Mitogen-activated Protein Kinase 1 Expression Research Articles

Inhibiting Effect and Mechanism of Aconitum tanguticum (Maxim.) Stapf on Intestinal Fibrosis of CCD-18Co Cells

Abstract Objective: The objective of the study was to explore the potential signaling mechanism of Aconitum tanguticum (Maxim.) Stapf (ATS) and its impact on intestinal fibrosis in vitro. Methods: Network pharmacology was used to screen the active components of ATS and predict their potential targets in intestinal fibrosis. The protein–protein interaction network graph was constructed using drug–disease intersection targets retrieved from the Search Tool for Retrieval of Interacting Genes/Proteins database. The network diagram was analyzed using Cytoscape 3.6.1’stopology function. The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were conducted using the database for annotation, visualization, and integrated discovery platform. Intestinal fibroblast model in vitro was constructed using transforming growth factor-β1 (TGF-β1)-induced CCD-18Co cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the network pharmacology-predicted antifibrotic signaling pathway of ATS and the traditional antifibrotic signaling pathway. Results: Network pharmacology revealed that there were 19 active components in ATS, suggesting that ATS could be involved in the regulation of mitogen-activated protein kinase 1 (MAPK1), PIK3CA, MAPK3, and other important targets that are present in cancer pathways, including proteoglycans, influenza A, and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) signaling pathways. The results of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) demonstrated that 0.1–1 μg/mL ATS may suppress tissue inhibitor of metalloproteinase 1, collagen I, and alpha-smooth muscle actin expression levels. The expression of the TGF-β/suppressor of the mother against decapentaplegic (Smad) and PI3K/Akt signaling pathways was controlled by ATS. In addition, ATS inhibited the PI3K/Akt pathway by reducing the expression of MAPK1, HSP90AA1, and PIK3CA. Conclusions: It has been confirmed that ATS is a multipathway and multitarget treatment for intestinal fibrosis. This study suggested that ATS might alleviate intestinal fibrosis by blocking both the TGF-β/Smad and the PI3K/Akt pathway.

Expression and promoter methylation of mitogen-activated protein kinase 1 in tumor and marginal cells of breast cancer.

In the present study, we sought to explore potential differences in the expression and promoter methylation of mitogen-activated protein kinase 1 (MAPK1) between tumor and marginal cells of breast cancer lesions. A total of 50 randomly selected patients with breast cancer (BCa) undergoing needle biopsy were enrolled. Clinical specimens containing both tumor and marginal cells were collected and preserved. After DNA extraction using specific primers, MAPK1 mRNA and promoter methylation were measured with spectrophotometry at 260/280 nm absorption wavelengths. To deliver a comparative analysis, data from The Cancer Genome Atlas (TCGA) program regarding breast cancer (BRCA), were downloaded from Xena Functional Genomics Explorer and separately analyzed. The suitability of MAPK1 expression and promoter methylation as biomarkers for BCa was analyzed with receiver operating characteristic (ROC) curves. We found a positive correlation between tumor stage and MAPK1 expression (P-value: 0.029) in BCa. Likewise, MAPK1 expression was significantly associated with lymph node metastasis (P-value: 0.018). There was a significant difference in the expression of MAPK1 mRNA between tumor and marginal cells of BCa and BRCA (P-value < 0.001). However, we did not find any statistically significant difference in MAPK1 promoter methylation between tumor and marginal cells of both BCa and BRCA. With an area under the curve (AUC) of 0.71, the diagnostic accuracy of MAPK1 expression in BCa and BRCA was validated. However, MAPK1 promoter methylation was not found to be a suitable biomarker. Our findings suggest that while MAPK1 expression, might be a promising biomarker for evaluating oncogenic activity in patients suspected of BCa. We were not able to detect a prognostic/diagnostic role for MAPK1 promoter methylation.

