Circular RNA circVAPA modulates macrophage pyroptosis in sepsis-induced acute lung injury through targeting miR-212-3p/Sirt1/Nrf2/NLRP3 axis.

Sepsis-induced acute lung injury (ALI) is an inflammatory condition involving the pyroptosis of macrophages. This study investigated the role of circular RNA hsa_circ_0006990 (circVAPA) in regulating macrophage pyroptosis in ALI and the underlying mechanisms. The expression pattern of circVAPA was examined in the mouse model of ALI and in the LPS-treated RAW264.7 macrophage cell line. Lung tissue damage was evaluated by haematoxylin and eosin staining, immunohistochemistry and a myeloperoxidase activity assay. The molecular mechanisms were investigated by luciferase reporter assay, western blot, RT-qPCR and ELISA. circVAPA was down-regulated in the lung tissues of ALI mice and LPS-induced RAW264.7 cells. circVAPA over-expression alleviated lung tissue injury and dampened LPS-induced pyroptosis and Th17-associated inflammatory responses. miR-212-3p was identified as a target of circVAPA, and miR-212-3p negatively regulated the expression of Sirt1. Sirt1 knockdown largely abolished the effect of circVAPA over-expression on pyroptosis. CircVAPA/miR-212-3p/Sirt1 axis also regulates Nrf2 and NLRP3 expression upon LPS challenge. By targeting miR-212-3p, circVAPA over-expression negatively regulates the expression of Sirt1 and pyroptosis-related factors (Nrf2 and NLRP3), which alleviates the inflammatory damages in sepsis-induced ALI.

Osteoclast-derived exosomal miR-212-3p suppressed the anabolism and accelerated the catabolism of chondrocytes in osteoarthritis by targeting TGF-ß1/Smad2 signaling.

Osteoarthritis (OA) is a common aging-related disease affecting entire joint structures, encompassing articular cartilage and subchondral bone. Although senescence and dysfunction of chondrocytes are considered crucial factors in the occurrence of OA, the exact pathogenesis remains to be investigated. In our study, chondrocytes were incubated with a conditioned medium obtained from osteoclasts at different differentiation stages, suggesting that osteoclasts and osteoclast precursors suppressed anabolism and promoted the catabolism of chondrocytes in vitro. In contrast, the function of osteoclasts was more significant than osteoclast precursors. Further blocking of osteoclast exosome secretion by using GW4869 abolished the effect of osteoclasts on chondrocytes. Functionally, exosomal transfer of osteoclast-derived miR-212-3p inhibited Smad2 to mediate chondrocyte dysfunction, thus accelerating cartilage matrix degradation in OA via TGF-ß1/Smad2 signaling. The mechanism was also confirmed within the articular cartilage in OA patients and surgery-induced OA mice. Our study provides new information on intercellular interactions in the bone microenvironment within articular cartilage and subchondral bone during OA progression. The miR-212-3p/Smad2 axis is a potential target for the prevention and therapy of OA.

LncRNA XIST Protects Against Polycystic Ovary Syndrome via the Regulation of miR-212-3p/RASA1 Axis.

The polycystic ovary syndrome (PCOS), a common endocrine disorder, is mainly related to infertility. Moreover, it is characterized by promoted androgen, suppressed ovulation and insulin resistance. Long non-coding RNA X inactive specific transcript (lncRNA XIST), known as an oncogene or a cancer inhabited factor, is involved in several disease. However, the diagnostic mechanisms of lncRNA XIST in PCOS have not been clarified. Our study aimed to explain whether lncRNA XIST regulates KGN cells proliferation and apoptosis via microRNA (miR)-212-3p/RASA1 axis in PCOS. Levels of lncRNA XIST, miR-212-3p and RASA1 in KGN cells were detected through reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. Fluorescence in situ Hybridization (FISH) was performed to confirm the expression of lncRNA XIST and miR-212-3p in KGN cells. StarBase and dual-luciferase reporter assay were applied for exploring the interaction between miR-212-3p and RASA1. Cell viability, apoptosis, protein expression of Bcl-2 and Bax were assessed by MTT, flow cytometry analysis, RT-qPCR and western blot, respectively. We found that lncRNA XIST was low-expressed, miR-212-3p was over-expressed, and RASA1 was dramatically down-regulated in KGN cells. LncRNA XIST negatively regulated miR-212-3p expression in KGN cells. MiR-212-3p interacted with RASA1 and negatively regulated RASA1 levels in KGN cells. Up-regulation of lncRNA XIST signally decreased cells viability, stimulated more apoptotic cells, enhanced Bax expression, and depressed Bcl-2 level in KGN cells. However, these observations were abolished after miR-212-3p mimic treatment. Furthermore, miR-212-3p inhibitor significantly inhibited cell proliferation, enhanced more apoptotic cells, increased Bax expression, and decreased Bcl-2 level in KGN cells, and these effects were eliminated by RASA1-siRNA transfection. Our observations revealed that lncRNA XIST protects against PCOS through regulating miR-212-3p/RASA1 axis, suggesting that lncRNA XIST may be a promising therapeutic target for PCOS therapy.

