MiR-30a-5p isoform -1|1 promotes the progression of gastric cancer by inhibiting TMEM66 and reducing intratumoral cytotoxic T cells.

Gastric cancer is histologically classified into the intestinal subtype, which forms tubular structures, and the aggressive diffuse subtype, characterized by rapid invasion and poor prognosis. The variety and quantity of miRNA isoforms between different histological subtypes of gastric cancer were unknown. Through systematic filtering, we found that more diverse miR-30a-5p isoforms was present in the diffuse subtype of gastric cancer, and was associated with patients' worse survival independent of tumor stage based on the TCGA miRNA-seq data. Among all nine isoforms of miR-30a-5p, miR-30a-5p -1|1 was more abundant than the archetype of miR-30a-5p. Higher expression of miR-30a-5p -1|1 was observed in patients with advanced tumor stage and poor survival. Furthermore, miR-30a-5p -1|1 could promote the metastasis of gastric cancer cells both in vitro and in vivo by down-regulating TMEM66. In clinical samples, decreased expression of TMEM66 was characteristic of gastric cancer, and the low level of TMEM66 correlated with deceased CD8 positive cells in the tumor microenvironment probably due to decreased cytokines production. In conclusion, the variety of miR-30a-5p isoforms correlates with worse survival in gastric cancer patients. Moreover, miR-30a-5p -1|1 could promote gastric cancer metastasis by inhibiting TMEM66 and the infiltration of intratumoral CD8 positive cells.

Modified bone marrow mesenchymal stem cells derived exosomes loaded with MiRNA ameliorates non-small cell lung cancer.

The study aimed to reveal the function of LXY30 peptide-modified bone marrow mesenchymal stem cell-derived exosomes (LXY30-Exos) in NSCLC. LXY30 peptide is a peptide ligand targeting α3ß1 integrin, and LXY30 specifically binds to Exos derived from different cells. We use transmission electron microscopy to identify LXY30-Exos and tracking analysis for particles, and the LXY30-Exos internalized by NSCLC cells in vitro and targeted NSCLC tumours in vivo were verified by multiple molecular technologies. The functions of LXY30-Exos-encapsulated miR-30c, miR-181b or miR-613 were assessed using cell proliferation, migration and cell apoptosis assays. Meanwhile, the safety of the above engineered Exos was evaluated in vivo. After LXY30-Exos were isolated and identified, LXY30-Exos were confirmed to be internalized by NSCLC cells in vitro and specifically targeted NSCLC tumours in vivo. Functionally, LXY30-Exos-encapsulated miR-30c, miR-181b or miR-613 weakened the proliferation, migration and cell cycle of NSCLC cells induced cellular apoptosis in vitro and restrained the tumour progression in vivo. Meanwhile, the safety of LXY30-Exos-encapsulated miR-30c, miR-181b or miR-613 was confirmed in vivo. Overall, miR-30c, miR-181b and miR-613 encapsulated in LXY30 peptide-modified BMSC-Exos relieved NSCLC.

circ_PPAPDC1A promotes Osimertinib resistance by sponging the miR-30a-3p/ IGF1R pathway in non-small cell lung cancer (NSCLC).

BACKGROUND: Recent evidence has demonstrated that abnormal expression and regulation of circular RNA (circRNAs) are involved in the occurrence and development of a variety of tumors. The aim of this study was to investigate the effects of circ_PPAPDC1A in Osimertinib resistance in NSCLC. METHODS: Human circRNAs microarray analysis was conducted to identify differentially expressed (DE) circRNAs in Osimertinib-acquired resistance tissues of NSCLC. The effect of circ_PPAPDC1A on cell proliferation, invasion, migration, and apoptosis was assessed in both in vitro and in vivo. Dual-luciferase reporter assay, RT-qPCR, Western-blot, and rescue assay were employed to confirm the interaction between circ_PPAPDC1A/miR-30a-3p/IGF1R axis. RESULTS: The results revealed that circ_PPAPDC1A was significantly upregulated in Osimertinib acquired resistance tissues of NSCLC. circ_PPAPDC1A reduced the sensitivity of PC9 and HCC827 cells to Osimertinib and promoted cell proliferation, invasion, migration, while inhibiting apoptosis in Osimertinib-resistant PC9/OR and HCC829/OR cells, both in vitro and in vivo. Silencing circ_PPAPDC1A partially reversed Osimertinib resistance. Additionally, circ_PPAPDC1A acted as a competing endogenous RNA (ceRNA) by targeting miR-30a-3p, and Insulin-like Growth Factor 1 Receptor (IGF1R) was identified as a functional gene for miR-30a-3p in NSCLC. Furthermore, the results confirmed that circ_PPAPDC1A/miR-30a-3p/IGF1R axis plays a role in activating the PI3K/AKT/mTOR signaling pathway in NSCLC with Osimertinib resistance. CONCLUSIONS: Therefore, for the first time we identified that circ_PPAPDC1A was significantly upregulated and exerts an oncogenic role in NSCLC with Osimertinib resistance by sponging miR-30a-3p to active IGF1R/PI3K/AKT/mTOR pathway. circ_PPAPDC1A may serve as a novel diagnostic biomarker and therapeutic target for NSCLC patients with Osimertinib resistance.

