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.

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.

Transcriptome sequencing analysis reveals miR-30c-5p promotes ferroptosis in cervical cancer and inhibits growth and metastasis of cervical cancer xenografts by targeting the METTL3/KRAS axis.

Cervical cancer is the most common malignant tumor in the female reproductive system worldwide, and its molecular mechanisms remain complex and poorly understood. Various techniques, including transcriptome sequencing, RT-qPCR, ELISA, immunofluorescence, Western blot, CCK-8 assay, Transwell assay, and xenograft models, were employed to investigate gene/miRNA expression, cellular proliferation, migration, and the interactions between miR-30c-5p, METTL3, and KRAS. Our transcriptome sequencing results demonstrated a significant downregulation of miR-30c-5p in cervical cancer cells. Further investigations using RNA pull-down, dual-luciferase reporter assay, Me-RIP, and PAR-CLIP confirmed METTL3 as one of the downstream targets of miR-30c-5p, while KRAS was identified as an iron-death suppressor gene susceptible to m6A modification. Notably, our Me-RIP analysis demonstrated the involvement of METTL3 in m6A modification of KRAS. In vitro experiments revealed that miR-30c-5p facilitated ferroptosis in cervical cancer cells by inhibiting the METTL3/KRAS axis, thus suppressing proliferation and migration. Additionally, in vivo studies demonstrated that miR-30c-5p repressed the growth and metastasis of cervical cancer xenografts through the inhibition of the METTL3/KRAS axis. Overall, this study highlights the critical role of miR-30c-5p in modulating cervical cancer progression by targeting the METTL3/KRAS axis, providing new insights into the molecular mechanisms underlying cervical cancer growth and metastasis.

LncRNA DLEU2 contributes to Taxol resistance of gastric cancer cells through regulating the miR-30c-5p-LDHA axis.

Gastric cancer (GC) is a human malignancy which is associated with high mortality rate and poor prognosis. In addition to surgery, chemo- and radio-therapies are effective strategies against GC at advanced or metastatic stage. Taxol is a traditionally anti-cancer drug which is applied to various types of cancer. However, development of drug resistance limited the anti-cancer effects of Taxol. Currently, the biological roles and mechanisms of non-coding RNA DLEU2 in Taxol resistant GC remain unclear. This study reported that DLEU2 was significantly upregulated and miR-30c-5p was remarkedly downregulated in gastric tumours and cell lines. Silencing DLEU2 or overexpression of miR-30c-5p effectively increased the Taxol sensitivity of GC cells. Through bioinformatics analysis, RNA pull-down and luciferase assay, we demonstrated that DLEU2 sponged miR-30c-5p to block its expression in GC cells. Moreover, from the established Taxol resistant GC cell line, we detected remarkedly upregulated DLEU2 and downregulated miR-30c-5p expressions and significantly elevated glucose metabolism. Under low glucose condition, Taxol resistant cells were more susceptible to Taxol. In addition, we showed overexpression of miR-30c-5p blocked glucose metabolism through inhibiting the LDHA, a glucose metabolism key enzyme by direct targeting the 3'UTR of LDHA. Finally, rescue experiments validated that restoration of miR-30c-5p in DLEU2-overexpressing Taxol resistant GC cells effectively overcame the DLEU2-promoted Taxol resistance. In summary, this study uncovered new roles and molecular mechanisms of the lncRNA DLEU2-promoted Taxol resistance of gastric cancer cells, presenting the DLEU2-miR-30c-5p-LDHA-glucose metabolism axis a potentially therapeutic target for treatment of Taxol resistant GC.

CircTrim37 Ameliorates Intracerebral Hemorrhage Outcomes by Modulating Microglial Polarization via the miR-30c-5p/SOCS3 Axis.

Circular RNAs (circRNAs) have been progressively recognized as critical regulators in the pathology and pathophysiology of central nervous system disease. However, the potential role of circRNAs in intracerebral hemorrhage (ICH) is still largely unclear. Here, we demonstrate that circTrim37 expression was significantly upregulated at 3 days after ICH by circular RNA microarray and qPCR assays. Overexpression of circTrim37 could significantly ameliorate brain injury volume, brain edema, neurologic deficits, and inflammation in vivo after ICH. CircTrim37 promotes M2 polarization while restrains M1 polarization in vitro. Furthermore, circTrim37 acts as an endogenous sponge for miR-30c-5p, thereby inhibiting miR-30c-5p activity, leading to the upregulation of SOCS3 and making the balance of microglial response towards an M2 phenotype. Taken together, our results indicate the participation of circTrim37 and its coupling mechanism in ICH and provide a novel therapeutic target for ICH.