MAPK1 promotes the metastasis and invasion of gastric cancer as a bidirectional transcription factor

BackgroundThe Mitogen-activated protein kinase 1 (MAPK1) has both independent functions of phosphorylating histones as a kinase and directly binding the promoter regions of genes to regulate gene expression as a transcription factor. Previous studies have identified elevated expression of MAPK1 in human gastric cancer, which is associated with its role as a kinase, facilitating the migration and invasion of gastric cancer cells. However, how MAPK1 binds to its target genes as a transcription factor and whether it modulates related gene expressions in gastric cancer remains unclear.ResultsHere, we integrated biochemical assays (protein interactions and chromatin immunoprecipitation (ChIP)), cellular analysis assays (cell proliferation and migration), RNA sequencing, ChIP sequencing, and clinical analysis to investigate the potential genomic recognition patterns of MAPK1 in a human gastric adenocarcinoma cell-line (AGS) and to uncover its regulatory effect on gastric cancer progression. We confirmed that MAPK1 promotes AGS cells invasion and migration by regulating the target genes in different directions, up-regulating seven target genes (KRT13, KRT6A, KRT81, MYH15, STARD4, SYTL4, and TMEM267) and down-regulating one gene (FGG). Among them, five genes (FGG, MYH15, STARD4, SYTL4, and TMEM267) were first associated with cancer procession, while the other three (KRT81, KRT6A, and KRT13) have previously been confirmed to be related to cancer metastasis and migration.ConclusionOur data showed that MAPK1 can bind to the promoter regions of these target genes to control their transcription as a bidirectional transcription factor, promoting AGS cell motility and invasion. Our research has expanded the understanding of the regulatory roles of MAPK1, enriched our knowledge of transcription factors, and provided novel candidates for cancer therapeutics.

Circ_0005736 promotes tenogenic differentiation of tendon-derived stem cells through the miR-636/MAPK1 axis

BackgroundTendon-derived stem cells (TDSCs) are one of stem cells characterized by greater clonogenicity, tenogenesis, and proliferation capacity. Circ_0005736 has been shown to be decreased in Rotator cuff tendinopathy. Here, we investigated the function and relationship of circ_0005736 in TDSC tenogenic differentiation.MethodsTransforming growth factor β1 (TGF-β1) was used to induce the tenogenic differentiation in TDSC. Cell proliferation, invasion and migration were evaluated by Cell Counting Kit-8, 5-Ethynyl-2′-deoxyuridine, transwell, and wound healing assays, respectively. The detection of the levels of genes and proteins was performed by qRT-PCR and Western blot. The binding between miR-636 and circ_0005736 or MAPK1 (Mitogen-Activated Protein Kinase 1) was verified using dual-luciferase reporter assay and RIP assays.ResultsTGF-β1 induced tenogenic differentiation by enhancing the production of tendon-specific markers and TDSC proliferation, invasion and migration. TGF-β1 treatment promoted circ_0005736 expression, knockdown of circ_0005736 abolished TGF-β1-induced tenogenic differentiation in TDSCs. Mechanistically, circ_0005736 acted as a sponge for miR-636 to up-regulate the expression of MAPK1, which was confirmed to be a target of miR-636 in TDSCs. Further rescue assays showed that inhibition of miR-636 could rescue circ_0005736 knockdown-induced suppression on TGF-β1-caused tenogenic differentiation in TDSCs. Moreover, forced expression of miR-636 abolished TGF-β1-caused tenogenic differentiation in TDSCs, which was rescued by MAPK1 up-regulation.ConclusionCirc_0005736 enhanced TGF-β1-induced tenogenic differentiation in TDSCs via increasing the production of tendon-specific markers and TDSC proliferation, invasion and migration through miR-636/MAPK1 axis.

LncRNA HCG27 Promotes Glucose Uptake Ability of HUVECs by MiR-378a-3p/MAPK1 Pathway.