Silencing of LINC00707 Alleviates Brain Injury by Targeting miR-30a-5p to Regulate Microglia Inflammation and Apoptosis.

The role of microglia in traumatic brain injury (TBI) has gained considerable attention. The present study aims to elucidate the potential mechanisms of Long intergenic non-protein coding RNA 707 (LINC00707) in TBI-induced microglia activation and inflammatory factor release. An in vivo model of rat TBI and in vitro microglia model was established using Controlled cortex injury (CCI) and lipopolysaccharide (LPS) stimulation. RT-qPCR to detect LINC00707 levels in rat cerebral cortex or cells. Modified Neurological Impairment Score (mNSS) and Morris Water Maze test was conducted to assess the neurological deficits and cognitive impairment. ELISA analysis of pro-inflammatory factors levels. CCK-8 and flow cytometry for cell viability and apoptosis levels. Dual-luciferase report and RIP assay to validate the targeting relationship between LINC00707 and miR-30a-5p. LINC00707 was elevated in the TBI rat cerebral cortex and LPS-induced microglia, while miR-30a-5p was noticeably decreased (P < 0.05). Increased mNSS, cognitive dysfunction, and brain edema in TBI rats were all prominently reversed by silencing of LINC00707, but this reversal was partially abrogated by decreasing miR-30a-5p (P < 0.05). Inhibition of LINC00707 suppressed the overproduction of inflammatory factors in TBI rats (P < 0.05). LPS decreased microglial cell viability, increased apoptosis, and promoted inflammatory overproduction than control, but the silencing of LINC00707 reversed its effect. Suppression of miR-30a-5p attenuated this reversal (P < 0.05). miR-30a-5p was the target miRNA of LINC00707. All in all, the results suggested that inhibiting LINC00707/miR-30a-5p axis could alleviate the progression of TBI by suppressing the inflammation and apoptosis of microglia cells.

Short-term changes in serum miRNA levels and patient-reported clinical outcomes in myasthenia gravis.

INTRODUCTION/AIMS: The circulating microRNAs (miRNAs) miR-150-5p, miR-30e-5p, and miR-21-5p have been suggested as potential biomarkers for myasthenia gravis (MG); however, the relationships between short-term natural changes of the miRNAs and patient-reported MG outcome scores have not been well-studied. We assessed the short-term fluctuations in miRNA levels and patient-reported outcome measures in MG. METHODS: This prospective cohort study included 39 MG patients with regular follow-ups and unchanged medications at the Neurology outpatient clinic at Uppsala University Hospital. Patients had weekly follow-up visits for 1 month, at which blood samples were drawn, and scores from MG activities of daily living (MG-ADL), MG quality-of-life-15 (MG-QoL15), and Fatigue Severity Scale (FSS) were assessed. Serum levels of miRNA miR-150-5p, miR-30e-5p, and miR-21-5p were analyzed using quantitative real-time PCR. RESULTS: Intra-individual levels of miR-30e-5p and miR-150-5p were stable, whereas a significant reduction in miR-21-5p was observed from week 1 to week 2 (p = .0024) and from week 2 to week 3 (p < .0001). There were intra-individual differences over a short time in MG-ADL, with higher scores in female patients (p = .0281) and a significant reduction from the first to the second weeks (p = .0281), whereas MG-QoL15 and FSS scores were stable. DISCUSSION: The suggested MG biomarkers miR-30e-5p and miR-150-5p were more stable than miR-21-5p over a short time, indicating their short-term stability as biomarkers. Prospective multi-center studies with longer periods of follow-up and matched controls are needed to validate these miRNAs as biomarkers in MG.