MiR-30c-5p-Targeted Regulation of GNAI2 Improves Neural Function Injury and Inflammation in Cerebral Ischemia-Reperfusion Injury.

MiRNAs are related to neuronal proliferation and apoptosis following cerebral ischemia-reperfusion injury (CIRI). This study focused on miR-30c-5p in the disease. An oxygen-glucose deprivation/re-oxygenation (OGD/R) model was prepared in HT22 cells and transfected to overexpress miR-30c-5p and G Protein Subunit Alpha I2 (GNAI2) respectively or co-transfected to silence miR-30c-5p and GNAI2. Meanwhile, a middle cerebral artery occlusion (MCAO) model was constructed in mice, and miR-30c-5p and GNAI2 were silenced in vivo simultaneously. The mice were evaluated for neurological damage, apoptosis, and inflammation. HT22 cells were tested for cytotoxicity, proliferation, apoptosis, and inflammatory factors. The interaction between miR-30c-5p and GNAI2 was predicted, analyzed, and confirmed. MiR-30c-5p was found to be downregulated in both experimental models. miR-30c-5p reduced lactate dehydrogenase production, inflammatory response, inhibit apoptosis, and enhanced neuronal proliferation, while GNAI2 overexpression showed the opposite results. Downregulated miR-30c-5p worsened neurological function, apoptosis, and inflammation of MCAO mice while silencing GNAI2 attenuated the influence of downregulated miR-30c-5p. MiR-30c-5p can improve neuronal apoptosis and inflammatory response caused by CIRI and is neuroprotective by targeting GNAI2, providing a new target for treating CIRI.

High expression of miR-30c-5p in satellite cells of high-fat diet-induced obese rabbits inhibited satellite cell proliferation and promoted differentiation.

It was revealed in our previous study that the expression of miR-30c-5p in the skeletal muscle of rabbits fed high-fat diet was highly expressed. In the present study, we further investigated the function of miR-30c-5p in proliferation and differentiation of skeletal muscle satellite cell (SMSC). The results obtained in the present study showed that the skeletal muscle fibers of the rabbits fed the standard normal diet (SND) were orderly, regular, and uniform after HE staining, however, the muscle fibers of the rabbits fed the high-fat diet (HFD) were generally atrophied, some were arranged disorderly, the intercellular space was enlarged, the nucleus was increased, and the morphology and position were abnormal. Compared with the SND group, it was observed that the weekly weight gain and fat percentage were relatively higher, and also the levels of the serum biochemical indexes such as glucose, cholesterol, and triglyceride increased significantly in the rabbits fed with HFD. In addition, the results after the transfection of miR-30c-5p mimic, mimic NC (negative control), miR-30c-5p inhibitor, and inhibitor NC into the SMSCs showed that the cell counting kit-8 (CCK-8) proliferation experiment suggested that the number of cells in the over expression group was significantly lower than that in the mimic NC group at 48, 72, 96 h of cell proliferation. At 48, 72, 120 h, the number of cells in the inhibitor group was significantly higher than that in the mimic NC group. The number of EdU positive cells decreased significantly in the over expression group compared with the mimic NC group, however, it increased significantly in the inhibitor group compared with the inhibitor NC group. Moreover, compared with the mimic NC group, the myotube area increased significantly in the miR-30c-5p mimic group, whereas it decreased significantly in the miR-30c-5p inhibitor group as compared with the inhibitor NC group. In addition, we found that trinucleotide repeat containing adaptor 6A (TNRC6A) was successfully validated as a target gene for miR-30c-5p. The expression of TNRC6A in the miR-30c-5p mimic group was significantly lower than that in the mimic NC group. It was further observed that the expression of TNRC6A increased significantly in the miR-30c-5p inhibitor group as compared to that in the inhibitor NC group. Taken together, the results obtained in this study showed that miR-30c-5p promotes the differentiation as well as inhibits the proliferation of rabbit skeletal muscle satellite cells, and TNRC6A is a target gene of miR-30c-5p.

Adipocyte-derived exosomal miR-30c-5p promotes ovarian angiogenesis in polycystic ovary syndrome via the SOCS3/STAT3/VEGFA pathway.

