Profile of plasma microRNAs as a potential biomarker of Wilson's disease.

BACKGROUND: Wilson's disease (WD) is a rare condition resulting from autosomal recessive mutations in ATP7B, a copper transporter, manifesting with hepatic, neurological, and psychiatric symptoms. Timely diagnosis and appropriate treatment yield a positive prognosis, while delayed identification and/or insufficient therapy lead to a poor outcome. Our aim was to establish a prognostic method for WD by characterising biomarkers based on circulating microRNAs. METHODS: We conducted investigations across three cohorts: discovery, validation (comprising unrelated patients), and follow-up (revisiting the discovery cohort 3 years later). All groups were compared to age- and gender-matched controls. Plasma microRNAs were analysed via RNA sequencing in the discovery cohort and subsequently validated using quantitative PCR in all three cohorts. To assess disease progression, we examined the microRNA profile in Atp7b-/- mice, analysing serum samples from 6 to 44 weeks of age and liver samples at three time points: 20, 30, and 40 weeks of age. RESULTS: In patients, elevated levels of the signature microRNAs (miR-122-5p, miR-192-5p, and miR-885-5p) correlated with serum activities of aspartate transaminase, alanine aminotransferase and gamma-glutamyl transferase. In Atp7b-/- mice, levels of miR-122-5p and miR-192-5p (miR-885-5p lacking a murine orthologue) increased from 12 weeks of age in serum, while exhibiting fluctuations in the liver, possibly attributable to hepatocyte regenerative capacity post-injury and the release of hepatic microRNAs into the bloodstream. CONCLUSIONS: The upregulation of the signature miR-122-5p, miR-192-5p, and miR-885-5p in patients and their correlation with liver disease progression in WD mice support their potential as biomarkers of WD.

Serum miR-192-5p is a promising biomarker for lethal radiation injury.

In the event of a nuclear or radiation accident, rapid identification is required for those who exposed to potentially lethal dose irradiation. However, existing techniques are not adequate for the classification of lethal injury. Several studies have explored the potential of miRNAs as biomarkers for ionizing radiation injury, however, there are few miRNAs with specific expression for lethal radiation injury. Therefore, the aim of this study was to screen and validate the possibility of serum miRNAs as biomarkers of lethal radiation injury. We found the specific expression of mmu-miR-374c-5p / mmu-miR-194-5p on first day and mmu-miR-192-5p / mmu-miR-223-3p on third day in the mouse serum only under 10 Gy irradiation by miRNA sequencing and all significantly correlated with lymphocyte counts by Pearson's correlation analysis. In addition, it was found that among the 4 candidate serum miRNAs, only highly-expressed mmu-miR-192-5p in mouse serum irradiated at lethal doses was returned to sham-like expression levels at 3 days post-irradiation with amifostine pretreatment and closely correlated with survival rate. We demonstrated for the first time that mmu-miR-192-5p screened from lethally irradiated mice sera can be used as a potential biomarker for lethal irradiation injury, which will be helpful to improve efficiency of medical treatment to minimize casualties after a large-scale nuclear accident.

Revitalizing gut barrier integrity: role of miR-192-5p in enhancing autophagy via Rictor in enteritis.

Intestinal inflammation and compromised barrier function are critical factors in the pathogenesis of gastrointestinal disorders. This study aimed to investigate the role of miR-192-5p in modulating intestinal epithelial barrier (IEB) integrity and its association with autophagy. A DSS-induced colitis model was used to assess the effects of miR-192-5p on intestinal inflammation. In vitro experiments involved cell culture and transient transfection techniques. Various assays, including dual-luciferase reporter gene assays, quantitative real-time PCR, Western blotting, and measurements of transepithelial electrical resistance, were performed to evaluate changes in miR-192-5p expression, Rictor levels, and autophagy flux. Immunofluorescence staining, H&E staining, TEER measurements, and FITC-dextran analysis were also used. Our findings revealed a reduced expression of miR-192-5p in inflamed intestinal tissues, correlating with impaired IEB function. Overexpression of miR-192-5p alleviated TNF-induced IEB dysfunction by targeting Rictor, resulting in enhanced autophagy flux in enterocytes (ECs). Moreover, the therapeutic potential of miR-192-5p was substantiated in colitis mice, wherein increased miR-192-5p expression ameliorated intestinal inflammatory injury by enhancing autophagy flux in ECs through the modulation of Rictor. Our study highlights the therapeutic potential of miR-192-5p in enteritis by demonstrating its role in regulating autophagy and preserving IEB function. Targeting the miR-192-5p/Rictor axis is a promising approach for mitigating gut inflammatory injury and improving barrier integrity in patients with enteritis.NEW & NOTEWORTHY We uncover the pivotal role of miR-192-5p in fortifying intestinal barriers amidst inflammation. Reduced miR-192-5p levels correlated with compromised gut integrity during inflammation. Notably, boosting miR-192-5p reversed gut damage by enhancing autophagy via suppressing Rictor, offering a potential therapeutic strategy for fortifying the intestinal barrier and alleviating inflammation in patients with enteritis.

