ST3GAL1 Promotes Malignant Phenotypes in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis, and elucidation of the molecular mechanisms underlying iCCA malignancy is of great significance. Glycosylation, an important post-translational modification, is closely associated with tumor progression. Altered glycosylation, including aberrant sialylation resulting from abnormal expression of sialyltransferases (STs) and neuraminidases (NEUs), is a significant feature of cancer cells. However, there is limited information on the roles of STs and NEUs in iCCA malignancy. Here, utilizing our proteogenomic resources from a cohort of 262 patients with iCCA, we identified ST3GAL1 as a prognostically relevant molecule in iCCA. Moreover, overexpression of ST3GAL1 promoted proliferation, migration, and invasion and inhibited apoptosis of iCCA cells in vitro. Through proteomic analyses, we identified the downstream pathway potentially regulated by ST3GAL1, which was the NF-κB signaling pathway, and further demonstrated that this pathway was positively correlated with malignancy in iCCA cells. Notably, glycoproteomics showed that O-glycosylation was changed in iCCA cells with high ST3GAL1 expression. Importantly, the altered O-glycopeptides underscored the potential utility of O-glycosylation profiling as a discriminatory marker for iCCA cells with ST3GAL1 overexpression. Additionally, miR-320b was identified as a post-transcriptional regulator of ST3GAL1, capable of suppressing ST3GAL1 expression and then reducing the proliferation, migration, and invasion abilities of iCCA cell lines. Taken together, these results suggest ST3GAL1 could serve as a promising therapeutic target for iCCA.

THUMPD3-AS1 inhibits ovarian cancer cell apoptosis through the miR-320d/ARF1 axis.

Ovarian cancer is one of the most common gynecologic malignancies that has a poor prognosis. THUMPD3-AS1 is an oncogenic long noncoding RNA (lncRNA) in several cancers. Moreover, miR-320d is downregulated and inhibited proliferation in ovarian cancer cells, whereas ARF1 was upregulated and promoted the malignant progression in epithelial ovarian cancer. Nevertheless, the role of THUMPD3-AS1 in ovarian cancer and the underlying mechanism has yet to be elucidated. Human normal ovarian epithelial cells (IOSE80) and ovarian cancer cell lines (CAVO3, A2780, SKOV3, OVCAR3, and HEY) were adopted for in vitro experiments. The functional roles of THUMPD3-AS1 in cell viability and apoptosis were determined using CCK-8, flow cytometry, and TUNEL assays. Western blot was performed to assess the protein levels of ARF1, Bax, Bcl-2, and caspase 3, whereas RT-qPCR was applied to measure ARF1 mRNA, THUMPD3-AS1, and miR-320d levels. The targeting relationship between miR-320d and THUMPD3-AS1 or ARF1 was validated with dual luciferase assay. THUMPD3-AS1 and ARF1 were highly expressed in ovarian cancer cells, whereas miR-320d level was lowly expressed. THUMPD3-AS1 knockdown was able to repress cell viability and accelerate apoptosis of OVCAR3 and SKOV3 cells. Also, THUMPD3-AS1 acted as a sponge of miR-320d, preventing the degradation of ARF1. MiR-320d downregulation reversed the tumor suppressive function induced by THUMPD3-AS1 depletion. Additionally, miR-320d overexpression inhibited ovarian cancer cell viability and accelerated apoptosis, which was overturned by overexpression of ARF1. THUMPD3-AS1 inhibited ovarian cancer cell apoptosis by modulation of miR-320d/ARF1 axis. The discoveries might provide a prospective target for ovarian cancer treatment.