Melatonin Regulates Rheumatoid Synovial Fibroblasts-Related Inflammation: Implications for Pathological Skeletal Muscle Treatment.

Melatonin has been reported to regulate circadian rhythms and have anti-inflammatory characteristics in various inflammatory autoimmune diseases, but its effects in diseases-associated muscle atrophy remain controversial. This study is aimed to determine the evidence of melatonin in rheumatoid arthritis (RA)-related pathological muscle atrophy. We used initially bioinformatics results to show that melatonin regulated significantly the correlation between pro-inflammation and myogenesis in RA synovial fibroblasts (RASF) and myoblasts. The conditioned medium (CM) from melatonin-treated RASF was incubated in myoblasts with growth medium and differentiated medium to investigate the markers of pro-inflammation, atrophy, and myogenesis. We found that melatonin regulated RASF CM-induced pathological muscle pro-inflammation and atrophy in myoblasts and differentiated myocytes through NF-κB signaling pathways. We also showed for the first time that miR-30c-1-3p is negatively regulated by three inflammatory cytokines in human RASF, which is associated with murine-differentiated myocytes. Importantly, oral administration with melatonin in a collagen-induced arthritis (CIA) mouse model also significantly improved arthritic swelling, hind limb grip strength as well as pathological muscle atrophy. In conclusion, our study is the first to demonstrate not only the underlying mechanism whereby melatonin decreases pro-inflammation in RA-induced pathological muscle atrophy but also increases myogenesis in myoblasts and differentiated myocytes.

Plasma exosomal miR-30a-5p inhibits osteogenic differentiation of bone marrow mesenchymal stem cells from a chronic unpredictable mild stress-induced depression rat model.

With rising society stress, depression-induced osteoporosis is increasing. However, the mechanism involved is unclear. In this study, we explored the effect of plasma exosomal miRNAs on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation in a chronic unpredictable mild stress (CUMS)-induced depression rat model. After 12 weeks of CUMS-induced depression, the pathological changes in the bone tissue and markers of osteogenic differentiation were tested by micro-computed tomography, hematoxylin-eosin staining, and quantitative real-time reverse transcription PCR (qRT-PCR). Plasma exosomes from rats were isolated and co-incubated with BMSCs for 14 d to detect the effect on osteogenic markers. Next-generation sequencing identified the miRNAs in the plasma exosomes, and the differential miRNAs were analyzed and verified by qRT-PCR. BMSCs were infected with lentivirus to upregulate miRNA-30a-5p and incubated in a medium that induced osteogenic differentiation for 14 d. The effect of miR-30a-5p on osteogenic differentiation was determined by qPCR and alizarin red staining. CUMS-induced depression rat model was established successfully, and exhibited reduced bone mass and damaged bone microstructure compared to that of the controls. The observed pathological changes suggested the occurrence of osteoporosis in the CUMS group, and the mRNA expression of osteogenic markers was also significantly reduced. Incubation of BMSCs with plasma exosomes from the CUMS group for 14 d resulted in a significant decrease in the expression of osteogenic markers. Twenty-five differentially expressed miRNAs in plasma exosomes were identified and upregulation of miR-30a-5p was observed to significantly inhibit the expression of osteogenic markers in BMSCs. Our findings contributed to a comprehensive understanding of the mechanism of osteoporosis caused by depression, and demonstrated the potential of miR-30a-5p as a novel biomarker or therapeutic target for the treatment of osteoporosis.

MicroRNA-30e-3p regulates the inflammatory response by targeting the gimap8 gene in Ctenopharyngodon idella kidney (CIK) cells.