MiRNA-192-5p-targeted activated leukocyte cell adhesion molecule improved inflammatory injury of neonatal necrotizing enterocolitis.

BACKGROUND: Neonatal necrotizing enterocolitis (NEC) is a common gastrointestinal emergency in neonates. MiRNA-192-5p was found associated with ulcerative colitis (UC) progression, also with aberrant expression in intestinal cancer tissue. However, the effects of miRNA-192-5p on NEC have not been reported. METHODS: Based on the bioinformatics analysis of the GEO dataset, miR-192-5p was identified as the differentially expressed miRNA in NEC, and activated leukocyte cell adhesion molecule (ALCAM) was predicted as its target. After that, in vitro, rat intestinal epithelial cell-6 (IEC-6) were stimulated with LPS to construct a cell model of NEC. IEC-6 cells were transfected with miRNA-192-5p mimics, miRNA-192-5p inhibitors, or miRNA-192-5p inhibitors + sh-ALCAM, and relevant negative control. In vivo, SD rats were treated with artificial feeding, hypoxic reoxygenation, cold stimulation, and LPS gavage to induce NEC, followed by injection of agomiR-NC or agomiRNA-192-5p. Then effects of miRNA-192-5p on NEC model IEC-6 cell viability, apoptosis, ALCAM expression, Interleukin (IL)-1ß and IL-6 levels, intestinal injury, intestinal permeability were detected. RESULTS: MiRNA-192-5p expression was downregulated in NEC IEC-6 cells, whose overexpression increased IEC-6 cell viability. MiRNA-192-5p inhibitors increased IL-1ß, IL-6 levels and promoted IEC-6 cell apoptosis. MiRNA-192-5p targeting of ALCAM decreased ALCAM expression, IL-1ß, and IL-6 levels. AgomiRNA-192-5p decreased ALCAM, IL-1ß, and IL-6 levels in intestinal tissue and pathological damage and increased miRNA-192-5p levels. CONCLUSION: MiR-192-5p protects against intestinal injury by inhibiting ALCAM-mediated inflammation and intestinal epithelial cells, which would provide a new idea for NEC treatment.

miR-192 family in breast cancer: Regulatory mechanisms and diagnostic value.

There is a growing interest in the role of the miRNA family in human cancer. The miRNA-192 family is a group of conserved small RNAs, including miR-192, miR-194, and miR-215. Recent studies have shown that the incidence and mortality of breast cancer have been increasing epidemiologically year by year, and it is urgent to clarify the pathogenesis of breast cancer and seek new diagnostic and therapeutic methods. There is increasing evidence that miR-192 family members may be involved in the occurrence and development of breast cancer. This review describes the regulatory mechanism of the miRNA-192 family affecting the malignant behavior of breast cancer cells and evaluates the value of the miRNA-192 family as a diagnostic and prognostic biomarker for breast cancer. It is expected that summarizing and discussing the relationship between miRNA-192 family members and breast cancer, it will provide a new direction for the clinical diagnosis and treatment of breast cancer and basic medical research.

Clinical predictive value of control attenuation parameters in combination with miR-192-5p in patients with acute pancreatitis in nonalcoholic fatty liver disease.