Gestational diabetes mellitus (GDM) is the most common metabolic disorder during pregnancy. LncRNA HLA complex group 27 (HCG27) plays a crucial role in various metabolic diseases. However, the relationship between lncRNA HCG27 and GDM is not clear. This study aimed to verify a competing endogenous RNA (ceRNA) interaction regulation axis of miR-378a-3p/mitogen-activated protein kinase 1 (MAPK1) regulated by HCG27 in GDM. LncRNA HCG27 and miR-378a-3p were detected by RT-qPCR. The expression of MAPK1 in umbilical vein endothelial cells (HUVECs) was detected by RT-qPCR and that in the placenta by Western blotting. To explore the relationship among lncRNA HCG27, miR-378a-3p, MAPK1 and the glucose uptake ability of HUVECs, vector HCG27, si-HCG27, miR-378a-3p mimic and inhibitor were transfected to achieve overexpression and inhibition of HCG27 or miR-378a-3p. The interaction between miR-378a-3p and lncRNA HCG27 or MAPK1 was confirmed by the dual-luciferase reporter assay. Besides, glucose consumption by HUVECs was detected by the glucose assay kit. HCG27 expression was significantly decreased in both the placenta and primary umbilical vein endothelial cells, while the expression of miR-378a-3p was significantly increased in GDM tissues, and the expression of MAPK1 was decreased in GDM tissues. This ceRNA interaction regulation axis was proved to affect the glucose uptake function of HUVECs. The transfection of si-HCG27 could significantly reduce the expression of the MAPK1 protein. If the MAPK1 overexpression plasmid was transfected simultaneously with si-HCG27 transfection, the reduced glucose uptake in HUVECs resulting from the decrease in lncRNA HCG27 was reversed. MiR-378a-3p mimic can significantly reduce the mRNA expression of MAPK1 in HUVECs, whereas miR-378a-3p inhibitor can significantly increase the mRNA expression of MAPK1. The inhibition of miR-378a-3p could restore the decreased glucose uptake of HUVECs treated with si-HCG27. Besides, overexpression of lncRNA HCG27 could restore the glucose uptake ability of the palmitic acid-induced insulin resistance model of HUVECs to normal. LncRNA HCG27 promotes glucose uptake of HUVECs by miR-378a-3p/MAPK1 pathway, which may provide potential therapeutic targets for GDM. Besides, the fetal umbilical cord blood and umbilical vein endothelial cells collected from pregnant women with GDM after delivery could be used to detect the presence of adverse molecular markers of metabolic memory, so as to provide guidance for predicting the risk of cardiovascular diseases and health screening of offspring.

Development and validation of an oxidative stress‑related prognostic signature in osteosarcoma: A combination of molecular experiments and bioinformatics.

Osteosarcoma (OS) is one of the most prevalent malignancies with a bad prognosis. Oxidative stress is closely associated with various type of cancer. The present study aimed to establish an oxidative stress-related gene prognostic signature. Supported by The Cancer Genome Atlas and Gene Expression Omnibus, the least absolute shrinkage and selection operator regression, Cox regression, receiver operating characteristic curves and Kaplan-Meier survival analysis were used to construct and validate a prognostic signature and the derived risk score. Tumor microenvironment scores and immune infiltration levels in OS were calculated. Correlation between these parameters and risk score was analyzed. In addition, single analysis of each hub gene was performed. Finally, a series of molecular experiments was used to detect the role of MAP3K5 (one of the hub genes) in OS. A total of five genes most associated with OS prognosis were identified as independent predictors, namely catalase (CAT), mitogen-activated protein kinase 1 (MAPK1), glucose-6-phosphate dehydrogenase (G6PD), mitogen-activated protein kinase kinase kinase 5 (MAP3K5) and C-C motif chemokine ligand 2 (CCL2). Based on the signature, higher risk score indicated poorer prognosis. Nomogram performed well and reliably predicted 3- and 5-year survival rate in OS. Patients with increasing risk scores had higher tumor purity and lower immune infiltration levels. Compared with an osteoblast cell line, the expression of CAT, CCL2, MAPK1 and G6PD was upregulated and MAP3K5 was downregulated. MAP3K5 inhibited cellular proliferation and motility, promoted cellular apoptosis and induced reactive oxygen species generation. Overall, the signature could effectively predict the prognosis of patients with OS and were expected to be potential biomarkers. And it provided new ideas for understanding the interactions between oxidative stress and OS.

Mechanism of Zhinao Capsule in Treating Alzheimer's Disease Based on Network Pharmacology Analysis and Molecular Docking Validation.

Objective This study aimed to determine the active components of Zhinao capsule (ZNC) and the targets in treating Alzheimer's disease (AD) so as to investigate and explore the mechanism of ZNC for AD. Methods The active components and targets of ZNC were determined from the traditional Chinese medicine systems pharmacology database (TCMSP). The target genes of AD were searched for in GeneCards. Cytoscape was used to construct an herb-component-target-disease network. A protein-protein interaction (PPI) network was constructed by STRING. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the OmicShare. UCSF Chimera and SwissDock were used for molecular docking verification. Finally, four key target genes were validated by Western blotting. Results In total, 55 active components, 287 targets of active components, 1197 disease genes, and 134 common genes were screened, which were significantly enriched in 3975 terms of biological processes (BP), 284 terms of cellular components (CC), 433 terms of molecular functions (MF), and 245 signaling pathways. Caspase-3 (CASP3) and beta-sitosterol, tumor necrosis factor-alpha (TNF-α) and quercetin, vascular endothelial growth factor A (VEGFA) and baicalein, and mitogen-activated protein kinase 1 (MAPK1) and quercetin showed good-to-better docking. Moreover, ZNC not only downregulated CASP3 and TNF-α protein expression but also upregulated the protein expression of VEGFA and MAPK1. Conclusions The active components of ZNC, such as beta-sitosterol, quercetin, and baicalein may act on multiple targets like CASP3, VEGFA, MAPK1, and TNF-α to affect T cell receptor (TCR), TNF, and MAPK signaling pathway, thereby achieving the treatment of AD. This study provides a scientific basis for further exploring the potential mechanism of ZNC in the treatment of AD and a reference for its clinical application.