Decreased Serum Levels of the Insulin Resistance-Related microRNA miR-320a in Patients with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is often associated with metabolic abnormalities in the affected patients such as obesity or a dysregulated glucose metabolism/insulin resistance (IR). IR affects the serum levels of several circulating microRNAs; however, studies on the association between IR-related microRNAs and PCOS are scarce. Therefore, we quantified the serum levels of the IR-associated microRNAs miR-93, miR-148a, miR-216a, miR-224 and miR-320a via qPCR in a cohort of 358 infertility patients, of whom 136 were diagnosed with PCOS. In bivariate correlation analyses, the serum levels of miR-93 and miR-216a were inversely associated with dipeptidyl peptidase 4 serum concentrations, and the miR-320a serum levels were significantly downregulated in PCOS patients (p = 0.02, Mann-Whitney U test). Interestingly, in all patients who achieved pregnancy after Assisted Reproductive Technology (ART) cycles, the serum levels of the five IR-associated microRNAs were significantly elevated compared to those of non-pregnant patients. In cell culture experiments, we detected a significant upregulation of miR-320a expression following testosterone stimulation over 24 and 48 h in KGN and COV434 granulosa carcinoma cells. In conclusion, we demonstrated a significantly reduced serum level of the IR-associated miR-320a in our patient cohort. This result once again demonstrates the close relationship between metabolic disorders and the dysregulation of microRNA expression patterns in PCOS.

Human umbilical cord mesenchymal stem cells-derived exosomal circDLGAP4 promotes angiogenesis after cerebral ischemia-reperfusion injury by regulating miR-320/KLF5 axis.

Accumulating evidence suggests that human umbilical cord mesenchymal stem cell-derived exosomes (hUC-MSCs-Exos) are a promising therapeutic strategy for cerebral ischemia-reperfusion injury (CIRI). However, the underlying mechanism remains unclear. hUC-MSCs-Exos were identified by electron microscopy, NTA, and Western blotting. In the hypoxia/reoxygenation (H/R) cell model, human brain microvascular endothelial cells (HBMECs) were cocultured with hUC-MSCs-Exos. Then, cell viability, migration, apoptosis, and tube formation were measured by MTT, flow cytometry, transwell, and tube formation assays. RT-qPCR and Western blotting were used to detect the changes in RNA and protein. RNA pull-down and dual luciferase reporter assays confirmed the relationship between circDLGAP4, miR-320, and KLF5. Ischemia-reperfusion (I/R) rat model was established for in vivo experiments. hUC-MSCs-Exos increased the expression levels of circDLGAP4 and KLF5 but decreased miR-320 in H/R-treated HBMECs by transferring exosomal circDLGAP4. Knockdown of circDLGAP4 in hUC-MSCs-Exos reversed the promoting effects of hUC-MSCs-Exos on cell viability, migration, and tube formation in H/R-treated HBMECs in vitro and also abolished the protective effects of hUC-MSCs-Exos on cerebrovascular injury in I/R rats. Mechanistically, exosomal circDLGAP4 negatively regulated miR-320 in HBMECs, which directly bound to KLF5. In addition, the downregulation of miR-320 could reverse the regulatory effect of exosomal shcircDLGAL5 in H/R-treated HBMECs by upregulating KLF5. hUC-MSCs-Exos-derived circDLGAP4 reduced cerebrovascular injury by regulating miR-320/KLF5 signaling. These results provide a stem cell-based approach to treat CIRI.

Tumor-derived small extracellular vesicles facilitate omental metastasis of ovarian cancer by triggering activation of mesenchymal stem cells.