Hypoxia Inhibits Cell Cycle Progression and Cell Proliferation in Brain Microvascular Endothelial Cells via the miR-212-3p/MCM2 Axis.

Hypoxia impairs blood-brain barrier (BBB) structure and function, causing pathophysiological changes in the context of stroke and high-altitude brain edema. Brain microvascular endothelial cells (BMECs) are major structural and functional elements of the BBB, and their exact role in hypoxia remains unknown. Here, we first deciphered the molecular events that occur in BMECs under 24 h hypoxia by whole-transcriptome sequencing assay. We found that hypoxia inhibited BMEC cell cycle progression and proliferation and downregulated minichromosome maintenance complex component 2 (Mcm2) expression. Mcm2 overexpression attenuated the inhibition of cell cycle progression and proliferation caused by hypoxia. Then, we predicted the upstream miRNAs of MCM2 through TargetScan and miRanDa and selected miR-212-3p, whose expression was significantly increased under hypoxia. Moreover, the miR-212-3p inhibitor attenuated the inhibition of cell cycle progression and cell proliferation caused by hypoxia by regulating MCM2. Taken together, these results suggest that the miR-212-3p/MCM2 axis plays an important role in BMECs under hypoxia and provide a potential target for the treatment of BBB disorder-related cerebrovascular disease.

LncRNA NEAT1 Regulates the Development of Parkinson's Disease by Targeting AXIN1 Via Sponging miR-212-3p.

Long non-coding RNA (lncRNA) nuclear-enriched assembly transcript 1 (NEAT1) has been reported to be highly expressed in Parkinson's disease (PD). However, the mechanism of NEAT1 in PD progression has not been fully elucidated. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine injection (MPTP) was used to construct PD mouse models in vivo, and 1-methyl-4-phenyl pyridine (MPP+) was used to build PD cell models in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to test the expression of NEAT1, microRNA (miR)-212-3p and axis inhibition protein 1 (AXIN1). The viability, apoptosis and inflammation of cells were determined using cell counting kit 8 (CCK8) assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Then, the protein levels of apoptosis-related markers and AXIN1 were measured by western blot (WB) analysis. Furthermore, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between miR-212-3p and NEAT1 or AXIN1. NEAT1 was upregulated in PD mouse models and cell models. Function experiments confirmed that NEAT1 knockdown could promote the viability, suppress the apoptosis and inflammation of MPP+-stimulated SK-N-SH cells to restrain PD progression. MiR-212-3p was downregulated in PD, and its inhibitor could reverse the suppression effect of NEAT1 knockdown on PD progression. Additionally, AXIN1 was a target of miR-212-3p, and its overexpression could invert the inhibition effect of miR-212-3p mimic on PD progression. Furthermore, AXIN1 expression was inhibited by NEAT1 silencing and promoted by NEAT1 overexpression, while these effect could be recovered by miR-212-3p inhibitor and mimic, respectively. Our results demonstrated that NEAT1 knockdown suppressed PD progression through regulating the miR-212-3p/AXIN1 pathway, indicating that NEAT1 might be a therapeutic target for neuroprotection in PD.

Negative feedback loop of ERK/CREB/miR-212-3p inhibits HBeAg-induced macrophage activation.