Polycystic ovary syndrome (PCOS) is a systemic endocrine disease affecting women's reproductive health. Ovarian angiogenesis in PCOS patients is abnormal, manifested by increased ovarian stromal vascularization and upregulated proangiogenic factors such as vascular endothelial growth factor (VEGF). However, the specific mechanisms underlying these changes in PCOS remain unknown. In this study, we induced the adipogenic differentiation in preadipocyte 3T3-L1 cells and found that adipocyte-derived exosomes promoted proliferation, migration, tube formation, and VEGFA expression in human ovarian microvascular endothelial cells (HOMECs) by delivering miR-30c-5p. Mechanistically, dual luciferase reporter assay demonstrated that miR-30c-5p directly targeted the 3'- untranslated region (UTR) of suppressor of cytokine signaling 3 (SOCS3) mRNA. In addition, adipocyte-derived exosomal miR-30c-5p activated signal transducer and activator of transcription 3 (STAT3)/VEGFA pathway in HOMECs via targeting SOCS3. In vivo experiments indicated that tail vein injection of adipocyte-derived exosomes exacerbated endocrine and metabolic disorders and ovarian angiogenesis in mice with PCOS via miR-30c-5p. Taken together, the study revealed that adipocyte-derived exosomal miR-30c-5p promotes ovarian angiogenesis via the SOCS3/STAT3/VEGFA pathway, thereby participating in the development of PCOS.

Knockdown of the long non­coding RNA MALAT1 ameliorates TNF­α­mediated endothelial cell pyroptosis via the miR­30c­5p/Cx43 axis.

Long noncoding RNAs (lncRNAs) are related to the development of atherosclerosis (AS). However, the role of lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in tumor necrosis factor­α (TNF­α)­induced rat aortic endothelial cell (RAOEC) pyroptosis, as well as the underlying mechanisms, remain unclear. RAOEC morphology was assessed using an inverted microscope. The mRNA and/or protein expression levels of MALAT1, microRNA(miR)­30c­5p and connexin 43 (Cx43) were assessed using reverse transcription­quantitative PCR (RT­qPCR) and/or western blotting, respectively. The relationships among these molecules were validated by dual­luciferase reporter assays. Biological functions, such as LDH release, pyroptosis­associated protein levels and the proportion of PI­positive cells, were evaluated using a LDH assay kit, western blotting and Hoechst 33342/PI staining, respectively. The present study demonstrated that compared with the control group, the mRNA expression levels of MALAT1 and protein expression levels of Cx43 were significantly up­regulated, whereas miR­30c­5p mRNA expressions levels were significantly decreased in TNF­α­treated RAOEC pyroptosis. Knockdown of MALAT1 or Cx43 significantly attenuated the increase in LDH release, pyroptosis­associated protein expression and PI­positive cell numbers among RAOEC treated using TNF­α, whereas an miR­30c­5p mimic exerted the opposite effect. Furthermore, miR­30c­5p was demonstrated to be a negative regulator of MALAT1 and could also target Cx43. Finally, co­transfection with siMALAT1 and miR­30c­5p inhibitor could attenuate the protective effect of MALAT1 knockdown against TNF­α­mediated RAOEC pyroptosis by upregulation of Cx43 expression. In conclusion, MALAT1 might serve an important role in TNF­α­mediated RAOEC pyroptosis by regulating the miR­30c­5p/Cx43 axis, which would provide a potential novel diagnostic and therapeutic target for AS.

MALAT1-miR-30c-5p-CTGF/ATG5 axis regulates silica-induced experimental silicosis by mediating EMT in alveolar epithelial cells.

Epithelial-mesenchymal transdifferentiation of alveolar type Ⅱ epithelial cells is a vital source of pulmonary myofibroblasts, and myofibroblasts formation is recognized as an important phase in the pathological process of silicosis. miR-30c-5p has been determined to be relevant in the activation of the epithelial-mesenchymal transition (EMT) in numerous disease processes. However, elucidating the role played by miR-30c-5p in the silicosis-associated EMT process remains a great challenge. In this work, based on the establishment of mouse silicosis and A549 cells EMT models, miR-30c-5p was interfered with in vivo and in vitro models to reveal its effects on EMT and autophagy. Moreover, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), connective tissue growth factor (CTGF), autophagy-related gene 5 (ATG5), and autophagy were further interfered with in the A549 cells models to uncover the possible molecular mechanism through which miR-30c-5p inhibits silicosis associated EMT. The results demonstrated the targeted binding of miR-30c-5p to CTGF, ATG5, and MALAT1, and showed that miR-30c-5p could prevent EMT in lung epithelial cells by acting on CTGF and ATG5-associated autophagy, thereby inhibiting the silicosis fibrosis process. Furthermore, we also found that lncRNA MALAT1 might competitively absorb miR-30c-5p and affect the EMT of lung epithelial cells. In a word, interfering with miR-30c-5p and its related molecules (MALAT1, CTGF, and ATG5-associated autophagy) may provide a reference point for the application of silicosis intervention-related targets.