Recent studies have increasingly linked miRNAs with the modulation of inflammatory responses and immunosuppressive activities. This investigation reveals that mir-30e-3p selectively binds to and modulates gimap8, as demonstrated by luciferase reporter assays and qPCR analyses. Upon LPS stimulation of CIK cells, mir-30e-3p expression was notably elevated, inversely correlating with a decrease in gimap8 mRNA levels. Overexpression of mir-30e-3p attenuated the mRNA levels of pro-inflammatory cytokines beyond the effect of LPS alone, suggesting a regulatory role of mir-30e-3p in inflammation mediated by the gimap8 gene. These insights contribute to our understanding of the complex mechanisms governing inflammatory and immune responses.

circMTO1/miR-30c-5p/SOCS3 axis alleviates oral submucous fibrosis through inhibiting fibroblast-myofibroblast transition.

BACKGROUND: circRNAs have been shown to participate in diverse diseases; however, their role in oral submucous fibrosis (OSF), a potentially malignant disorder, remains obscure. Our preliminary experiments detected the expression of circRNA mitochondrial translation optimization 1 homologue (circMTO1) in OSF tissues (n = 20) and normal mucosa tissues (n = 20) collected from Hunan Xiangya Stomatological Hospital, and a significant decrease of circMTO1 expression was showed in OSF tissues. Therefore, we further explored circMTO1 expression in OSF. METHODS: Target molecule expression was detected using RT-qPCR and western blotting. The migration and invasion of buccal mucosal fibroblasts (BMFs) were assessed using wound healing and Transwell assays. The interaction between miR-30c-5p, circMTO1, and SOCS3 was evaluated using dual luciferase, RNA immunoprecipitation (RIP), and RNA pull-down assays. The colocalisation of circMTO1 and miR-30c-5p was observed using fluorescence in situ hybridisation (FISH). RESULTS: circMTO1 and SOCS3 expression decreased, whereas miR-30c-5p expression increased in patients with OSF and arecoline-stimulated BMFs. Overexpression of circMTO1 effectively restrained the fibroblast-myofibroblast transition (FMT), as evidenced by the increase in expression of Coll I, α-SMA, Vimentin, and the weakened migration and invasion functions in BMFs. Mechanistic studies have shown that circMTO1 suppresses FMT by enhancing SOCS3 expression by sponging miR-30c-5p and subsequently inactivating the FAK/PI3K/AKT pathway. FMT induced by SOCS3 silencing was reversed by the FAK inhibitor TAE226 or the PI3K inhibitor LY294002. CONCLUSION: circMTO1/miR-30c-5p/SOCS3 axis regulates FMT in arecoline-treated BMFs via the FAK/PI3K/AKT pathway. Expanding the sample size and in vivo validation could further elucidate their potential as therapeutic targets for OSF.

CircCUL1 inhibits trophoblast cell migration and invasion and promotes cell autophagy by sponging hsa-miR-30e-3p in fetal growth restriction via the ANXA1/PI3K/AKT axis.

Fetal growth restriction (FGR) severely affects the health outcome of newborns and represents a major cause of perinatal morbidity. The precise involvement of circCULT1 in the progression of FGR remains unclear. We performed next-generation sequencing and RT-qPCR to identify differentially expressed circRNAs in placental tissues affected by FGR by comparing them with unaffected counterparts. Edu, flow cytometry, and transwell assay were conducted to detect HTR8/SVneo cell's function in regard to cell proliferation, migration, and invasion. The interaction between circCUL1 and hsa-miR-30e-3p was assessed through dual-luciferase reporter assays, validation of the interaction between circCUL1 and ANXA1 was performed using RNA pulldown and immunoprecipitation assays. Western blot analysis was performed to evaluate protein levels of autophagy markers and components of the PI3K/AKT signaling pathway. A knockout (KO) mouse model was established for homologous mmu-circ-0001469 to assess fetal mouse growth and development indicators. Our findings revealed an upregulation of circCUL1 expression in placental tissues from patients with FGR. We found that suppression of circCUL1 increased the trophoblast cell proliferation, migration, and invasion, circCUL1 could interact with hsa-miR-30e-3p. Further, circCUL1 stimulated autophagy, modulating trophoblast cell autophagy via the ANXA1/PI3K/AKT pathway, and a notable disparity was observed, with KO mice displaying accelerated embryo development and exhibiting heavier placentas in comparison to wild-type C57BL/6 mice. By modulating the ANXA1/PI3K/AKT signaling pathway through the interaction with hsa-miR-30e-3p, circCUL1 promotes autophagy while concurrently suppressing trophoblast cell proliferation, migration, and invasion. These findings offer novel insights into potential diagnostic markers and therapeutic targets for FGR research.