The activation of liver macrophages is closely related to liver injury after HBV infection. Our previous results demonstrated that HBeAg played a key role in inducing macrophage activation. As we all know, miRNAs are involved in the regulation of multiple immune cell functions. Meanwhile, we have shown that miR-155 positively regulates HBeAg-induced macrophage activation and accelerates liver injury. Subsequently, based on our previous miRNA sequencing results, we further evaluated the role of miR-212-3p called 'neurimmiR' in HBeAg-induced macrophages in this study. First, miR-212-3p expression was significantly elevated in HBeAg-treated macrophages. Meanwhile, we found up-regulation of miR-212-3p significantly decreased the production of cytokines, whereas knockdown of miR-212-3p held the opposite effect by gains and losses of function. Mechanically, although MAPK signal pathway, including ERK, JNK and p38, was activated in HBeAg-induced macrophages, only ERK promoted the expression of miR-212-3p via transcription factor CREB, which was able to bind to the promoter of miR-212-3p verified by ChIP assay. Moreover, we further indicated that up-regulated miR-212-3p inhibited HBeAg-induced inflammatory cytokine production through targeting MAPK1. In conclusion, miR-212-3p was augmented in HBeAg-stimulated macrophages via ERK/CREB signal pathway and the elevated miR-212-3p suppressed inflammatory cytokine production induced by HBeAg through targeting MAPK1.

MUC13 promotes intrahepatic cholangiocarcinoma progression via EGFR/PI3K/AKT pathways.

BACKGROUND & AIMS: Mucin 13 (MUC13) is reportedly overexpressed in human malignancies. However, the clinicopathological and biological significance of MUC13 in human intrahepatic cholangiocarcinoma (iCCA) remain unclear. The aim of this study was to define the role of MUC13 in the progression of iCCA. METHODS: Expression levels of MUC13 in human iCCA samples were evaluated by immunohistochemistry, western blot, and real-time PCR. In vitro and in vivo experiments were used to assess the effect of MUC13 on iCCA cell growth and metastasis. Crosstalk between MUC13 and EGFR/PI3K/AKT signaling was analyzed by molecular methods. The upstream regulatory effects of MUC13 were evaluated by Luciferase and DNA methylation assays. RESULTS: MUC13 was overexpressed in human iCCA specimens and iCCA cells. MUC13 overexpression positively correlated with clinicopathological characteristics of iCCA, such as vascular invasion and lymph node metastasis, and was independently associated with poor survival. Results from loss-of-function and gain-of-function experiments suggested that knockdown of MUC13 attenuated, while overexpression of MUC13 enhanced, the proliferation, motility, and invasiveness of iCCA cells in vitro and in vivo. Mechanistically, we found that the phosphatidylinositol 3-kinase-AKT signal pathway and its downstream effectors, such as tissue inhibitor of metalloproteinases 1 and matrix metallopeptidase 9, were required for MUC13-mediated tumor metastasis of iCCA. MUC13 interacted with epidermal growth factor receptor (EGFR) and subsequently activated the EGFR/PI3K/AKT signaling pathway by promoting EGFR dimerization and preventing EGFR internalization. We also found that MUC13 was directly regulated by miR-212-3p, whose downregulation was related to aberrant CpG hypermethylation in the promoter area. CONCLUSIONS: These findings suggest that aberrant hypermethylation-induced downregulation of miR-212-3p results in overexpression of MUC13 in iCCA, leading to metastasis via activation of the EGFR/PI3K/AKT signaling pathway. LAY SUMMARY: Mucin 13 overexpression has been implicated in the development of malignancies, although its role in intrahepatic cholangiocarcinoma has not been studied. Herein, we show that mucin 13 plays a critical role in intrahepatic cholangiocarcinoma. Mucin 13 could have therapeutic value both as a prognostic marker and as a treatment target.

ZLM-7 inhibits the occurrence and angiogenesis of breast cancer through miR-212-3p/Sp1/VEGFA signal axis.