Evaluation of the Relationship between Aromatase/Sirtuin1 Interaction and miRNA Expression in Human Neuroblastoma Cells.

BACKGROUND: Changes in activation/inhibition of Sirtuin-1 (SIRT1) and aromatase play an important role in a plethora of diseases. MicroRNAs (miRNAs) modulate multiple molecular pathways and affect a substantial number of physiological and pathological processes. OBJECTIVE: The aim of this study was to investigate any possible interaction between aromatase and SIRT1 in SH-SY5Y cells and to see how there is a connection between this interaction and miRNA expression, if there is an interaction. METHODS: In this study, cells were incubated in serum-deprived media for 6, 12, and 24 h. Aromatase and SIRT1 expressions were evaluated by Western blot. The IC50 concentration of SIRT1 activator (SRT1720), SIRT1 inhibitor (EX527), and aromatase inhibitors (letrozole and fadrozole) was determined by the XTT method. Then, CYP19A1 and SIRT1 levels were evaluated in the presence of SIRT1 siRNA or IC50 values for each activator/inhibitor. Finally, CYP19A1, SIRT1 expression and miRNA target gene were assessed with bioinformatic approaches. RESULTS: Aromatase and SIRT1 protein levels were significantly elevated in the cells incubated at 24 h in serum-deprived media (p ≤ 0.05). SIRT1 also positively regulated CYP19A1 in SH-SY5Y cells in media with/without FBS. Serum deprivation depending on time course caused changes in the oxidant/ antioxidant system. While oxidative stress index tended to decrease in the absence of FBS at 24 h compared to the control, it showed a significant decrease at 48 h in a serum-deprived manner (p ≤ 0.001). As a result of bioinformatics analysis, we determined 3 miRNAs that could potentially regulate SIRT1 and CYP19A1. hsa-miR-27a-3p and hsa-miR-181a-5p correlated in terms of their expressions at 24 h compared to 12 h, and there was a significant decrease in the expression of these miRNAs. On the contrary, the expression of hsa-miR-30c-5p significantly increased at 24 h compared to 12 h. CONCLUSION: Considering the results, a direct link between aromatase and SIRT1 was observed in human neuroblastoma cells. The identification of key miRNAs, hsa-miR-27a-3p, hsa-miR-30c-5p, and hsa-miR-181a-5p targeting both aromatase and SIRT1, provides an approach with novel insights on neurology-associated diseases.

Identification of miR-30c-5p as a tumor suppressor by targeting the m6 A reader HNRNPA2B1 in ovarian cancer.

BACKGROUND: microRNAs (miRNAs) and N6-methyladenosine (m6 A) play important roles in ovarian cancer (OvCa). However, the mechanisms by which miRNAs regulate m6 A in OvCa have not been elucidated so far. METHODS: To screen m6 A-related miRNAs, Pearson's correlation analysis of miRNAs and m6 A regulators was implemented using The Cancer Genome Atlas database (TCGA). To determine the level of m6 A, RNA m6 A quantitative assays were used. Then, colony formation assays, EdU assays, wound healing assays, and Transwell assays were performed. The dual-luciferase reporter assay was used to confirm the miRNA target genes. Protein-protein interaction (PPI) analysis of the target genes was performed, and hub genes were discovered using the cytoHubba/Cytoscape software. The underlying molecular mechanisms were explored by bioinformatics and RNA stability assays. RESULTS: A total of 126 miRNAs were identified as m6 A-related miRNAs by Pearson's correlation analysis. Among them, the high level of miR-30c-5p was associated with good prognosis in OvCa patients. In vitro, the miR-30c-5p agomir lowered the m6 A level and inhibited OvCa cell proliferation, migration, and invasion. The hub target genes of miR-30c-5p were identified as (i) XPO1, (ii) AGO1, (iii) HNRNPA2B1, of which m6 A reader HNRNPA2B1 was highly expressed in OvCa tissues and related with poor prognosis. In vitro, knockdown of HNRNPA2B1 significantly reduced m6 A level and hampered the proliferation and migration of OvCa cells. The inhibition of m6 A reader HNRNPA2B1 attenuated the suppression of proliferation and migration and the low m6 A level induced by the miR-30c-5p downregulation. Mechanistically, m6 A reader HNRNPA2B1 might regulate CDK19 mRNA stability to alter m6 A level. CONCLUSIONS: miR-30c-5p inhibits OvCa progression and reduces the m6 A level by inhibiting m6 A reader HNRNPA2B1, thus providing new insights into the m6 A regulatory mechanism in OvCa.