Binding site redundancy is critical for the regulation of fas by miR-30c in blunt snout bream (Megalobrama amblycephala).

MiR-30c and fatty acid synthase (fas) both play important roles in physiological processes such as lipid synthesis and fat metabolism. Predictive analysis revealed that fas is a target gene of miR-30c with multiple seed sites. Seed sites are useful to predict miRNA targeting relationships; however, detailed analyses of seed sites in fish genomes remain poorly studied. In this study, the regulatory relationship between miR-30c and fas, number and effect of seed regions, and mechanism by which miR-30c regulates lipid metabolism were evaluated in blunt snout bream (Megalobrama amblycephala). Four miR-30c target sites for fas were identified using various prediction tools. miR-30c mimics were transfected into 293 T cells, and dual-luciferase reporter assays were used to evaluate the roles of different fas target sites. When a single target site was mutated, relative luciferase activity was higher than that in the control group, with different activity levels depending on the mutation site. When multiple target sites were mutated, relative luciferase activity increased significantly as the number of mutation sites increased and was the highest when the four sites were mutated simultaneously. The miR-30c agomir was injected into the abdominal cavity of M. amblycephala at various concentrations for analyses of physiological and biochemical parameters in the liver and blood and the expression of genes related to lipid metabolism in the liver. Total cholesterol, free fatty acid, triglyceride, and low density lipoprotein levels were significantly lower after miR-30c agomir injection comparing to the control (P < 0.05). Additionally, the expression levels of genes related to lipid metabolism were significantly lower after miR-30c agomir injection than in the control (P < 0.05). In summary, this study identified four specific miR-30c target sites in the 3' UTR of fas mRNA; the effects of these sites are cumulative, and the redundancy ensures the accurate regulation of fas during evolution. In addition, miR-30c has a negative regulatory effect on fas and regulates lipid metabolism via various genes related to this process. Therefore, the regulation of miR-30c can effectively ameliorate the side effects of a high-fat diet on liver function in M. amblycephala.

Cisplatin induces acute kidney injury by downregulating miR-30e-5p that targets Galnt3 to activate the AMPK signaling pathway.

Cisplatin nephrotoxicity is an etiological factor for acute kidney injury (AKI). MicroRNA (miRNA) expression is dysregulated in cisplatin-induced AKI (cAKI) although the underlying mechanisms are unclear. A cAKI model was established by intraperitoneally injecting cisplatin, and key miRNAs were screened using high-throughput miRNA sequencing. The functions of key miRNAs were determined using the cell viability, live/dead, reactive oxygen species (ROS), and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays. Additionally, the macrophage membrane was wrapped around a metal-organic framework (MOF) loaded with miRNA agomir to develop a novel composite material, macrophage/MOF/miRNA agomir nanoparticles (MMA NPs). High-throughput miRNA sequencing revealed that miR-30e-5p is a key miRNA that is downregulated in cAKI. The results of in vitro experiments demonstrated that miR-30e-5p overexpression partially suppressed the cisplatin-induced or lipopolysaccharide (LPS)-induced downregulation of cell viability, proliferation, upregulation of ROS production, and cell death. Meanwhile, the results of in vivo and in vitro experiments demonstrated that MMA NPs alleviated cAKI by exerting anti-inflammatory effects. Mechanistically, cisplatin downregulates the expression of miR-30e-5p, and the downregulated miR-30e-5p can target Galnt3 to activate the adenosine 5'-monophosphate activated protein kinase (AMPK) signaling pathway, which promotes the progression of AKI. Our study found that miR-30e-5p is a key downregulated miRNA in cAKI. The downregulated miR-30e-5p promotes AKI progression by targeting Galnt3 to activate the AMPK signaling pathway. The newly developed MMA NPs were found to have protective effects on cAKI, suggesting a potential novel strategy for preventing cAKI.

hsa_circ_0005991 promotes epithelial-mesenchymal transition by regulating miR-30b-3p/Cdc42EP1 axis in ovary endometriosis.