INTRODUCTION: Control attenuation parameters (CAP) can detect nonalcoholic fatty liver disease (NAFLD). Our previous study found that miR-192-5p could screen for acute pancreatitis (AP) in NAFLD patients. This study focused on the role of CAP and miR-192-5p in NAFLD of acute AP. MATERIAL AND METHODS: AP patients and controls were enrolled. Classification of AP patients into NAFLD/AP patients and non-NAFLD/AP was made based on the CAP value. CAP was measured by liver transient elastography. Serum miR-192-5p was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Logistic regression analysis was conducted to examine the risk factors for the development of NAFLD. Receiver operating characteristic (ROC) was assessed for the predictive value of AP severity. RESULTS: NAFLD was more common in the AP group than in the controls (35.00% vs. 8.75%). The CAP value was higher in AP patients with NAFLD than in non-NAFLD, whereas miR-192-5p was significantly lower in AP patients with NAFLD. Additionally, AP patients with NALFD are more likely to experience respiratory failure, systemic inflammatory response syndrome (SIRS), and pancreatic necrosis with longer hospitalisation and exacerbate the incidence of moderate to severe AP. Both miR-192-5p and TG are potential risk factors for the development of NAFLD in patients with AP. Furthermore, the CAP value gradually increased with increasing AP severity, while miR-192-5p gradually decreased. Finally, the sensitivity and specificity of CAP combined with miR-192-5p for the prediction of moderate to severe AP were scored as 82.61% and 82.43%, respectively. CONCLUSIONS: NAFLD exacerbated the progression of AP, and CAP combined with miR-192-5p could predict the severity of AP. Our study may provide more reference for AP disease progression and treatment.

LncRNA Gm15232 Promotes Lipogenesis in Aging Mice Through the miR-192-3p/GCR Pathway.

Recent scientific studies have highlighted the importance of long-chain noncoding RNAs (lncRNAs) in a variety of metabolic diseases, but the specific functions and mechanisms of lncRNAs in aberrant lipid synthesis associated with aging are unknown. In this work, we inspected the effects of lncRNAs on the lipid metabolism in aging mice, as substantial evidence suggests that aging disturbs lipid metabolism. The results revealed that the expression of lncRNA Gm15232 was significantly elevated in the epididymal white adipose tissue of aging mice compared to adult mice. This upregulation of Gm15232 functioned as a competitive endogenous RNA by inhibiting the expression of miR-192-3p, and the ensuing downregulation of miR-192-3p increased the expression of the glucocorticoid receptor gene, which ultimately stimulated fat synthesis. The upregulation of Gm15232 thus increased lipogenesis through this mechanism. This study reveals a potential target for the treatment of age-related abnormalities of lipid metabolism.

A large-scale microRNA transcriptome-wide association study identifies two susceptibility microRNAs, miR-1307-5p and miR-192-3p, for colorectal cancer risk.

Transcriptome-wide association studies (TWAS) have identified many putative susceptibility genes for colorectal cancer (CRC) risk. However, susceptibility miRNAs, critical dysregulators of gene expression, remain unexplored. We genotyped DNA samples from 313 CRC East Asian patients and performed small RNA sequencing in their normal colon tissues distant from tumors to build genetic models for predicting miRNA expression. We applied these models and data from genome-wide association studies (GWAS) including 23 942 cases and 217 267 controls of East Asian ancestry to investigate associations of predicted miRNA expression with CRC risk. Perturbation experiments separately by promoting and inhibiting miRNAs expressions and further in vitro assays in both SW480 and HCT116 cells were conducted. At a Bonferroni-corrected threshold of P < 4.5 × 10-4, we identified two putative susceptibility miRNAs, miR-1307-5p and miR-192-3p, located in regions more than 500 kb away from any GWAS-identified risk variants in CRC. We observed that a high predicted expression of miR-1307-5p was associated with increased CRC risk, while a low predicted expression of miR-192-3p was associated with increased CRC risk. Our experimental results further provide strong evidence of their susceptible roles by showing that miR-1307-5p and miR-192-3p play a regulatory role, respectively, in promoting and inhibiting CRC cell proliferation, migration, and invasion, which was consistently observed in both SW480 and HCT116 cells. Our study provides additional insights into the biological mechanisms underlying CRC development.

The tRF-3024b hijacks miR-192-5p to increase BCL-2-mediated resistance to cytotoxic T lymphocytes in Esophageal Squamous Cell Carcinoma.

