiveness, healing of cells, and apoptosis rate of OC cells. Furthermore, the interactions between LINC00665 and miR-148b-3p and between miR-148b-3p and KLF5 were verified by the luciferase reporter assay, and the correlations among these three genes were analyzed. LINC00665 expression was upregulated both in OC cell lines and tissues. Si-LINC00665 inhibited cell proliferation, invasion, and migration and induced apoptosis to a certain extent. The subcellular fraction assay revealed LINC00665 to be located mainly in the cytoplasm. miR-148b-3p was a target of LINC00665, and KLF5 was directly targeted by miR-148b-3p. Si-LINC00665 inhibited KLF5 expression, miR-148b-3p inhibitor promoted KLF5 expression, and si-KLF5 inhibited LINC00665 expression. Interestingly, the expression of LINC00665 was reversely associated with miR-148b-3p expression but positively correlated with KLF5. Furthermore, miR-148b-3p expression was negatively correlated with KLF5. In addition, si-KLF5 inhibited the malignant biological behavior of OC cells, whereas miR-148b-3p inhibitor had the opposite effect. Most importantly, the si-LINC00665 could reverse the promotion effect of the miR-148b-3p inhibitor on the malignant biological behavior of OC cells. LINC00665 can be used as an effective prognostic indicator of OC, which has the potential to be a new therapeutic target.

Circular RNA circ_0021001 regulates miR-148b-3p/GREM1 axis to modulate proliferation and apoptosis of vascular smooth muscle cells.

Intracranial aneurysm (IA) is an abnormal expression in the intracranial arteries, which is related to the growth and apoptosis of vascular smooth muscle cells (VSMCs). Circular RNA (circRNA) circ_0021001 (also named circARFIP2) has been identified to mediate the regulation of VSMCs proliferation. However, the molecular mechanism of circ_0021001 involved in VSMC dysfunction in IA is poorly defined. The expression levels of circ_0021001, microRNA-148b-3p (miR-148b-3p), and Gremlin 1 (GREM1) were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, proliferation, cell cycle progression, and apoptosis were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Protein levels of proliferating cell nuclear antigen (PCNA), p21, B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), and GREM1 were examined by western blot assay. The binding relationship between miR-148b-3p and circ_0021001 or GREM1 was predicted by StarBase and then verified using a dual-luciferase reporter assay. The expression levels of circ_0021001 and GREM1 were increased, and that of miR-148b-3p was decreased in IA tissues and HUASMCs. Moreover, the downregulation of circ_0021001 could repress proliferation ability and induce apoptosis of HUASMCs. The mechanical analysis uncovered that circ_0021001 served as a sponge of miR-148b-3p to regulate GREM1 expression. Circ_0021001 silencing could suppress cell growth and induce apoptosis of HUASMCs partially through modulating the miR-148b-3p/GREM1, presented circ_0021001 as a promising therapeutic target for IA.

Renal Megalin mRNA Downregulation Is Associated with CKD Progression in IgA Nephropathy.

INTRODUCTION: Megalin plays an important role in proximal tubule uptake of filtered proteins. Downregulation and dysfunction of megalin were previously demonstrated in IgA nephropathy (IgAN); however, its relationship to IgAN progression remains unclear. METHODS: We measured renal megalin mRNA and miR-148b, previously identified as a regulator of megalin, in a retrospective cohort of 417 IgAN patients at the time of biopsy, and evaluated their associations with chronic kidney disease (CKD) progression event, defined as end-stage renal disease or ≥40% decline in estimated glomerular filtration rate, using Cox proportional hazard models. Risk classification statistics were calculated for CKD progression. RESULTS: During a median follow-up of 43 months, 121 (29.0%) patients reached the CKD progression event. Patients in the highest tertile of renal megalin mRNA had a lower risk for CKD progression than in the lowest tertile (hazard ratio (HR): 0.407, 95% confidence interval (CI) 0.231-0.719; p = 0.002). Log megalin mRNA was independent and negatively associated with CKD progression in IgAN (HR: 0.529, 95% CI 0.377-0.742; p < 0.001). The addition of renal megalin mRNA to a model with traditional risk factors improved risk prediction of disease progression (C statistic from 0.76 to 0.80; integrated discrimination index: 0.04 [95% CI: 0.02-0.07]). Moreover, patients in the highest tertile of renal miR-148b had a 2.3-fold higher risk for CKD progression compared with those in the lowest tertile. CONCLUSIONS: Lower renal megalin mRNA levels were associated with a greater risk of CKD progression in IgAN independent of clinical and pathological characteristics, suggesting that renal megalin could be an important prognostic factor for IgAN.

circHIPK3 regulates apoptosis and mitochondrial dysfunction induced by ischemic stroke in mice by sponging miR-148b-3p via CDK5R1/SIRT1.