BACKGROUND: Omental metastasis is the major cause of ovarian cancer recurrence and shortens patient survival, which can be largely attributed to the dynamic evolution of the fertile metastatic microenvironment driven by cancer cells. Previously, we found that adipose-derived mesenchymal stem cells (ADSCs) undergoing a phenotype shift toward cancer-associated fibroblasts (CAFs) participated in the orchestrated omental premetastatic niche for ovarian cancer. Here, we aim to elucidate the underlying mechanisms. METHODS: Small extracellular vesicles were isolated from ovarian cancer cell lines (ES-2 and its highly metastatic subline, ES-2-HM) and patient ascites using ultracentrifugation. Functional experiments, including Transwell and EdU assays, and molecular detection, including Western blot, immunofluorescence, and RT-qPCR, were performed to investigate the activation of ADSCs in vitro. High-throughput transcriptional sequencing and functional assays were employed to identify the crucial functional molecules inducing CAF-like activation of ADSCs and the downstream effector of miR-320a. The impact of extracellular vesicles and miR-320a-activated ADSCs on tumor growth and metastasis was assessed in subcutaneous and orthotopic ovarian cancer xenograft mouse models. The expression of miR-320a in human samples was evaluated using in situ hybridization staining. RESULTS: Primary human ADSCs cocultured with small extracellular vesicles, especially those derived from ES-2-HM, exhibited boosted migration, invasion, and proliferation capacities and elevated α-SMA and FAP levels. Tumor-derived small extracellular vesicles increased α-SMA-positive stromal cells, fostered omental metastasis, and shortened the survival of mice harboring orthotopic ovarian cancer xenografts. miR-320a was abundant in highly metastatic cell-derived extracellular vesicles, evoked dramatic CAF-like transition of ADSCs, targeted the 3'-untranslated region of integrin subunit alpha 7 and attenuated its expression. miR-320a overexpression in ovarian cancer was associated with omental metastasis and shorter survival. miR-320a-activated ADSCs facilitated tumor cell growth and omental metastasis. Depletion of integrin alpha 7 triggered CAF-like activation of ADSCs in vitro. Video Abstract CONCLUSIONS: miR-320a in small extracellular vesicles secreted by tumor cells targets integrin subunit alpha 7 in ADSCs and drives CAF-like activation, which in turn facilitates omental metastasis of ovarian cancer.

MiR-320a Acts as a Tumor Suppressor in Somatotroph Pituitary Neuroendocrine Tumors by Targeting BCAT1.

INTRODUCTION: Aberrant miR-320a has been reported to be involved in the tumorigenesis of several cancers. In our previous study, we identified the low expression of circulating miR-320a in patients with somatotroph pituitary neuroendocrine tumor (PitNET); however, the role of miR-320a in somatotroph PitNET proliferation is still unclear. METHODS: Cell viability and colony formation assays were used to detect the effect of miR-320a and BCAT1 on GH3 cells. TargetScan was used to identify the target genes of miR-320a. Dual-luciferase reporter gene assay was used to explore the relation between miR-320a and BCAT1. Transcriptome and proteome analyses were performed between somatotroph PitNETs and healthy controls. The expression level of miR-320a in somatotroph PitNETs were detected by RT-qPCR and Western blot. RESULTS: miR-320a mimics inhibit cell proliferation, while miR-320a inhibitors promote cell proliferation in GH3 cells. An overlap analysis using a Venn diagram revealed that BCAT1 is the only target gene of miR-320a overexpressed in somatotroph PitNETs compared to healthy controls, as revealed by both microarray and proteomics results. A dual-luciferase reporter gene assay showed that miR-320a may bind to the BCAT1-3'UTR. The transfection of miR-320a mimics downregulated the expression and miR-320a inhibitors and upregulated the expression of BCAT1 in GH3 cells. The interference of BCAT1 expression in GH3 cells downregulated cell proliferation and growth. Pan-cancer analyses demonstrated that high BCAT1 expression often indicates a poor prognosis. CONCLUSION: Our findings illustrate that miR-320a may function as a tumor suppressor and BCAT1 may promote tumor progression. miR-320a may inhibit the growth of somatotroph PitNETs by targeting BCAT1.

MiR-320 inhibits PRRSV replication by targeting PRRSV ORF6 and porcine CEBPB.

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), results in huge economic losses to the world pig industry. MiRNAs have been reported to be involved in regulation of viral infection. In our study, miR-320 was one of 21 common differentially expressed miRNAs of Meishan, Pietrain, and Landrace pig breeds at 9-h post-infection (hpi). Bioinformatics and experiments found that PRRSV replication was inhibited by miR-320 through directly targeting PRRSV ORF6. In addition, the expression of CCAAT enhancer binding protein beta (CEBPB) was also inhibited by miR-320 by targeting the 3' UTR of CEBPB, which significantly promotes PRRSV replication. Intramuscular injection of pEGFP-N1-miR-320 verified that miR-320 significantly inhibited the replication of PRRSV and alleviated the symptoms caused by PRRSV in piglets. Taken together, miR-320 have significant roles in the infection and may be promising therapeutic target for PRRS.