BACKGROUND: Breast cancer (BC) is a common malignant tumor with poor prognosis. Angiogenesis is related to the growth and progression of solid tumors and associated with prognosis. ZLM-7, SP1, VEGFA and miR-212-3p were associated with BC angiogenesis and proliferation, however the detailed mechanism was not clear. This study aimed to reveal the regulatory mechanism of angiogenesis of BC. METHODS: BC cell lines were treated with 10 nM ZLM-7 for 8 h. We detected protein expression level by western blot and RNA expression level by qRT-PCR. Overexpression or inhibition of miR-212-3p is performed using miR-212-3p mimics or miR-212-3p inhibitor, Sp1 overexpression using pcDNA3.1 vector. Angiogenesis was analyzed by co-culturing BC cell lines and HUVEC cells. To evaluate regulatory relationship between miR-212-3p and Sp1, dual luciferase assay was performed. Besides, the direct interaction between Sp1 and VEGFA was analyzed by ChIP. Migration and invasion were analyzed by transwell assay and proliferation was detected by clone formation assay. In mice xenograft model developed using BC cells, we also detected angiogenesis marker CD31 through immunohistochemistry. RESULTS: ZLM-7 up-regulated miR-212-3p and inhibited invasion, migration, proliferation and angiogenesis of BC, while miR-212-3p inhibitor antagonized such effects. Binding sequence was revealed between miR-212-3p and Sp1, and expression of Sp1 was inhibited by miR-212-3p on both protein and mRNA level. Sp1 could interact with VEGFA and promoted its expression. Overexpression of miR-212-3p inhibited migration, invasion, proliferation and angiogenesis of BC cell lines, while Sp1 overexpression showed the opposite effect and could antagonize these effects of miR-212-3p overexpression. ZLM-7 decreased VEGFA expression, which was rescued by co-transfection with miR-212-3p inhibitor. Similar, ZLM-7 could inhibit tumor growth and angiogenesis through the miR-212-3p/Sp1/VEGFA axis in vivo. CONCLUSIONS: ZLM-7 could directly up-regulate miR-212-3p in BC. MiR-212-3p could inhibit VEGFA expression through Sp1, thereby inhibiting angiogenesis and progression of BC.

Pro-inflammatory cytokine-induced microRNA-212-3p expression promotes myocyte contraction via methyl-CpG-binding protein 2: a novel mechanism for infection-related preterm parturition.

Preterm labour is a common pregnancy complication contributing to major maternal and fetal morbidity and mortality. We have found microRNA (miR)-212-3p, a potential infection-associated molecule, was significantly over-expressed during human preterm labour. However, the mechanism remains unknown. In this study, we have adopted a lipopolysaccharide (LPS)-induced Institute of Cancer Research murine preterm model to examine the role of miR-212-3p in the infection-induced preterm labour. Myometrial miR-212-3p expression was increased by nearly 4-fold in the term labour group (P = 0.10) and 12-fold (P = 0.03) in the LPS-induced preterm labour group compared with the non-labour group. In vitro cellular experiments confirmed that a series of pro-inflammatory cytokines, including interleukin (IL)1B (P = 0.02) and IL-6 (P = 0.01), rather than LPS (P = 0.08) itself could significantly upregulate miR-212-3p expression in human myometrial smooth muscle cells. Methyl-CpG-binding protein 2 (MeCP2), as a target gene of miR-212-3p confirmed by our dual luciferase assay, influenced myocyte contractility and connexin 43 expression which is an important contraction-associated protein. Therefore, we conclude that miR-212-3p may be involved in infection-induced preterm labour through MeCP2 and it is a promoting molecule and novel target for the diagnosis and treatment of preterm labour in the future.

MicroRNA-212 activates hepatic stellate cells and promotes liver fibrosis via targeting SMAD7.

There has been an increasing number of researches about microRNAs (miRNAs) in the progression of liver fibrosis from the point of their comprehensive functions in regulating the activation of hepatic stellate cells (HSCs). Among them, it has been reported that miR-212 is up-regulated in activated rat primary HSCs. However, its mechanism has not been determined yet. Here, we confirmed that the level of miR-212-3p was up-regulated in livers of carbon tetrachloride (CCl4)-treated mice compared with the normal control, which is a classical model of chronically damaged fibrotic liver. In vitro, we demonstrated that TGF-ß, a master fibrogenic cytokine, could induce the level of miR-212. In turn, overexpression of miR-212 could induce the activation marker of HSC including α-smooth muscle actin (α-SMA) and collagens by activating TGF-ß signaling pathway. Furthermore, SMAD7, a dominant suppressor of TGF-ß pathway, was identified as a direct target of miR-212-3p. Our results indicate that miR-212-3p facilitates the activation of HSCs and TGF-ß pathway by targeting SMAD7, highlighting that it can be served as a novel biomarker or therapeutic target for liver fibrosis.