Network pharmacology analysis and experimental verification reveal the mechanism of the traditional Chinese medicine YU-Pingfeng San alleviating allergic rhinitis inflammatory responses.

YU-Pingfeng San (YPFS) can regulate inflammatory response to alleviate the symptoms of nasal congestion and runny rose in allergic rhinitis (AR). However, the mechanism of action remains unclear. In this study, 30 active ingredients of three effective herbs included in YPFS and 140 AR/YPFS-related genes were identified by database analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the targets were mainly enriched in immune inflammatory-related biological processes and pathways. Finally, three hub gene targeting epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B1 (AKT1) related to YPFS and AR were identified by network pharmacology analysis. YPFS treatment decreased the expression of EGFR, MAPK1, and AKT1 in ovalbumin (OVA)-induced AR mice and impaired the production of inflammatory factors interleukin (IL)-4, IL-5, and IL-13, thus alleviating immunoglobulin E (IgE) production and the symptoms of scratching nose in AR. Through molecular docking analysis, we found that the active ingredients decursin, anomalin, and wogonin of YPFS could bind to EGFR, MAPK1, and AKT1 proteins. Moreover, decursin treatment impaired the expression of IL-4 and IL-5 in human PBMCs. These results suggested that YPFS could alleviate the AR inflammatory responses by targeting EGFR, MAPK1, and AKT1, showing the mechanism of action of YPFS in AR treatment.

LncRNA LINC00680 Acts as a Competing Endogenous RNA and Is Associated With the Severity of Myasthennia Gravis.

Background and PurposeMyasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune disorder that can seriously affect patients' quality of life. However, few studies have focused on the severity of MG. Moreover, existing therapeutic efforts, including those targeting biomarkers for MG, remain unsatisfactory. Therefore, it is vital that we investigate the pathogenesis of MG and identify new biomarkers that can not only evaluate the severity of the disease but also serve as potential therapeutic targets. Long noncoding RNA LINC00680 has been found to be associated with the progression of a variety of diseases as a competing endogenous RNA (ceRNA). However, the specific role of LINC00680 in MG has yet to be clarified. Here, we aimed to investigate the association between LINC00680 and the severity of MG.MethodsBioinformatics tools, quantitative real-time PCR, Western blotting, and luciferase assays were selected to investigate key signaling pathways and RNA expression in patients with MG. The Quantitative MG Score scale and the MG Composite scale were used to evaluate the severity of MG in the included patients. Cell viability assays and flow cytometry analysis were selected to analyze cell proliferation and apoptosis.ResultsCompared with control subjects, the expression levels of LINC00680 and mitogen-activated protein kinase 1 (MAPK1) in peripheral blood mononuclear cells of patients with MG were both upregulated; the levels of miR-320a were downregulated. A positive correlation was detected between LINC00680 expression and the severity of MG. Luciferase reporter assays identified that LINC00680 acts as a target for miR-320a. The in vitro analysis confirmed that LINC00680 regulates the expression of MAPK1 by sponging miR-320a. Finally, the functional analysis indicated that LINC00680 promoted Jurkat cell proliferation and inhibited cellular apoptosis by sponging miR-320a.ConclusionLINC00680 may be associated with the severity of MG as a ceRNA by sponging miR-320a to upregulate MAPK1. These findings suggest that LINC00680 may represent a potential biomarker which evaluates the severity of MG and may serve as a therapeutic target.