Icariin alleviates diabetic renal interstitial fibrosis aggravation by inhibiting miR-320a-3p targeting BMP6.

Diabetic nephropathy is a common complication of diabetes, accumulating evidence underscores the pivotal role of tubulointerstitial fibrosis in the progression of diabetic nephropathy. However, the underlying mechanisms remain incompletely understood. Although the mechanisms in diabetic nephropathy fibrosis have been the focus of many studies, only limited information is currently available concerning microRNA regulation in tubulointerstitial fibrosis. In this study, we aimed to investigate the roles of miR-320a-3p and bone morphogenetic protein-6 (BMP6) in tubulointerstitial fibrosis. After inducing fibrosis with high glucose in HK-2 cells, we found that miR-320a-3p is significantly up-regulated, whereas BMP6 is markedly down-regulated. These changes suggest close link between miR-320a-3p and BMP6 in tubulointerstitial fibrosis. To elucidate this phenomenon, miR-320a-3p mimic, inhibitor and siBMP6 were employed. We observed in miR-320a-3p mimic group the fibrosis marker include alpha smooth muscle actin and type I collagen was significantly up-regulated, whereas BMP6 exhibited the opposite trend. Additionally, we found icariin could alleviate tubulointerstitial fibrosis by downregulation the miR-320a-3p expression. In conclusion, miR-320a-3p promotes tubulointerstitial fibrosis during the development of DN by suppressing BMP signal pathway activity via inhibiting BMP6 expression. Suggesting that miR-320a-3p represents a potential therapeutic target for tubulointerstitial fibrosis induced by diabetic nephropathy.

MiR-320a upregulation contributes to the effectiveness of pemetrexed by inhibiting the growth and invasion of human lung cancer cell line (Calu-6).

BACKGROUND: Lung cancer is a common and deadly disease. Chemotherapy is the most common treatment, which inhibits cancer cell growth. Pemetrexed (PMX) is often used with other drugs. Environmental stress can affect regulatory non-coding RNAs such as MicroRNAs that modify gene expression. This study investigates the effect of PMX on the hsa-miR-320a-3p expression in the Calu-6 lung cancer cell line. METHODS AND RESULT: Calu-6 cells were cultured in an incubator with 37 °C, 5% CO2, and 98% humidity. The MTT test was performed to determine the concentration of PMX required to inhibit 50% of cell growth. To examine growth inhibition and apoptosis, release of lactate dehydrogenase (LDH), cell assays and caspase 3 and 7 enzyme activity were used. Finally, molecular studies were conducted to compare the expression of hsa-miR-320a-3p and genes including VDAC1, DHFR, STAT3, BAX and BCL2 before and after therapy. RESULTS: According to a study, it has been observed that PMX therapy significantly increases LDH release after 24 h. The study found that PMX's IC50 on Calu-6 is 8.870 µM. In addition, the treated sample showed higher expression of hsa-miR-320a-3p and BAX, while the expression of VDAC1, STAT3, DHFR and BCL2 decreased compared to the control sample. CONCLUSION: According to the findings of the current research, hsa-miR-320a-3p seems to have the potential to play an important role in the development of novel approaches to the treatment of lung cancer.

Diagnostic and prognostic significance of miR-320a-3p in patients with chronic heart failure.