LncRNA SNHG5 promotes the progression of osteosarcoma by sponging the miR-212-3p/SGK3 axis.

BACKGROUND: Long non-coding RNA (lncRNA) SNHG5 has been found to play an important role in tumors. Nevertheless, the function and mechanism of lncRNA SNHG5 in osteosarcoma (OS) remains unclear. The purpose of this study was to investigate whether lncRNA SNHG5 can regulate the occurrence and development of OS cells. METHODS: We performed quantitative real time PCR to detect the expression of lncRNA SNHG5 in OS cells. 143B, MG63 (knockdown) and U2OS, U2R (overexpression) cell lines were chosen for the function study of SNHG5. The effect of SNHG5, miR-212-3p, and SGK3 in OS cells was explored by MTT assays, clony formation, flow cytometry, transwell assays, wound healing assays, and cell spreading assays. Quantitative real-time PCR, Western blot analysis and luciferase assays were used to detect the interaction between lncRNA SNHG5 and miR-212-3p. RESULTS: In this study, knockdown of lncRNA SNHG5 suppressed the growth and metastasis of OS cells, whereas the overexpression of SNHG5 produced an opposite result. Mechanistically, lncRNA SNHG5 functions as a sponger against miR-212-3p and suppresses the miR-212-3p/SGK3 signaling pathway. Introduction of miR-212-3p mimics or inhibitors reverses SNHG5 overexpression or silences the exerted tumor promoting or suppressing effect. In addition, our results showed that the function of SNHG5 can be rescued by miR-212-3p and can regulate the growth and metastasis of OS cells via SGK3, the downstream target of miR-212-3p. CONCLUSIONS: In summary, our study demonstrated that lncRNA SNHG5 can regulate the proliferation and metastasis of OS cells through the miR-212-3p/SGK3 axis. This axis may provide a new target for future clinical treatment.

MiR-212-3p inhibits LPS-induced inflammatory response through targeting HMGB1 in murine macrophages.

Sepsis is a major cause of mortality in seriously ill patients characterized by a series of severe systemic inflammatory responses due to an infection. Thus, there is a critically need to search more accurate biomarkers and targets for diagnosis and treatment of sepsis. Our study showed that miR-212-3p was up-regulated in LPS-treated macrophage RAW264.7 cells. Overexpression of miR-212-3p in RAW264.7 cells led to suppression of pro-inflammatory cytokines (TNF-α and IL-6) induced by LPS. Bioinformatic predictions and experimental researches both revealed that HMGB1 was a direct target of miR-212-3p. Meanwhile, the results showed that overexpression of miR-212-3p inhibited the cytoplasmic translocation of HMGB1 in LPS-induced RAW264.7 cells. Subsequently, transfection of the pcDNA3.1/HMGB1 plasmid, which produced HMGB1 overexpression, exhibited similar effects as the LPS-induced macrophage inflammatory response and markedly activated the MAPKs including p38, ERK and JNK phosphorylation. Furthermore, we also found that the phosphorylation of p38 MAPK and ERK was downregulated by miR-212-3p mimics upon LPS injection. In conclusion, these results reveal that miR-212-3p directly targets HMGB1 to suppress inflammatory response in LPS-induced RAW264.7 cells. All our findings indicate that miR-212-3p may act as a potential pharmacological target for promising and effective therapeutic intervention in microbial infection in the future.

SRSF3-regulated miR-132/212 controls cell migration and invasion by targeting YAP1.