The limited efficacy of immune checkpoint inhibitors (ICIs) in the treatment of advanced Esophageal Squamous Cell Carcinoma (ESCC) poses a challenge. Recent evidence suggests that tumor cells' insensitivity to cytotoxic T lymphocytes (CTLs) contributes to drug resistance against ICIs. Here, a particular tRNA-derived fragment called tRF-3024b has been identified as playing a significant role in tumor cell resistance to CTLs. Through tRF sequencing (tRF-seq), we observed a high expression of tRF-3024b in ESCC cells that survived co-culture with CTLs. Further in vitro studies demonstrated that tRF-3024b reduced the apoptosis of tumor cells when co-cultured with CTLs. The mechanism behind this resistance involves tRF-3024b promoting the expression of B-cell lymphoma-2 (BCL-2) by sequestering miR-192-5p, a microRNA that would normally inhibit BCL-2 expression. This means that tRF-3024b indirectly enhances the protective effects of BCL-2, reducing apoptosis in tumor cells. Rescue assays confirmed that the suppressive function of tRF-3024b relies on BCL-2. In summary, the tRF-3024b/miR-192-5p/BCL-2 axis sheds light on the crucial role of tRF-3024b in regulating BCL-2 expression. These findings offer valuable insights into strategies to enhance the response of ESCC to CTLs and improve the effectiveness of immunotherapy approaches in treating ESCC.

MicroRNA-192-5p downregulates Fat Mass and Obesity-associated Protein to aggravate renal ischemia/reperfusion injury.

Acute kidney injury (AKI) is a common disorder without effective therapy yet. Renal ischemia/reperfusion (I/R) injury is a common cause of AKI. MicroRNA miR-192-5p has been previously reported to be upregulated in AKI models. However, its functional role in renal I/R injury is not fully understood. This study aimed to investigate the effects and the underlying mechanism of miR-192-5p in renal I/R progression. Hypoxia/reoxygenation (H/R)-induced cell injury model in HK-2 cells and I/R-induced renal injury model in mice were established in this study. Cell counting kit-8 assay was performed to determine cell viability. Quantitative real-time PCR and western blot analysis were performed to detect gene expressions. Hematoxylin-eosin and periodic acid-Schiff staining were performed to observe the histopathological changes. Enzyme-linked immunosorbent assay was performed to detect the kidney markers' expression. In vivo and in vitro results showed that miR-192-5p was up-regulated in the I/R-induced mice model and H/R-induced cell model, and miR-192-5p overexpression exacerbated I/R-induced renal damage. Then, the downstream target of miR-192-5p was analyzed by combining the differentially expressed mRNAs and the predicted genes and confirmed using a dual-luciferase reporter assay. It was found that miR-192-5p was found to regulate fat mass and obesity-associated (FTO) protein expression by directly targeting the 3' untranslated region of FTO mRNA. Moreover, in vivo and in vitro studies unveiled that FTO overexpression alleviated renal I/R injury and promoted HK-2 cell viability via stimulating autophagy flux. In conclusion, miR-192-5p aggravated I/R-induced renal injury by blocking autophagy flux via down-regulating FTO.

Circ_0001602 aggravates the progression of acute myeloid leukemia by regulating the miR-192-5p/ZBTB20 axis.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant blood cancer with a poor prognosis and complex pathogenesis. Recently, the critical role of circular RNAs (circRNAs) has been demonstrated in the malignant progression of AML. This study aimed to investigate the functional role and underlying mechanism of circ_0001602 in AML development. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) assay was conducted for detecting the expression of circ_0001602, CCND3, microRNA-192-5p (miR-192-5p), and Zinc Finger and BTB Domain-Containing Protein 20 (ZBTB20) mRNA. RNase R assay and Actinomycin D assay were implemented to determine the characteristics of circ_0001602. Cell counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Flow cytometry was employed for assessing cell cycle distribution and apoptosis. Dual-luciferase reporter assay and RIP assay were utilized for confirming the interactions between miR-192-5p and circ_0001602 or ZBTB20. RESULTS: Circ_0001602 and ZBTB20 were upregulated and miR-192-5p level was reduced in AML tissues and cells. Depletion of circ_0001602 repressed cell proliferation and induced cell cycle arrest and apoptosis in AML cells. Functionally, circ_0001602 was identified to be the sponge of miR-192-5p, and miR-192-5p silence restored the suppressive effects of circ_0001602 knockdown on AML cell progression. Furthermore, ZBTB20 was a target of miR-192-5p, and ZBTB20 overexpression neutralized the miR-192-5p-mediated inhibiting actions on the malignant phenotypes of AML cells. Besides, circ_0001602 could sponge miR-192-5p to positively regulate ZBTB20 expression. CONCLUSION: Circ_0001602 contributed to AML cell development at least partially through modulating the miR-192-5p/ZBTB20 axis, which provided new insights for AML treatment.