Endometriosis is a common gynecological disease with an enigmatic pathogenesis. This work explored the function of hsa_circ_0005991 in ovarian endometriosis. High-throughput RNA-Seq was conducted in five matched ectopic (EC) and eutopic (EU) samples. Further, several types of cell function experiments were conducted. According to bioinformatics analysis, a competing endogenous RNA network was established. It included 5 circRNAs, 13 miRNAs, and 551 mRNAs. The expression levels of hsa_circ_0005991 and Cdc42EP1 were significantly elevated, while miR-30b-3p was reduced in the EC group. Upregulation of hsa_circ_0005991 raised Cdc42EP1 levels, induced EMT, and boosted Ishikawa cell proliferation, migration, and invasion. hsa_circ_0005991 knockdown indicated the opposite effects. When co-transfected with miR-30b-3p mimics or inhibitors, these effects could be reversed, respectively. Western blot assays showed alterations of EMT markers in EC samples. hsa_circ_0005991/miR-30b-3p/Cdc42EP1 axis promotes the EMT process in endometriosis, which may offer a theoretical foundation for the mechanism exploration and therapy of this disease.

Identification of miR-30c-5p microRNA in Serum as a Candidate Biomarker to Diagnose Endometriosis.

The diagnosis of endometriosis by laparoscopy is delayed until advanced stages. In recent years, microRNAs have emerged as novel biomarkers for different diseases. These molecules are small non-coding RNA sequences involved in the regulation of gene expression and can be detected in peripheral blood. Our aim was to identify candidate serum microRNAs associated with endometriosis and their role as minimally invasive biomarkers. Serum samples were obtained from 159 women, of whom 77 were diagnosed with endometriosis by laparoscopy and 82 were healthy women. First, a preliminary study identified 29 differentially expressed microRNAs between the two study groups. Next, nine of the differentially expressed microRNAs in the preliminary analysis were evaluated in a new cohort of 67 women with endometriosis and 72 healthy women. Upon validation by quantitative real-time PCR technique, the circulating level of miR-30c-5p was significantly higher in the endometriosis group compared with the healthy women group. The area under the curve value of miR-30c-5p was 0.8437, demonstrating its diagnostic potential even when serum samples registered an acceptable limit of hemolysis. Dysregulation of this microRNA was associated with molecular pathways related to cancer and neuronal processes. We concluded that miR-30c-5p is a potential minimally invasive biomarker of endometriosis, with higher expression in the group of women with endometriosis diagnosed by laparoscopy.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

MicroRNA-30e-3p reduces coronary microembolism-induced cardiomyocyte pyroptosis and inflammation by sequestering HDAC2 from the SMAD7 promoter.

This study investigates the mechanism by which microRNA (miR)-30e-3p reduces coronary microembolism (CME)-induced cardiomyocyte pyroptosis and inflammation. Cardiac function tests, histological staining, and transmission electron microscopy were performed on CME-model rats injected with adeno-associated viral vectors. Cardiomyocytes were transfected 24 h before a cellular model of pyroptosis was established via treatment with 1 µg/mL lipopolysaccharide (LPS) for 4 h and 5 mM ATP for 30 min. Pyroptosis, inflammation, and Wnt/ß-catenin signaling in cardiomyocytes were detected. Dual-luciferase reporter assays and/or RNA pull-down assays were performed to verify the binding of miR-30e-3p to HDAC2 mRNA or HDAC2 to the SMAD7 promoter. Chromatin immunoprecipitation was used to assess the level of H3K27 acetylation at the SMAD7 promoter. miR-30e-3p and SMAD7 expression levels were downregulated and HDAC2 expression was upregulated with CME. The overexpression of miR-30e-3p restored cardiac functions in CME-model rats and reduced serum cTnI, IL-18, and IL-1ß levels, microinfarcts, inflammatory cell infiltration, apoptosis, collagen content, and GSDMD-N, cleaved caspase-1, and NLRP3 expression in the myocardium, but these effects were reversed by SMAD7 knockdown. The overexpression of miR-30e-3p or knockdown of HDAC2 reduced LDH, IL-18, and IL-1ß secretion, propidium iodide intake, and GSDMD-N, NLRP3, cleaved caspase-1, Wnt3a, Wnt5a, and ß-catenin expression in the cardiomyocyte model. miR-30e-3p inhibited the expression of HDAC2 by binding HDAC2 mRNA. HDAC2 repressed the expression of SMAD7 by catalyzing H3K27 deacetylation at the SMAD7 promoter. miR-30e-3p, by binding HDAC2 to promote SMAD7 expression, reduces CME-induced cardiomyocyte pyroptosis and inflammation.