Although SRSF3 (Serine/arginine-rich splicing factor 3) plays a significant role in various biological processes, many of its functions still remain unclear. More particularly, little is known about SRSF3's involvement in the regulation of miRNA. In this report, we found that invasive and migratory abilities were inhibited in SRSF3-silenced U2OS and HeLa cells. We also found that a knockdown of SRSF3 results in a decreased expression level of REST (RE1-silencing transcription factor). The silencing of REST increased the expression of primary miR-132/212 as well as their mature forms. In particular, miR-132-3p and miR-212-3p possess an identical seed sequences and a common target gene. Overexpression of miR-132-3p and miR-212-3p suppressed the expression of YAP1 (Yes-associated protein 1) by directly binding to the 3՚UTR of its mRNA. CCND1 (Cyclin D1), which acts downstream of YAP1, was downregulated in both miR-132-3p and miR-212-3p-overexpressed cells, in correlation with diminished YAP1 levels. Taken together, our results reveal that SRSF3 controls the expression of the miR-132/212 cluster through regulating REST expression, and that the REST-elicited alteration of miRNA expression is implicated in enabling the migratory and invasive abilities of cancer cells.

SOX11 identified by target gene evaluation of miRNAs differentially expressed in focal and non-focal brain tissue of therapy-resistant epilepsy patients.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control the expression of their target genes via RNA interference. There is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including epilepsy, a chronic neurological disorder characterized by spontaneous recurrent seizures. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy in which disease-induced abnormalities of hippocampal neurogenesis in the subgranular zone as well as gliosis and neuronal cell loss in the cornu ammonis area are reported. We hypothesized that in MTLE altered miRNA-mediated regulation of target genes could be involved in hippocampal cell remodeling. A miRNA screen was performed in hippocampal focal and non-focal brain tissue samples obtained from the temporal neocortex (both n=8) of MTLE patients. Out of 215 detected miRNAs, two were differentially expressed (hsa-miR-34c-5p: mean increase of 5.7 fold (p=0.014), hsa-miR-212-3p: mean decrease of 76.9% (p=0.0014)). After in-silico target gene analysis and filtering, reporter gene assays confirmed RNA interference for hsa-miR-34c-5p with 3'-UTR sequences of GABRA3, GRM7 and GABBR2 and for hsa-miR-212-3p with 3'-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2. Reporter gene assays with mutated 3'-UTR sequences of the transcription factor SOX11 identified two different binding sites for hsa-miR-212-3p and its primary transcript partner hsa-miR-132-3p. Additionally, there was an inverse time-dependent expression of Sox11 and miR-212-3p as well as miR-132-3p in rat neonatal cortical neurons. Transfection of neurons with anti-miRs for miR-212-3p and miR-132-3p suggest that both miRNAs work synergistically to control Sox11 expression. Taken together, these results suggest that differential miRNA expression in neurons could contribute to an altered function of the transcription factor SOX11 and other genes in the setting of epilepsy, resulting not only in impaired neural differentiation, but also in imbalanced neuronal excitability and accelerated drug export.

LncRNA MIR155HG functions as a ceRNA for inhibition of lung adenocarcinoma growth and prediction of prognosis.

Introduction: Long non-coding RNAs (lncRNAs) functioning as competing endogenous RNAs (ceRNAs) play critical roles in tumour progression. However, prognosis-related ceRNA networks in lung adenocarcinoma (LUAD) have not been well characterised. Material and methods: LUAD datasets were downloaded from the TCGA database, and the patients were divided into metastasis and non-metastasis groups. The differential expression of lncRNAs (DELs), miRNAs (DEMs), and mRNAs (DEGs) was analysed using the Limma package. Next, interactions between miRNA, lncRNA, and mRNA were predicted by miRcode, miRTar-Base, miRDB, and TargetScan. The ceRNA network was constructed based on these interactions using Cytoscape software. DEG enrichment analysis was performed by GO and KEGG. After the prognosis analysis, we further screened molecules and constructed the prognosis-related ceRNA network. Moreover, the interactions between lncRNA, miRNA, and mRNA were validated by biological experiments. Results: 854 DELs, 150 DEMs, and 2211 DEGs between metastasis and non-metastasis LUAD patients were identified. Functional enrichment analysis suggested that DEGs were closely related to key biological processes involved in LUAD progression. The prognosis-related ceRNA network included 1 miRNA, 2 lncRNAs, and 4 mRNAs. In this network, MIR155HG and ADAMTS9-AS2 can function as ceRNAs of miR-212 to regulate EPM2AIP1, LAX1, PRICKLE2, and CD226. Moreover, our study confirmed that MIR155HG inhibited the proliferation, migration, and invasion of LUAD cells by sponging miR-212-3p to regulate CD226. Conclusions: This ceRNA network contributes to understanding the pathogenesis of LUAD. Furthermore, the molecules in the network are valuable predictive factors for LUAD prognosis as well as potential therapeutic biomarkers.