Resveratrol plays an anti-fibrotic and anti-autophagy role by stimulating miR-192-5p expression in urethral fibrosis.

BACKGROUND: Resveratrol (RSV) exerts anti-fibrotic effects on various fibrotic diseases. Whereas the biological role of RSV on urethral fibrosis remains to be elucidated. This study aimed to determine the mechanisms by which RSV affects urethral fibrosis and autophagy. METHODS: Sprague‒Dawley rats and primary fibroblasts were treated with transforming growth factor-ß1 (TGFß1) to generate in vivo and in vitro fibrosis models. Then, those were treated with RSV, and autophagy and fibrosis-related indicators were tested. RESULTS: Firstly, we found that RSV reversed the upregulation of indicators related to TGFß1-induced fibrosis (TGFß1, α-smooth muscle actin, collagen type I, and collagen type III), autophagy (TFEB and LC3), and TGFßR1/Smad4 pathway, as well as the downregulation of p62 and miR-192-5p expression both in vivo and in vitro. Overexpression of miR-192-5p suppressed the upregulation of fibrosis-related markers expression, as well as TFEB and LC3 expression, induced by TGFß1, while the expression trend of p62 was the opposite. Inhibiting miR-192-5p reversed the effects of RSV on the model group cells. It was also shown that RSV combined with sh-Smad4 inhibited autophagy more effectively than RSV alone. CONCLUSION: These results suggest that RSV inhibits urinary fibrosis and autophagy via the miR-192-5p/TGFßR1/Smad4 pathway. RAV may be a potential drug for alleviating urethral fibrosis.

Serum-Circulating microRNAs in Sporadic Inclusion Body Myositis.

BACKGROUND: Sporadic inclusion body myositis (s-IBM) represents a unique disease within idiopathic inflammatory myopathies with a dual myodegenerative-autoimmune physiopathology and a lack of an efficacious treatment. Circulating miRNA expression could expand our knowledge of s-IBM patho-mechanisms and provide new potential disease biomarkers. To evaluate the expression of selected pre-amplified miRNAs in the serum of s-IBM patients compared to those of a sex- and age-matched healthy control group, we enrolled 14 consecutive s-IBM patients and 8 sex- and age-matched healthy controls. By using two different normalization approaches, we found one downregulated and three upregulated miRNAs. hsa-miR-192-5p was significantly downregulated, while hsa-miR-372-3p was found to be upregulated more in the s-IBM patients compared to the level of the controls. The other two miRNAs had a very low expression levels (raw Ct data > 29). hsa-miR-192-5p and hsa-miR-372-3p were found to be significantly dysregulated in the serum of s-IBM patients. These miRNAs are involved in differentiation and regeneration processes, thus possibly reflecting pathological mechanisms in s-IBM muscles and potentially representing disease biomarkers.

Promising renoprotective effect of gold nanoparticles and dapagliflozin in diabetic nephropathy via targeting miR-192 and miR-21.

Diabetic nephropathy (DN) is a worldwide issue that eventually leads to end-stage renal failure, with limited therapeutic options. Prior research has revealed that gold nanoparticles (AuNPs) have a substantial antidiabetic impact. In addition, sodium-glucose cotransporter2 (SGLT2) inhibitors, including dapagliflozin (DAPA), had renoprotective impact on DN. Therefore, this research attempted to determine the potential AuNPs and DAPA impacts in ameliorating experimentally DN induction and the underlying mechanisms focusing on miR-192 and miR-21, correlating them with autophagy, apoptosis, fibrosis, and oxidative stress. Diabetes induction was through a single intraperitoneal streptozotocin (55 mg/kg) injection, and rats with diabetes received AuNPs (2.5 mg/kg/day) as well as DAPA (2 mg/kg/day) for 7 weeks as a treatment. AuNPs and DAPA treatment for 7 weeks substantially alleviated DN. AuNPs and DAPA significantly increased catalase (CAT) activity as well as serum total antioxidant capacity (TAC), along with a substantial decline in malondialdehyde (MDA). AuNPs and DAPA treatment alleviated renal fibrosis as they decreased transforming growth factorß1(TGF-ß1) as well as matrix metalloproteinase-2 (MMP-2) renal expression, decreased apoptosis through alleviating the proapoptotic gene (caspase-3) renal expression and increased the antiapoptotic gene (Bcl-2) renal expression, and increased autophagy as they increased LC-3 as well as Beclin-1 renal expression. Autophagy activation, inhibition of apoptosis, and renal fibrosis could be due to their inhibitory impact on miR-192 and miR-21 renal expression. AuNPs and DAPA have a protective effect on DN in rats by targeting miR-192 and miR-21 and their downstream pathways, including fibrosis, apoptosis, autophagy, and oxidative stress.