Effect of LINC00707 on proliferation, migration and inflammatory factors of human vascular smooth muscle cells induced by ox-LDL by targeting miR-30c-5p / 中国药理学通报

Aim To explore the effect of LINC00707 on the proliferation, migration and inflammatory factors of human vascular smooth muscle cells induced by oixidized low-density lipoprotein (ox-LDL) and its possible mechanism. Methods ox-LDL was used to induce human vascular smooth muscle cells (HVSMCs) to establish an atherosclerotic cell model. si-NC, siLINC00707, miR-NC, miR-30c-5p mimic were trans-fected into HVSMCs, and then 100 mg • L~ ox-LDL was added to the cells. si-LINC00707 and anti-miRNC, or si-LINC00707 and miR-30c-5p Inhibitor were co-transfected into HVSMCs and then treated with 100 mg • L ox-LDL. qRT-PCR was used to detect the expression of LINC00707 and miR-30c-5p. CCK-8 and Transwell test were used to detect cell proliferation and migration. ELISA was used to detect the levels of IL-6, TNF-a, and IL-10. The dual-luciferase reporter experiment was used to detect the targeting relationship between LINC00707 and miR-30c-5p. Western blot was used to detect the protein expression of E-cadherin and N-cadherin. Results The expression of LINC00707 in HVSMCs induced by ox-LDL increased (P <0. 05), while the expression of miR-30c-5p decreased (P < 0. 05). After transfection with siLINC00707 or miR-30c-5p mimic, cell viability, the protein level of N-cadherin, the levels of IL-6 and TNF-a decreased (P < 0. 05), and the number of im grating cells decreased (P<0. 05), while the protein level of E-coadherin and the level of IL-10 increased (P <0. 05). LINC00707 could target miR-30c-5p. After co-transfection with si-LINC00707 and miR-30c-5p inhibitor, cell survival rate, the protein level of N-cadherin, the levels of IL-6 and TNF-α increased (P < 0.05), and the number of migrating cells increased (P <0. 05), while the protein level of E-cadherin and the level of IL-10 decreased (P < 0. 05). Conclusion Down-regulation of the expression of LINC00707 could inhibit the proliferation, migration and inflammation of human vascular smooth muscle cells induced by ox-LDL by promoting the expression of miR-30c-5p.

Identification of Epigenetic Interactions between MicroRNA-30c-5p and DNA Methyltransferases in Neuropathic Pain.

Neuropathic pain is a prevalent and severe chronic syndrome, often refractory to treatment, whose development and maintenance may involve epigenetic mechanisms. We previously demonstrated a causal relationship between miR-30c-5p upregulation in nociception-related neural structures and neuropathic pain in rats subjected to sciatic nerve injury. Furthermore, a short course of an miR-30c-5p inhibitor administered into the cisterna magna exerts long-lasting antiallodynic effects via a TGF-ß1-mediated mechanism. Herein, we show that miR-30c-5p inhibition leads to global DNA hyper-methylation of neurons in the lumbar dorsal root ganglia and spinal dorsal horn in rats subjected to sciatic nerve injury. Specifically, the inhibition of miR-30-5p significantly increased the expression of the novo DNA methyltransferases DNMT3a and DNMT3b in those structures. Furthermore, we identified the mechanism and found that miR-30c-5p targets the mRNAs of DNMT3a and DNMT3b. Quantitative methylation analysis revealed that the promoter region of the antiallodynic cytokine TGF-ß1 was hypomethylated in the spinal dorsal horn of nerve-injured rats treated with the miR-30c-5p inhibitor, while the promoter of Nfyc, the host gene of miR-30c-5p, was hypermethylated. These results are consistent with long-term protection against neuropathic pain development after nerve injury. Altogether, our results highlight the key role of miR-30c-5p in the epigenetic mechanisms' underlying neuropathic pain and provide the basis for miR-30c-5p as a therapeutic target.