Silencing LncRNA SNHG16 suppresses the diabetic inflammatory response by targeting the miR-212-3p/NF-κB signaling pathway.

BACKGROUND: Long noncoding RNAs (LncRNAs) have been identified to play an important role in diabetes. The aim of the present study was to determine the expression and function of small nucleolar RNA host gene 16 (SNHG16) in diabetic inflammation. METHODS: For the in vitro experiments, quantitative real-time PCR (qRT-PCR), Western blotting and immunofluorescence were used to detect LncRNA SNHG16 expression in the high-glucose state. The potential microRNA sponge target of LncRNA SNHG16, miR-212-3p, was detected by dual-luciferase reporter analysis and qRT-PCR. For the in vivo experiments, glucose changes in mice were detected after si-SNHG16 treatment, and SNHG16 and inflammatory factor expression in kidney tissues were detected by qRT-PCR and immunohistochemistry. RESULTS: LncRNA SNHG16 was upregulated in diabetic patients, HG-induced THP-1 cells, and diabetic mice. Silencing SNHG16 inhibited the diabetic inflammatory response and the development of diabetic nephropathy. miR-212-3p was found to be directly dependent on LncRNA SNHG16. miR-212-3p could inhibitor P65 phosphorylation in THP-1 cells. The miR-212-3p inhibitor reversed the action of si-SNHG16 in THP-1 cells and induced an inflammatory response in THP-1 cells. LncRNA SNHG16 was also found to be higher in the peripheral blood of diabetic patients than in the normal person. The area under the ROC curve is 0.813. CONCLUSION: These data suggested that silencing LncRNA SNHG16 suppresses diabetic inflammatory responses by competitively binding miR-212-3p to regulate NF-κB. LncRNA SNHG16 can be used as a novel biomarker for patients with type 2 diabetes.

Histone methyltransferase Setdb1 mediates osteogenic differentiation by suppressing the expression of miR-212-3p under mechanical unloading.

Emerging evidence indicates that multiple mechanisms are involved in bone loss induced by mechanical unloading. Thus far, few study has established the pathophysiological role of histone modification for osteogenic differentiation under mechanical unloading. Here we demonstrated that the histone H3 lysine 9 (H3K9) methyltransferase Setdb1, which was sensitive to mechanical unloading, was increased during osteogenic differentiation of MC3T3-E1 cells for the first time. Knockdown of Setdb1 significantly blocked osteoblast function in vivo and in vitro. Through bioinformatics analysis of candidate miRNAs regulated by H3K9me3, we further identified that Setdb1 inhibited the expression of miR-212-3p by regulating the formation of H3K9me3 in the promoter region. Mechanically, we revealed that miR-212-3p was upregulated under mechanical unloading and suppressed osteogenic differentiation by directly downregulating High mobility group box 1 protein (Hmgb1) expression. Furthermore, we verified the molecular mechanism of the SETDB1/miR-212-3p/HMGB1 pathway in hFOB cells under mechanical unloading. In summary, these data demonstrate the essential function of the Setdb1/miR-212-3p/Hmgb1 pathway in osteogenic differentiation under mechanical unloading, and present a potential protective strategies against bone loss induced by mechanical unloading.

Regulatory mechanisms of miR-212-3p on the secretion of inflammatory factors in monocyte-macrophages and the directed differentiation into osteoclasts in ankylosing spondylitis.