Gga-miR-30c-5p Suppresses Avian Reovirus (ARV) Replication by Inhibition of ARV-Induced Autophagy via Targeting ATG5.

Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection. IMPORTANCE Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.

Clostridioides difficile PCR ribotypes 001 and 084 can trigger autophagy process in human intestinal Caco-2 cells.

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro.

Specific Milk Composition of miR-30b Transgenic Mice Associated with Early Duodenum Maturation in Offspring with Lasting Consequences for Growth.

BACKGROUND: Milk composition is complex and includes numerous components essential for offspring growth and development. In addition to the high abundance of miR-30b microRNA, milk produced by the transgenic mouse model of miR-30b-mammary deregulation displays a significantly altered fatty acid profile. Moreover, wild-type adopted pups fed miR-30b milk present an early growth defect. OBJECTIVE: This study aimed to investigate the consequences of miR-30b milk feeding on the duodenal development of wild-type neonates, a prime target of suckled milk, along with comprehensive milk phenotyping. METHODS: The duodenums of wild-type pups fed miR-30b milk were extensively characterized at postnatal day (PND)-5, PND-6, and PND-15 using histological, transcriptomic, proteomic, and duodenal permeability analyses and compared with those of pups fed wild-type milk. Milk of miR-30b foster dams collected at mid-lactation was extensively analyzed using proteomic, metabolomic, and lipidomic approaches and hormonal immunoassays. RESULTS: At PND-5, wild-type pups fed miR-30b milk showed maturation of their duodenum with 1.5-fold (P < 0.05) and 1.3-fold (P < 0.10) increased expression of Claudin-3 and Claudin-4, respectively, and changes in 8 duodenal proteins (P < 0.10), with an earlier reduction in paracellular and transcellular permeability (183 ng/mL fluorescein sulfonic acid [FSA] and 12 ng/mL horseradish peroxidase [HRP], respectively, compared with 5700 ng/mL FSA and 90 ng/mL HRP in wild-type; P < 0.001). Compared with wild-type milk, miR-30b milk displayed an increase in total lipid (219 g/L compared with 151 g/L; P < 0.05), ceramide (17.6 µM compared with 6.9 µM; P < 0.05), and sphingomyelin concentrations (163.7 µM compared with 76.3 µM; P < 0.05); overexpression of 9 proteins involved in the gut barrier (P < 0.1); and higher insulin and leptin concentrations (1.88 ng/mL and 2.04 ng/mL, respectively, compared with 0.79 ng/mL and 1.06 ng/mL; P < 0.01). CONCLUSIONS: miR-30b milk displays significant changes in bioactive components associated with neonatal duodenal integrity and maturation, which could be involved in the earlier intestinal closure phenotype of the wild-type pups associated with a lower growth rate.

MiR-30 Family: A Novel Avenue for Treating Bone and Joint Diseases?

Bone and joint diseases are a group of clinically heterogeneous diseases characterized by various bone strength disorders, bone structural defects and bone mass abnormalities. Common bone diseases include osteoporosis, skeletal dysplasia, and osteosarcoma, and common joint diseases include osteoarthritis, rheumatoid arthritis, and degenerative disc disease. all of them lead to high medical costs. The miR-30 family consists of a total of 5 members: miR-30a, miR-30b, miR-30c, miR-30d and miR-30e. Accumulating evidence has indicated that the miR-30 family may be involved in the occurrence and development of bone and joint diseases. For example, miR-30a is highly expressed in blood samples of osteoporosis patients, miR-30a/b increases in cartilage tissue of osteoarthritis patients, and lower expression of miR-30c is associated with higher malignance and shorter survival time of osteosarcoma. Mechanistically, by targeting crucial transcription factors (RUNX2, SOX9, beclin-1, etc.), the miR-30 family regulates some critical pathways of bone homeostasis (Wnt/ß-Catenin, mTOR, PI3K/AKT, etc.). In view of the distinct actions of the miR-30 family on bone metabolism, we hypothesize that the miR-30 family may be a new remedy for the clinical treatment and prevention of some bone and joint diseases.