Toxicity mechanisms regulating bone differentiation and development defects following abnormal expressions of miR-30c targeted by triclosan in zebrafish.

As a ubiquitous environmental estrogen-disrupting chemical, triclosan (TCS) can induce severe osteotoxicity; however, the underlying molecular mechanisms remain uncertain. Herein, we evaluated the toxic effects of TCS on the development of cartilage and osteogenesis in 5-dpf zebrafish. Under TCS exposure from 62.5 to 250 µg/L, several osteodevelopmental malformations were observed, such as defect of craniofacial cartilage, pharyngeal arch cartilage dysplasia, and impairments on skeletal mineralization. Further, the morphology of mature chondrocytes became swollen and deformed, their number decreased, nucleus displacement occurred, and most immature chondrocytes were crowded at both ends of ceratobranchial. SEM observation of larval caudal fin revealed that, the layer of collagen fibers and the mineralized calcium nodules were significantly decreased, with the collagen fibers becoming shorter upon TCS exposure. The activity of bone-derived alkaline phosphatase significantly reduced, and marker functional genes related to cartilage and osteoblast development were abnormally expressed. RNA-seq and bioinformatics analysis indicated, that changes in marker genes intimately related to the negative regulation of miR-30c-5p overexpression targeted by TCS, and the up-regulation of miR-30c induced bone developmental defects by inhibiting the bone morphogenetic protein (BMP) signaling pathway. These findings were confirmed by artificially intervening the expression of miR-30c and using BMP pathway agonists in vivo. In sum, TCS induced osteototoxicity by targeting miR-30c up-regulation and interfering in the BMP signaling pathway. These findings enhance mechanistic understanding of TCS-induced spontaneous bone disorders and bone metastatic diseases. Further research is necessary to monitor chronic TCS-exposure levels in surrounding environments and develop relevant safety precautions based on TCS environmental risk.

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.

Gga-miR-30c-5p Enhances Apoptosis in Fowl Adenovirus Serotype 4-Infected Leghorn Male Hepatocellular Cells and Facilitates Viral Replication through Myeloid Cell Leukemia-1.

Fowl adenovirus serotype 4 (FAdV-4) is the primary causative agent responsible for the hepatitis-hydropericardium syndrome (HHS) in chickens, leading to considerable economic losses to stakeholders. Although the pathogenesis of FAdV-4 infection has gained attention, the underlying molecular mechanism is still unknown. Here, we showed that the ectopic expression of gga-miR-30c-5p in leghorn male hepatocellular (LMH) cells enhanced apoptosis in FAdV-4-infected LMH cells by directly targeting the myeloid cell leukemia-1 (Mcl-1), facilitating viral replication. On the contrary, the inhibition of endogenous gga-miR-30c-5p markedly suppressed apoptosis and viral replication in LMH cells. Importantly, the overexpression of Mcl-1 inhibited gga-miR-30c-5p or FAdV-4-induced apoptosis in LMH cells, reducing FAdV-4 replication, while the knockdown of Mcl-1 by RNAi enhanced apoptosis in LMH cells. Furthermore, transfection of LMH cells with gga-miR-30c-5p mimics enhanced FAdV-4-induced apoptosis associated with increased cytochrome c release and caspase-3 activation. Thus, gga-miR-30c-5p enhances FAdV-4-induced apoptosis by directly targeting Mcl-1, a cellular anti-apoptotic protein, facilitating FAdV-4 replication in host cells. These findings could help to unravel the mechanism of how a host responds against FAdV-4 infection at an RNA level.

NORAD modulates miR-30c-5p-LDHA to protect lung endothelial cells damage.