AIM: The purpose of this study was to investigate the diagnostic and prognostic value of miR-320a-3p in chronic heart failure (CHF). METHODS: A total of 103 patients with CHF and 95 healthy controls were included in the study population. The expression level of serum miR-320a-3p was detected by qRT-PCR. The diagnostic effect of miR-320a-3p on CHF was evaluated by receiver operating characteristic curve. Kaplan-Meier curve and Cox regression were used to analyze the risk factors for 4-year prognosis of CHF patients. Bioinformatics analysis was used to analyze the possible target genes of miR-320a-3p and related signaling pathways. RESULTS: Serum miR-320a-3p expression was increased in CHF patients, and the levels of BNP and LVEF were positively and negatively correlated with miR-320a-3p, respectively. The AUC value of ROC curve was 0.866, indicating that miR-320a-3p had high diagnostic accuracy for CHF. Survival curve and Cox analysis showed that high expression of miR-320a-3p was associated with poor prognosis in CHF patients, and age and miR-320a-3p were independent risk factors for prognosis in CHF patients. GO and KEGG analysis showed that the downstream target genes of miR-320a-3p were involved in biological processes such as cell adhesion, stem cell differentiation and neural development, and were enriched in mTOR, TNF, AMPK and other signaling pathways. CONCLUSIONS: miR-320a-3p increased abnormally in CHF and was related to the severity of CHF. miR-320a-3p has the potential to be a diagnostic and prognostic marker for CHF.

Circular RNA-circPan3 attenuates cardiac hypertrophy via miR-320-3p/HSP20 axis.

BACKGROUND: Circular RNAs are enriched in cardiac tissue and play important roles in the pathogenesis of heart diseases. In this study, we aimed to investigate the regulatory mechanism of a conserved heart-enriched circRNA, circPan3, in cardiac hypertrophy. METHODS: Cardiac hypertrophy was induced by isoproterenol. The progression of cardiomyocyte hypertrophy was assessed by sarcomere organization staining, cell surface area measurement, and expression levels of cardiac hypertrophy markers. RNA interactions were detected by RNA pull-down assays, and methylated RNA immunoprecipitation was used to detect m6A level. RESULTS: The expression of circPan3 was downregulated in an isoproterenol-induced cardiac hypertrophy model. Forced expression of circPan3 attenuated cardiomyocyte hypertrophy, while inhibition of circPan3 aggravated cardiomyocyte hypertrophy. Mechanistically, circPan3 was an endogenous sponge of miR-320-3p without affecting miR-320-3p levels. It elevated the expression of HSP20 by endogenously interacting with miR-320-3p. In addition, circPan3 was N6-methylated. Stimulation by isoproterenol downregulated the m6A eraser ALKBH5, resulting in N6-methylation and destabilization of circPan3. CONCLUSIONS: Our research is the first to report that circPan3 has an antihypertrophic effect in cardiomyocytes and revealed a novel circPan3-modulated signalling pathway involved in cardiac hypertrophy. CircPan3 inhibits cardiac hypertrophy by targeting the miR-320-3p/HSP20 axis and is regulated by ALKBH5-mediated N6-methylation. This pathway could provide potential therapeutic targets for cardiac hypertrophy.

Identification of MicroRNA Profiles in Fetal Spina Bifida: The Role in Pathomechanism and Diagnostic Significance.

Distinct miRNA expression patterns may reflect anomalies related to fetal congenital malformations such as spinal bifida (SB). The aim of this preliminary study was to determine the maternal miRNA expression profile of women carrying fetuses with SB. Therefore, six women carrying fetuses with SB and twenty women with euploid healthy fetuses were enrolled in this study. Using NanoString technology, we evaluated the expression level of 798 miRNAs in both plasma and amniotic fluid samples. A downregulation of miR-1253, miR-1290, miR-194-5p, miR-302d-3p, miR-3144-3p, miR-4536-5p, miR-548aa + miR-548t-3p, miR-548ar-5p, miR-548n, miR-590-5p, miR-612, miR-627-5p, miR-644a, and miR-122-5p, and an upregulation of miR-320e, let-7b-5p, miR-23a-3p, miR-873-3p, and miR-30d-5p were identified in maternal amniotic fluid samples in SB when compared to the control group. The target genes of these miRNAs play a predominant role in regulating the synthesis of several biological compounds related to signaling pathways such as those regulating the pluripotency of stem cells. Moreover, the maternal plasma expression of miR-320e was increased in pregnancies with SB, and this marker could serve as a valuable non-invasive screening tool. Our results highlight the SB-specific miRNA signature and the differentially expressed miRNAs that may be involved in SB pathogenesis. Our findings emphasize the role of miRNA as a predictive factor that could potentially be useful in prenatal genetic screening for SB.