Circular RNAs (circRNAs) are aberrantly expressed in the brain and play a role in a variety of central nervous system diseases. However, the essential role and therapeutic potential of circRNAs in ischemic stroke (IS) are poorly understood. Here, using circRNA sequencing, we showed that circRNA homeodomain-interacting protein kinase 3 (circHIPK3) was abundantly expressed in ischemic brain tissues in transient middle cerebral artery occlusion (tMCAO)-evoked stroke model mice. Knockdown of circHIPK3 markedly reduced the infarct volume, brain water content, neurological deficit scores, and blood-brain permeability and ameliorated brain microvascular endothelial cell (BMEC) apoptosis and mitochondrial dysfunction in tMCAO mice. Gain- and loss-of-function experiments were performed to verify the effects of miR-148b-3p on oxygen-glucose deprivation (OGD)-induced BMEC apoptosis and mitochondrial dysfunction. Mechanistically, circHIPK3 functions as an endogenous sponge of miR-148b-3p to decrease its activity, resulting in upregulation of CDK5R1 and CDK5 expression, downregulation of SIRT1 expression and subsequent BMEC apoptosis and mitochondrial dysfunction. Collectively, our findings suggest that circHIPK3 and its coupling mechanism are implicated in IS, providing translational evidence that circHIPK3 could be a key therapeutic target for IS.

Circular RNA_0120376 regulates microRNA-148b-3 and centrosomal protein 55 to promote non-small cell lung cancer development.

Circular RNAs (circRNAs) are non-coding RNAs with covalent closed-loop structures that are vital in regulating diverse pathological processes. This work is aimed to investigate the role of circ_0120376 in non-small cell lung cancer (NSCLC). Circ_0120376, microRNA (miR)-148b-3p, and centrosomal protein 55 (CEP55) mRNA expression in NSCLC tissues and cells were determined using qRT-PCR. The influences of circ_0120376 and miR-148b-3p on the proliferation of NSCLC cell lines were analyzed by CCK-8 and colony formation assays. Apoptosis was analyzed by flow cytometry. Cell migration and invasion were analyzed using the Transwell experiment. Binding relationships between circ_0120376 and miR-148b-3p and between miR-148b-3p and CEP55 3'UTR were investigated using the dual-luciferase reporter experiment and the RIP experiment. Western blot was conducted to analyze the regulatory effect of circ_0120376 and miR-148b-3p on CEP55 expression. We found that circ_0120376 was markedly overexpressed in NSCLC, and its overexpression was positively associated with increased T stage and lymph node metastasis of the patients. Functional experiments unveiled that circ_0120376 enhanced the proliferation, migration and invasion of NSCLC cells and impeded apoptosis, while knocking down circ_0120376 remarkably suppressed the malignant features of NSCLC cells mentioned above. Circ_0120376 could adsorb miR-148b-3p to reduce miR-148b-3p expression, and circ_0120376 could increase CEP55 expression via adsorbing miR-148b-3p. In summary, circ_0120376 contributes to the malignancy of NSCLC cells through a ceRNA mechanism via regulating miR-148b-3p/CEP55 axis. Circ_0120376 is likely to be a potential diagnostic biomarker and therapeutic target for NSCLC.

miR-148b-3p, as a tumor suppressor, targets son of sevenless homolog 1 to regulate the malignant progression in human osteosarcoma.

Osteosarcoma (OS) is a malignant tumor that occurs in children and adolescents. Previous studies reported a low expression of miR-148b-3p in OS, but its biological function in OS remains obscure. This study aimed to explore the role of miR-148b-3p in OS progression. Herein, the expression of miR-148b-3p and son of sevenless homolog 1 (SOS1) both in OS tissues and cells were examined using quantitative real-time polymerase chain reaction and Western blotting assay. miR-148b-3p mimic or inhibitor, pcDH-SOS1 plasmid or si-SOS1 and agomir-miR-148b-3p were constructed for cell transfection. In vitro, the biological effect of miR-148b-3p was determined employing MTT, EdU, colony formation, flow cytometry, transwell and wound healing assay, separately. The target relationship between SOS1 3'-untranslated region (3'-UTR) and miR-148b-3p was analyzed using dual-luciferase reporter gene. In vivo, the inhibition of agomir-miR-148b-3p in mice was evaluated via a xenograft mouse model. miR-148b-3p was noticeably low-expressed in OS tissues and cells, and miR-148b-3p over-expression in OS cells suppressed the growth, migration and invasion, induced apoptosis. The effect of miR-148b-3p-inhibitor on cell biological behavior is opposite to that of miR-148b-3p over-expression. Conversely, The expression of SOS1 was significant higher in OS tissues and cells, miR-148b-3p targeted and was negatively associated with the expression level of SOS1. In addition, the anti-tumor effect of miR-148b-3p was reversed by SOS1. Importantly, we demonstrated that the tumor growth of stably over-expressed miR-148b-3p human MG-63 cells was obviously reduced in tumor-bearing mice. These data highlighted that miR-148b-3p might be as a promising therapeutic target for OS.