Rapid and sensitive detection of miRNA via light scatter-aided emulsion-based isothermal amplification using a custom low-cost device.

MicroRNAs are likely to be a next-generation clinical biomarker for many diseases. While gold-standard technologies, e.g., reverse transcription-quantitative polymerase chain reaction (RT-qPCR), exist for microRNA detection, there is a need for rapid and low-cost testing. Here, an emulsion loop-mediated isothermal amplification (eLAMP) assay was developed for miRNA that compartmentalizes a LAMP reaction and shortens the time-to-detection. The miRNA was a primer to facilitate the overall amplification rate of template DNA. Light scatter intensity decreased when the emulsion droplet got smaller during the ongoing amplification, which was utilized to moitor the amplification non-invasively. A custom low-cost device was designed and fabricated using a computer cooling fan, a Peltier heater, an LED, a photoresistor, and a temperature controller. It allowed more stable vortexing and accurate light scatter detection. Three miRNAs, miR-21, miR-16, and miR-192, were successfully detected using the custom device. Specifically, new template and primer sequences were developed for miR-16 and miR-192. Zeta potential measurements and microscopic observations confirmed emulsion size reduction and amplicon adsorption. The detection limit was 0.01 fM, corresponding to 2.4 copies per reaction, and the detection could be made in 5 min. Since the assays were rapid and both template and miRNA + template could eventually be amplified, we introduced the success rate (compared to the 95% confidence interval of the template result) as a new measure, which worked well with lower concentrations and inefficient amplifications. This assay brings us one step closer to allowing circulating miRNA biomarker detection to become commonplace in the clinical world.

Serum exosomal miR-192 serves as a potential detective biomarker for early pregnancy screening in sows.

OBJECTIVE: The study was conducted to screen differentially expressed miRNAs in sows at early pregnancy by high-throughput sequencing and explore its mechanism of action on embryo implantation. METHODS: The blood serum of pregnant and non-pregnant Landrace×Yorkshire sows were collected 14 days after artificial insemination, and exosomal miRNAs were purified for high throughput miRNA sequencing. The expression patterns of 10 differentially expressed (DE) miRNAs were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR quantified the abundance of serum exosomal miR-192 in pregnant and control sows, and the diagnostic power was assessed by receiver operating characteristic (ROC) analysis. The target genes of DE miRNAs were predicted with bioinformatics software, and the functional and pathway enrichment analysis was performed on gene ontology and the Kyoto encyclopedia of genes and genomes terms. Furthermore, a luciferase reporter system was used to identify the target relation between miR-192 and integrin alpha 4 (ITGA4), a gene influencing embryo implantation in pigs. Finally, the expression levels of miRNAs and the target gene ITGA4 were analyzed by qRT-PCR, and western blot, with the proliferation of BeWo cells detected by cell counting kit-8 (CCK-8). RESULTS: A total of 221 known miRNAs were detected in the libraries of the pregnant and non-pregnant sows, of which 55 were up-regulated and 67 were down-regulated in the pregnant individuals compared with the non-pregnant controls. From these, the expression patterns of 10 DE miRNAs were validated. The qRT-PCR analysis further confirmed a significantly higher expression of miR-192 in the serum exosomes extracted from pregnant sows, when compared to controls. The ROC analysis revealed that miR-192 provided excellent diagnostic accuracy for pregnancy (area under the ROC curve [AUC] = 0.843; p>0.001). The dual-luciferase reporter assay indicated that miR-192 directly targeted ITGA4. The protein expression of ITGA4 was reduced in cells that overexpressed miR-192. Overexpression of miR-192 resulted in the decreased proliferation of BeWo cells and regulated the expression of cell cycle-related genes. CONCLUSION: Serum exosomal miR-192 could serve as a potential biomarker for early pregnancy in pigs. miR-192 targeted ITGA4 gene directly, and miR-192 can regulate cellular proliferation.