Platelet Microparticle-Derived MiR-320b Inhibits Hypertension with Atherosclerosis Development by Targeting ETFA.

Hypertension and atherosclerosis often occur simultaneously. This study aimed to explore the role and mechanism of platelet microparticle (PMP) -derived microRNA-320b (miR-320b) in patients with hypertension accompanied by atherosclerosis.We collected samples from 13 controls without hypertension and atherosclerosis and 20 patients who had hypertension accompanied by atherosclerosis. In vitro, platelets were activated by Thrombin receptor-activating peptide to produce PMPs. HUVECs were induced by CoCl2 to mimic a hypoxic environment in vitro. RT-qPCR was employed to detect the expression levels of CD61, miR-320b, and ETFA. The protein expression level of ETFA was evaluated via Western blotting. Furthermore, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, 5-ethynyl-2'-deoxyuridine, and wound healing assays were employed to assess the proliferation and migration of HUVECs. Enzyme-linked immunosorbent assay was used to measure the oxidative stress and inflammation-related factor expression.The expression of miR-320b was reduced in both platelets and PMPs but increased in plasma. MiR-320b promoted CoCl2-induced HUVEC viability, proliferation, and migration. The levels of the oxidative stress factors SOD and GSH as well as the inflammatory factor IL-10 were elevated in the CoCl2 + miR-320b mimics group compared with both the CoCl2 + mimics NC and CoCl2 groups. Conversely, the levels of the oxidative stress factors MDA and ROS as well as the inflammatory factors IL-6, TNF-α, and IL-1ß were decreased. These results were regulated by miR-320b targeting ETFA.PMP-derived miR-320b inhibits the development of hypertension accompanied by atherosclerosis by targeting ETFA.

Early miR-320b and miR-25-3p miRNA levels correlate with multiple sclerosis severity at 10 years: a cohort study.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating autoimmune disorder which may cause long-term disability. MicroRNA (miRNA) are stable, non-coding molecules that have been identified in our Comprehensive Longitudinal Investigation of Multiple Sclerosis at the Brigham and Women's Hospital (CLIMB)-cohort, as well as other international cohorts, as potential disease biomarkers in MS. However, few studies have evaluated the association of miRNA expression early in the MS disease course with long-term outcomes. Therefore, we aimed to evaluate the potential role of three candidate serum miRNAs previously correlated with MS disability in patients with MS, miR-320b, miR-25-3p and miRNA 486-5p, as early biomarkers of MS disability at 10-year follow-up. MAIN BODY: We included 144 patients with serum obtained within three years of MS onset. miRNA expression was measured by RNA extraction followed by RT-PCR. Demographic, clinical, brain MRI and other biomarkers were collected. The primary outcome was the association between early miRNA expression and retaining benign MS, defined as EDSS ≤ 2 at 10-year follow-up. Among the 144 patients, 104 were benign and 40 were not benign at 10-year follow-up. 89 (62%) were women, with mean age at onset 37.7 (SD: 9.6) years. Patients who retained benign MS had lower values of miR-25-3p (p = 0.047) and higher miR-320b (p = 0.025) values. Development of SPMS was associated with higher miR-320b (p = 0.002) levels. Brain parenchymal fraction at year 10 was negatively correlated with miR-25-3p (p = 0.0004) and positively correlated with miR-320b (p = 0.006). No association was found between miR-486-5p and any outcome, and 10-year T2-lesion volume was not associated with any miRNA. CONCLUSIONS: Our results show that miR-320b and miR-25-3p expression are early biomarkers associated with MS severity and brain atrophy. This study provides class III evidence of that miR-320b and miR-25-3p are associated with long-term MS disability which may be a potential tool to risk-stratify patients with MS for early treatment decisions.

Cold exposure protects against medial arterial calcification development via autophagy.