Resistin deletion protects against heart failure injury by targeting DNA damage response.

AIMS: Increased resistin (Retn) levels are associated with development of cardiovascular diseases. However, the role of Retn in heart failure (HF) is still unclear. Here we probed the functional and molecular mechanism underlying the beneficial effect of Retn deletion in HF. METHODS AND RESULTS: Wild-type (WT) and adipose tissue-specific Retn-knockout (RKO) mice were subjected to transverse aortic constriction (TAC)-induced HF. Cardiac function and haemodynamic changes were measured by echocardiography and left ventricular catheterization. Adipose tissue Retn deletion attenuated while Retn cardiac-selective overexpression, via a recombinant adeno-associated virus-9 vector, exacerbated TAC-induced hypertrophy, cardiac dysfunction, and myocardial fibrosis in WT and RKO mice. Mechanistically, we showed that Gadd45α was significantly increased in RKO HF mice while cardiac overexpression of Retn led to its downregulation. miR148b-3p directly targets Gadd45α and inhibits its expression. Retn overexpression upregulated miR148b-3p expression and triggered DNA damage response (DDR) in RKO-HF mice. Inhibition of miR148b-3p in vivo normalized Gadd45α expression, decreased DDR, and reversed cardiac dysfunction and fibrosis. In vitro Retn overexpression in adult mouse cardiomyocytes activated miR148b-3p and reduced Gadd45α expression. Gadd45α overexpression in H9C2-cardiomyoblasts protected against hydrogen peroxide- and Retn-induced DDR. CONCLUSION: These findings reveal that diminution in circulating Retn reduced myocardial fibrosis and apoptosis, and improved heart function in a mouse model of HF, at least in part, through attenuation of miR148b-3p and DDR. The results of this study indicate that controlling Retn levels may provide a potential therapeutic approach for treating pressure overload-induced HF.

miRNA-148b and its role in various cancers.

miRNA-148b belongs to the family miR-148/-152, with significant differences in nonseed sequences, which can target diverse mRNA molecules. Reportedly, it may undergo deregulation in lung and ovarian cancers and downregulation in gastric, pancreatic and colon cancers. However, there is a need for further studies to better characterize its mechanism of action and in different types of cancer. In this review, we focus on the aberrant expression of miR-148b in different cancer types and highlight its main target genes and signaling pathways, as well as its pathophysiologic role and relevance to tumorigenesis in several types of cancer.

Lay abstract miRNA-148b, or miR-148b, is a tumor suppressor that can regulate invasion-, apoptosis- and proliferation-related oncogenes. miR-148b prognostic and diagnostic potential has been the center of focus recent investigations and extensive studies have been performed on miR-148b regulation in carcinogenesis. Here, we review the role of miR-148b in various cancers and its potential therapeutic application as a target or biomarker.

LncRNA NEAT1 Acts as an miR-148b-3p Sponge to Regulate ROCK1 Inhibition of Retinoblastoma Growth.

BACKGROUND: It is reported that long non-coding RNA nuclear paraspeckle assembly transcript 1 (LncRNA NEAT1) is involved in the occurrence and development of various cancers. However, the detailed biological function and mechanism of LncRNA NEAT1 in retinoblastoma are still unclear. So we will explore the biological function and possible mechanism of LncRNA NEAT1 in retinoblastoma. MATERIALS AND METHODS: Quantitative real-time PCR (qRT-PCR) was used to detect LncRNA NEAT1 in retinoblastoma tissues and cell lines. Cell counting kit 8, Transwell and flow cytometry were applied to explore cell proliferation, invasion and apoptosis. The target miRNAs (miR) of LncRNA NEAT1 and miR and downstream target genes were predicted using Starbase3.0 software and confirmed by double luciferase reporting test and RNA binding protein immunoprecipitation (RIP). Western Blot was applied to explore ROCK1 in cells, and tumor allogeneic experiment was applied to study the role of LncRNA NEAT1 on tumor growth. RESULTS: It was found that LncRNA NEAT1 was up-regulated in retinoblastoma tissues, cells and serum, and the prognosis of patients with high expression of LNC RNA NEAT 1 was poor. Functional analysis showed that knocking down LncRNA NEAT1 could weaken proliferation and invasion, and accelerate apoptosis. Tumor allogeneic experiment showed that sh-NEAT1 injection can inhibit tumor growth. In addition, LncRNA NEAT1 inhibited proliferation and invasion, and promoted apoptosis through miR-148b-3p/ROCK1 axis. CONCLUSION: LncRNA NEAT1 can mediate miR-148b-3p/ROCK1 axis to weaken the proliferation and invasion of retinoblastoma.