Exosomal miR-192-5p secreted by bone marrow mesenchymal stem cells inhibits hepatic stellate cell activation and targets PPP2R3A.

Bone marrow mesenchymal stem cell (BSMC)-derived extracellular vehicles (EVs) have a pivotal therapeutic potential in hepatic fibrosis (HF). Activation of hepatic stellate cells (HSCs) is the key mechanism in HF progression. Downregulation of miR-192-5p was previously observed in activated HSCs. Nonetheless, the functions of BSMC-derived exosomal miR-192-5p in activated HSCs remain unclear. In this study, transforming growth factor (TGF)-ß1 was used to activate HSC-T6 cells to mimic HF in vitro. Characterization of BMSCs and BMSC-derived EVs was performed. Cell-counting kit-8 assay, flow cytometry, and western blotting revealed that TGF-ß1 increased cell viability, promoted cell cycle progression, and induced upregulation of fibrosis markers in HSC-T6 cells. Overexpression of miR-192-5p or BMSC-derived exosomal miR-192-5p suppressed TGF-ß1-triggered HSC-T6 cell activation. RT-qPCR revealed that protein phosphatase 2 regulatory subunit B'' alpha (PPP2R3A) was downregulated in miR-192-5p-overexpressed HSC-T6 cells. Luciferase reporter assay was used for verifying the relation between miR-192-5p and PPP2R3A, which showed that miR-192-5p targeted PPP2R3A in activated HSC-T6 cells. Collectively, BMSC-derived exosomal miR-192-5p targets PPP2R3A and inhibits activation of HSC-T6 cells.

Identification of miR-192 target genes in porcine endometrial epithelial cells based on miRNA pull-down.

INTRODUCTION: MicroRNAs (miRNAs)-a class of small endogenous non-coding RNAs-are widely involved in post-transcriptional gene regulation of numerous physiological processes. High-throughput sequencing revealed that the miR-192 expression level appeared to be significantly higher in the blood exosomes of sows at early gestation than that in non-pregnant sows. Furthermore, miR-192 was hypothesized to have a regulatory role in embryo implantation; however, the target genes involved in exerting the regulatory function of miR-192 required further elucidation. METHODS: In the present study, potential target genes of miR-192 in porcine endometrial epithelial cells (PEECs) were identified through biotin-labeled miRNA pull-down; functional and pathway enrichment analysis was performed via gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Bioinformatic analyses were concurrently used to predict the potential target genes associated with sow embryo implantation. In addition, double luciferase reporter vectors, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and Western blot were performed to verify the targeting and regulatory roles of the abovementioned target genes. RESULTS: A total of 1688 differentially expressed mRNAs were identified via miRNA pull-down. Through RT-qPCR, the accuracy of the sequencing data was verified. In the bioinformatics analysis, potential target genes of miR-192 appeared to form a dense inter-regulatory network and regulated multiple signaling pathways, such as metabolic pathways and the PI3K-Akt, MAPKs, and mTOR signaling pathways, that are relevant to the mammalian embryo implantation process. In addition, CSK (C-terminal Src kinase) and YY1 (Yin-Yang-1) were predicted to be potential candidates, and we validated that miR-192 directly targets and suppresses the expression of the CSK and YY1 genes. CONCLUSION: We screened 1688 potential target genes of miR-192 were screened, and CSK and YY1 were identified as miR-192 target genes. The outcomes of the present study provide novel insights into the regulatory mechanism of porcine embryo implantation and the identification of miRNA target genes.