Acute lung injury (ALI) is a devastating human malignancy characterized by excessively uncontrolled inflammation and lung endothelial dysfunction. Non-coding RNAs play essential roles in endothelial protections during the pathological processes of ALI. The precise functions and molecular mechanisms of the lncRNA-NORAD-mediated endothelial protection remain obscure. This study reports NORAD was significantly induced in human pulmonary microvascular endothelial cells (HPMECs) under lipopolysaccharide (LPS) treatment. Silencing NORAD effectively protected HPMECs against the LPS-induced cell dysfunction. In addition, RNA pull-down and luciferase assay validated that NORAD sponged miR-30c-5p, which showed reverse functions of NORAD in the LPS-induced cell injury of HPMECs. Furthermore, the glucose metabolism of HPMECs was significantly elevated under LPS stimulation which promoted the glucose consumption and extracellular acidification rate (ECAR) of HPMECs. Inhibiting NORAD or overexpressing miR-30c-5p suppressed glucose metabolism in HPMECs, leading to protective effects on HPMECs under LPS stimulation. The glycolysis key enzyme, lactate dehydrogenase-A (LDHA), was subsequently identified as a direct target of miR-30c-5p. Finally, recovery of miR-30c-5p in NORAD-overexpressing HPMECs effectively overrode the NORAD-promoted glycolysis and impaired endothelial dysfunction under LPS stimulation by targeting LDHA. Summarily, we demonstrated a NORAD-miR-30c-5p-LDHA-glycolysis axis in the LPS-induced HPMECs dysfunction in vitro and in vivo, contributing to the development of anti-ALI therapeutic approaches.

Circular RNA circRNF169 functions as a miR-30c-5p sponge to promote cellular senescence.

Circular RNAs (circRNAs), characterized as single-stranded closed circular RNA molecules, have been established to exert pivotal functions in various biological or pathological processes. Nonetheless, the effects and underlying mechanisms concerning circRNAs on the aging and aging-related diseases remain elusive. We herein compared the expression patterns of circRNAs in young and senescent mouse embryonic fibroblasts (MEFs), and uncovered that circRNF169 was dramatically up-regulated in senescent MEFs compared with that in young MEFs. Therefore, we further digged into the role and potential mechanisms of circRNF169 in the senescence of MEFs. The results of senescence-associate-ß-galactosidase staining and BrdU incorporation assay showed that silencing of circRNF169 significantly delayed MEFs senescence and promoted cell proliferation, while ectopic expression of circRNF169 exhibited the opposite effects. Moreover, the dual-luciferase reporter assay confirmed that circRNF169 acted as an endogenous miR-30c-5p sponge, which accelerated cellular senescence by sequestering and inhibiting miR-30c-5p activity. Taken together, our results suggested that circRNF169 exerted a crucial role in cellular senescence through sponging miR-30c-5p and represented a promising target for aging intervention.

Downregulation of lncRNA SNHG16 inhibits vascular smooth muscle cell proliferation and migration in cerebral atherosclerosis by targeting the miR-30c-5p/SDC2 axis.

Atherosclerosis (AS) is the basic lesion underlying the occurrence and development of cerebrovascular diseases. Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in AS. We aimed to explore the role of SNHG16 in AS and the molecular mechanism of VSMC involvement in the regulation of AS. The expression levels of SNHG16, miR-30c-5p and SDC2 were detected by qRT-PCR. CCK-8, wound healing and Transwell assays were used to assess ox-LDL-induced VSMC proliferation, migration, and invasion, respectively. Western blot analysis was used to detect SDC2 and MEK/ERK pathway-related protein levels. A dual-luciferase reporter assay confirmed the binding of SNHG16 with miR-30c-5p and miR-30c-5p with SDC2. SNHG16 and SDC2 expression was upregulated in patients with AS and ox-LDL-induced VSMCs, while miR-30c-5p was downregulated. Ox-LDL-induced VSMC proliferation and migration were increased, and the MEK/ERK signalling pathway was activated. MiR-30c-5p was targeted to SNHG16 and SDC2. Downregulating SNHG16 or upregulating miR-30c-5p inhibited ox-LDL-induced VSMC proliferation and migration and inhibited MEK/ERK signalling pathway activation. In contrast, downregulating miR-30c-5p or upregulating SDC2 reversed the effects of downregulating SNHG16 or upregulating miR-30c-5p. Furthermore, downregulating SDC2 inhibited ox-LDL-induced proliferation and migration of VSMCs and inhibited activation of the MEK/ERK signalling pathway, while upregulating lncRNA SNHG16 reversed the effects of downregulating SDC2. Downregulation of SNHG16 inhibited VSMC proliferation and migration in AS by targeting the miR-30c-5p/SDC2 axis. This study provides a possible therapeutic approach to AS.