Medial arterial calcification (MAC), a systemic vascular disease different from atherosclerosis, is associated with an increased incidence of cardiovascular events. Several studies have demonstrated that ambient temperature is one of the most important factors affecting cardiovascular events. However, there has been limited research on the effect of different ambient temperatures on MAC. In the present study, we showed that cold temperature exposure (CT) in mice slowed down the formation of vitamin D (VD)-induced vascular calcification compared with room temperature exposure (RT). To investigate the mechanism involved, we isolated plasma-derived exosomes from mice subjected to CT or RT for 30 days (CT-Exo or RT-Exo, respectively). Compared with RT-Exo, CT-Exo remarkably alleviated the calcification/senescence formation of vascular smooth muscle cells (VSMCs) and promoted autophagy by activating the phosphorylation of AMP-activated protein kinase (p-AMPK) and inhibiting phosphorylation of mammalian target of rapamycin (p-mTOR). At the same time, CT-Exo promoted autophagy in ß-glycerophosphate (ß-GP)-induced VSMCs. The number of autophagosomes and the expression of autophagy-related proteins ATG5 and LC3B increased, while the expression of p62 decreased. Based on a microRNA chip microarray assay and real-time polymerase chain reaction, miR-320a-3p was highly enriched in CT-Exo as well as thoracic aortic vessels in CT mice. miR-320a-3p downregulation in CT-Exo using AntagomiR-320a-3p inhibited autophagy and blunted its anti-calcification protective effect on VSMCs. Moreover, we identified that programmed cell death 4 (PDCD4) is a target of miR-320a-3p, and silencing PDCD4 increased autophagy and decreased calcification in VSMCs. Treatment with CT-Exo alleviated the formation of MAC in VD-treated mice, while these effects were partially reversed by GW4869. Furthermore, the anti-arterial calcification protective effects of CT-Exo were largely abolished by AntagomiR-320a-3p in VD-induced mice. In summary, we have highlighted that prolonged cold may be a good way to reduce the incidence of MAC. Specifically, miR-320a-3p from CT-Exo could protect against the initiation and progression of MAC via the AMPK/mTOR autophagy pathway.

Human Breast Milk miRNAs: Their Diversity and Potential for Preventive Strategies in Nutritional Therapy.

The endogenous miRNAs of breast milk are the products of more than 1000 nonprotein-coding genes, giving rise to mature small regulatory molecules of 19-25 nucleotides. They are incorporated in macromolecular complexes, loaded on Argonaute proteins, sequestrated in exosomes and lipid complexes, or present in exfoliated cells of epithelial, endothelial, or immune origins. Their expression is dependent on the stage of lactation; however, their detection depends on progress in RNA sequencing and the reappraisal of the definition of small RNAs. Some miRNAs from plants are detected in breast milk, opening the possibility of the stimulation of immune cells from the allergy repertoire. Each miRNA harbors a seeding sequence, which targets mRNAs, gene promoters, or long noncoding RNAs. Their activities depend on their bioavailability. Efficient doses of miRNAs are estimated to be roughly 100 molecules in the cytoplasm of target cells from in vitro and in vivo experiments. Each miRNA is included in networks of stimulation/inhibition/sequestration, driving the expression of cellular phenotypes. Three types of stress applied during lactation to manipulate miRNA supply were explored using rodent offspring: a foster mother, a cafeteria diet, and early weaning. This review presents the main mature miRNAs described from current mothers' cohorts and their bioavailability in experimental models as well as studies assessing the potential of miR-26 or miR-320 miRNA families to alter offspring phenotypes.

Circulatory miRNAs as Correlates of Elevated Intra-Pancreatic Fat Deposition in a Mixed Ethnic Female Cohort: The TOFI_Asia Study.