To explore the mechanisms of action of micro ribonucleic acid (miR)-212-3p in the secretion of inflammatory factors in monocyte-macrophages and the directed differentiation into osteoclasts (OCs) in ankylosing spondylitis (AS), proteoglycan was used to establish an AS mouse model. The mouse monocyte-macrophages were cultured in vitro, transfected with miR-212-3p mimic, and added with phosphorylated-extracellular signal-regulated kinase (p-ERK)1/2 agonist Ro67-7476 in vitro. After the cells were transfected with the miR-212-3p mimic in each group, the expressions of p-ERK1/2, matrix metalloproteinase-1 (MMP-1), MMP-3, interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) significantly declined, whereas those of tartrate-resistant acid phosphatase (TRAP), calcitonin, and p-nuclear factor of activated T cell 1 (NFATC1) significantly rose. After Ro67-7476 was added, the protein expressions of p-ERK1/2, MMP-1, MMP-3, IL-1ß, and TNF-α were significantly increased in each group, but they displayed decreasing trends in cells transfected with the miR-212-3p mimic. In contrast, the protein expressions of TRAP, calcitonin, and p-NFATC1 declined, but they showed increasing trends in cells transfected with the miR-212-3p mimic. miR-212-3p can, through inhibiting the phosphorylation of p-ERK1/2, prevent the aggregation of macrophages and the secretion of inflammatory factors. It also up-regulates the expression of OC marker proteins to facilitate the differentiation and maturation of OCs, ultimately relieving AS-induced inflammation and new bone growth-induced joint neoplasm.

Salvianolic acid A improves nerve regeneration and repairs nerve defects in rats with brain injury by downregulating miR-212-3p-mediated SOX7.

This study was to probe the protective effects and mechanisms of salvianolic acid A (SAA) on cerebral ischemia-reperfusion injury (CIRI). The middle cerebral artery occlusion model (MCAO) was established in rats. Rats' behavior, neurological deficits, brain injury, inflammation, and apoptosis in the brain tissue were evaluated. The inflammatory response and apoptosis of PC12 cells induced by oxygen glucose deprivation/reperfusion (OGD/R) were detected. SAA-mediated changes in miR-212-3p, SOX7, and Wnt/ß-catenin pathway were determined, and the targeting relationship between miR-212-3p and SOX7 was clarified. SAA alleviated the neurological deficits and brain injury of MCAO rats and inhibited the inflammatory response and apoptosis of OGD/R-conditioned PC-12 cells. SAA upregulated miR-212-3p, Wnt3a, and ß-catenin, whereas inhibited SOX7 levels. Silencing miR-212-3p counteracted the protective effect of SAA in the context of CIRI. SOX7 was a target protein of miR-212-3p. Silencing SOX7 based on SAA and miR-212-3p knockdown suppressed OGD/R-induced inflammation and apoptosis and increased Wnt3a and ß-catenin levels in PC12 cells. SAA can improve the brain and nervous system injury caused by cerebral ischemia-reperfusion by upregulating miR-212-3p, thereby inhibiting SOX7 and activating the Wnt/ßcatenin signaling pathway.

Long non-coding RNA SNHG5 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells via the miR-212-3p/GDF5/SMAD pathway.

BACKGROUND: The treatment of bone loss has posed a challenge to clinicians for decades. Thus, it is of great significance to identify more effective methods for bone regeneration. However, the role and mechanisms of long non-coding RNA small nucleolar RNA host gene 5 (SNHG5) during osteogenic differentiation remain unclear. METHODS: We investigated the function of SNHG5, Yin Yang 1 (YY1), miR-212-3p and growth differentiation factor 5 (GDF5) in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro and in vivo. Molecular mechanisms were clarified by chromatin immunoprecipitation assay and dual luciferase reporter assay. RESULTS: We found SNHG5 expression was upregulated during osteogenesis of hBMSCs. Knockdown of SNHG5 in hBMSCs inhibited osteogenic differentiation while overexpression of SNHG5 promoted osteogenesis. Moreover, YY1 transcription factor directly bound to the promoter region of SNHG5 and regulated SNHG5 expression to promote osteogenesis. Dual luciferase reporter assay confirmed that SNHG5 acted as a miR-212-3p sponge and miR-212-3p directly targeted GDF5 and further activated Smad1/5/8 phosphorylation. miR-212-3p inhibited osteogenic differentiation, while GDF5 promoted osteogenic differentiation of hBMSCs. In addition, calvarial defect experiments showed knockdown of SNHG5 and GDF5 inhibited new bone formation in vivo. CONCLUSION: Our results demonstrated that the novel pathway YY1/SNHG5/miR-212-3p/GDF5/Smad regulates osteogenic differentiation of hBMSCs and may serve as a potential target for the treatment of bone loss.