Up-regulation of miR-192-5p inhibits the ELAVL1/PI3Kδ axis and attenuates microvascular endothelial cell proliferation, migration and angiogenesis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a common complication of diabetes mellitus that poses a threat to adults. MicroRNAs (miRNAs) play a key role in DR progression. However, the role and mechanism of miR-192-5p in DR remain unclear. We aimed to investigate the effect of miR-192-5p on cell proliferation, migration and angiogenesis in DR. METHODS: Expression of miR-192-5p, ELAV-like RNA binding protein 1 (ELAVL1) and phosphoinositide 3-kinase delta (PI3Kδ) in human retinal fibrovascular membrane (FVM) samples and human retinal microvascular endothelial cells (HRMECs) was assessed using RT-qPCR. ELAVL1 and PI3Kδ protein levels were evaluated by Western blot. RIP and dual luciferase reporter assays were performed to confirm the miR-192-5p/ELAVL1/PI3Kδ regulatory networks. Cell proliferation, migration and angiogenesis were assessed by CCK8, transwell and tube formation assays. RESULTS: MiR-192-5p was decreased in FVM samples from DR patients and high glucose (HG)-treated HRMECs. Functionally, overexpressed miR-192-5p inhibited cell proliferation, migration and angiogenesis in HG-treated HRMECs. Mechanically, miR-192-5p directly targeted ELAVL1 and decreased its expression. We further verified that ELAVL1 bound to PI3Kδ and maintained PI3Kδ mRNA stability. Rescue analysis demonstrated that the suppressive effects of HG-treated HRMECs caused by miR-192-5p up-regulation were overturned by overexpressed ELAVL1 or PI3Kδ. CONCLUSION: MiR-192-5p attenuates DR progression by targeting ELAVL1 and reducing PI3Kδ expression, suggesting a biomarker for the treatment of DR.

Diagnostic Significance of hsa-miR-21-5p, hsa-miR-192-5p, hsa-miR-155-5p, hsa-miR-199a-5p Panel and Ratios in Hepatocellular Carcinoma on Top of Liver Cirrhosis in HCV-Infected Patients.

Early hepatocellular carcinoma (HCC) diagnosis is challenging. Moreover, for patients with alpha-fetoprotein (AFP)-negative HCC, this challenge is augmented. MicroRNAs (miRs) profiles may serve as potential HCC molecular markers. We aimed to assess plasma homo sapiens-(hsa)-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p-expression levels as a panel of biomarkers for HCC in chronic hepatitis C virus (CHCV) patients with liver cirrhosis (LC), especially AFP-negative HCC cases, as a step toward non-protein coding (nc) RNA precision medicine. SUBJECTS AND METHODS: 79 patients enrolled with CHCV infection with LC, subclassified into an LC group without HCC (n = 40) and LC with HCC (n = 39). Real-time quantitative PCR was used to measure plasma hsa-miR-21-5p, hsa-miR-155-5p, hsa-miR-192-5p, and hsa-miR-199a-5p. RESULTS: Plasma hsa-miR-21-5p and hsa-miR-155-5p demonstrated significant upregulation, while hsa-miR-199a-5p demonstrated significant downregulation in the HCC group (n = 39) when compared to the LC group (n = 40). hsa-miR-21-5p expression was positively correlated with serum AFP, insulin, and insulin resistance (r = 0.5, p < 0.001, r = 0.334, p = 0.01, and r = 0.303, p = 0.02, respectively). According to the ROC curves, for differentiating HCC from LC, combining AFP with each of hsa-miR-21-5p, hsa-miR-155-5p, and miR199a-5p improved the diagnostic sensitivity to 87%, 82%, and 84%, respectively, vs. 69% for AFP alone, with acceptable specificities of 77.5%, 77.5%, and 80%, respectively, and AUC = 0.89, 0.85, and 0.90, respectively vs. 0.85 for AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios discriminated HCC from LC at AUC = 0.76 and 0.71, respectively, with sensitivities = 94% and 92% and specificities = 48% and 53%, respectively. Upregulation of plasma hsa-miR-21-5p was considered as an independent risk factor for HCC development [OR = 1.198(1.063-1.329), p = 0.002]. CONCLUSIONS: Combining each of hsa-miR-21-5p, hsa-miR-155-5p, and hsa-miR-199a-5p with AFP made it possible to identify HCC development in the LC patients' cohort with higher sensitivity than using AFP alone. hsa-miR-21-5p/hsa-miR-199a-5p and hsa-miR-155-5p/hsa-miR-199a-5p ratios are potential HCC molecular markers for AFP-negative HCC patients. hsa-miR-21-5p was linked, clinically and via in silico proof, to insulin metabolism, inflammation, dyslipidemia, and tumorigenesis in the HCC patients' group as well as for an upregulated independent risk factor for the emergence of HCC from LC in the CHCV patients.