Ectopic lipid accumulation, including intra-pancreatic fat deposition (IPFD), exacerbates type 2 diabetes risk in susceptible individuals. Dysregulated circulating microRNAs (miRNAs) have been identified as correlating with clinical measures of pancreatitis, pancreatic cancer and type 1 diabetes. The aim of the current study was therefore to examine the association between circulating abundances of candidate miRNAs, IPFD and liver fat deposition as quantified using magnetic resonance imaging (MRI) and spectroscopy (MRS). Asian Chinese (n = 34; BMI = 26.7 ± 4.2 kg/m2) and European Caucasian (n = 34; BMI = 28.0 ± 4.5 kg/m2) females from the TOFI_Asia cohort underwent MRI and MRS analysis of pancreas (MR-%IPFD) and liver fat (MR-%liver fat), respectively, to quantify ectopic lipid deposition. Plasma miRNA abundances of a subset of circulatory miRNAs associated with IPFD and liver fat deposition were quantified by qRT-PCR. miR-21-3p and miR-320a-5p correlated with MR-%IPFD, plasma insulin and HOMA2-IR, but not MR-%liver fat. MR-%IPFD remained associated with decreasing miR-21-3p abundance following multivariate regression analysis. miR-21-3p and miR-320a were demonstrated to be negatively correlated with MR-%IPFD, independent of ethnicity. For miR-21-3p, this relationship persists with the inclusion of MR-%liver fat in the model, suggesting the potential for a wider application as a specific circulatory correlate of IPFD.

MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks.

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process-the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor-via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.

Circ_NNT suppresses the apoptosis and inflammation in glucose-induced human retinal pigment epithelium by regulating miR-320b/TIMP3 axis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a frequent complication of diabetes. Recent reports have showed that circular RNAs (circRNAs) play important roles in DR progression. Herein, the aim of this study was to explore the role and molecular mechanism of circ_NNT in DR process. METHODS: Human retinal pigment epithelial cells ARPE-19 were treated with high glucose (HG) in experimental group. The expression of circ_NNT, miR-320b, and TIMP3 (TIMP Metallopeptidase Inhibitor 3) were determined using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments were conducted by 5-ethynyl-2'-deoxyuridine (EdU) assay, MTT assay, flow cytometry, Western blot, and ELISA. The binding interaction was confirmed using dual-luciferase reporter and pull-down assays. RESULTS: HG stimulation led to a decrease of circ_NNT and TIMP3 expression, and an increase of miR-320b expression in ARPE-19 cells. Functionally, circ_NNT up-regulation reversed HG-evoked apoptosis and inflammation in ARPE-19 cells. Mechanistically, circ_NNT acted as a sponge for miR-320b to elevate TIMP3 expression. Further rescue experiments showed that miR-320b elevation attenuated the protective effects of circ_NNT on HG-induced ARPE-19 cells. Moreover, inhibition of miR-320b protected ARPE-19 cells against HG-evoked apoptosis and inflammation, which were abolished by TIMP3 knockdown. CONCLUSION: Circ_NNT protected ARPE-19 cells against HG-evoked apoptosis and inflammation via elevating TIMP3 through sequestering miR-320b, indicating that up-regulation of circ_NNT might contribute to the inhibition of DR process.

miR-320a promotes p53-dependent apoptosis of prostate cancer cells by negatively regulating TP73-AS1 invitro.

TP73 antisense RNA 1 (TP73-AS1) is an oncogenic long non-coding RNA that is activated in several types of cancers. It has been shown that the activity of TP73-AS1 is controlled by several miRNAs, but post-transcriptional mechanisms that regulate TP73-AS1 activity in prostate cancer remain highly elusive. Accordingly, in the present study, we aimed to determine the miRNAs that are involved in the regulation of TP73-AS1 in prostate cancer and to show the effects of these molecules on the malignant proliferation of prostate cancer cells. Remarkably, colony formation and cell migration were suppressed while cell cycle arrest and apoptosis were induced in prostate cancer cells overexpressing miR-200a and miR-320a. miR-200a and miR-320a were found to be upregulated in TP73-AS1 suppressed prostate cancer cells. Also, TP73-AS1 was shown to be downregulated following miR-200a and miR-320a overexpression. However, overexpression of miR-320a had no significant effect on the expression of TP73. Further analysis revealed that miR-320a induces p53-dependent apoptosis. Consequently, our findings indicate that miR-320a induces p53-dependent apoptosis by negatively regulating TP73-AS1 long non-coding RNA.