Suppression of MIR31HG affects the functional properties of thyroid cancer cells depending on the miR-761/MAPK1 axis

BackgroundThyroid cancer is the most prevalent endocrine malignancy. Long non-coding RNA (lncRNA) MIR31HG is abnormally expressed in thyroid cancer tissues. However, the precise, critical role of MIR31HG in thyroid cancer development remains unclear.MethodsMIR31HG, microRNA (miR)-761 and mitogen-activated protein kinase 1 (MAPK1) were quantified by quantitative real-time PCR (qRT-PCR) and immunoblotting. Cell viability, proliferation, apoptosis, invasion and migration abilities were evaluated by MTS, 5-Ethynyl-2′-Deoxyuridine (EdU), flow cytometry, transwell and wound-healing assays, respectively. Dual-luciferase reporter assays were used to validate the direct relationship between miR-761 and MIR31HG or MAPK1.ResultsMIR31HG was overexpressed in human thyroid cancer, and its overexpression predicted poor prognosis. Suppression of MIR31HG impeded cell proliferation, invasion and migration, as well as promoted cell apoptosis in vitro, and diminished the growth of xenograft tumors in vivo. Mechanistically, MIR31HG targeted and regulated miR-761. Moreover, miR-761 was identified as a molecular mediator of MIR30HG function in regulating thyroid cancer cell behaviors. MAPK1 was established as a direct and functional target of miR-761 and MAPK1 knockdown phenocopied miR-761 overexpression in impacting thyroid cancer cell behaviors. Furthermore, MIR31HG modulated MAPK1 expression by competitively binding to miR-761 via the shared binding sequence.ConclusionOur findings demonstrate that MIR31HG targets miR-761 to regulate the functional behaviors of thyroid cancer cells by upregulating MAPK1, highlighting a strong rationale for developing MIR31HG as a novel therapeutic target against thyroid cancer.

Knockdown of circ_0001679 alleviates lipopolysaccharide-induced MLE-12 lung cell injury by regulating the miR-338-3p/ mitogen-activated protein kinase 1 axis

ABSTRACT The upregulation of circ_0001679 was reported in lipopolysaccharide (LPS)-induced lung injury mouse model, but its functional roles and mechanisms in LPS-induced lung injury remain to be investigated. In this study, we aimed to explore the potential role of circ_0001679 in septic acute lung injury. We initially established an in vitro lung cell injury model using LPS-treated MLE-12 cells. siRNAs targeting circRNA_0001679 were employed to stably knock down circRNA_0001679, followed by functional assays to investigate the effect of circRNA_0001679 silencing. The levels of inflammatory cytokines such as IL-6, IL-β and TNF-α (Tumor necrosis factor-α) were detected by ELISA (Enzyme-linked immunosorbent assay). Meanwhile, protein levels of Bcl-2, cleaved-caspase 3, Bax, and MAPK1 (Mitogen-Activated Protein Kinase 1) proteins expression level were measured by Western blot. We found that Circ_0001679 was upregulated in LPS-induced MLE-12 cells, and silencing circ_0001679 attenuated the growth inhibition and suppressed apoptosis induced by LPS. Circ_0001679 knockdown also lowered levels of IL-6, IL-β and TNF-α, and prevent the activation of cleaved-caspase 3 protein. We further revealed that circ_0001679 functioned as a sponge of miR-338-3p to negatively regulate miR-338-3p activity. miR-338-3p downregulated its downstream target MAPK1, while the upregulation of circ_0001679 maintained a high-level expression of MAPK1 by suppressing miR-338-3p. Collectively, our study indicates that circ_0001679/miR-338-3p/MAPK1 axis may play an important role in the pathogenesis of acute lung injury (ALI).

Exosomes Containing LINC00636 Inhibit MAPK1 through the miR-450a-2-3p Overexpression in Human Pericardial Fluid and Improve Cardiac Fibrosis in Patients with Atrial Fibrillation

The purpose of this study was to investigate the regulatory mechanism of miR-450a-2-3p in myocardial fibrosis in patients with atrial fibrillation. For this purpose, the expression profile of GSE55296 was extracted from the GEO database, and differentially expressed lncRNAs were identified. Gene ontology analysis of the target genes of mir-450a-2-3p indicated that there was a regulatory relationship between LINC00636 and miR-450a-2-3p. Further, the expression levels of the analyzed RNAs were confirmed by RT-qPCR. TGF-β1-induced cardiac fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs) were used to establish a myocardial fibrosis model and endothelium-mesenchymal transformation (EMT) model in vivo. We hypothesized that exosomes containing LINC00636 regulate the expression of miR-450a-2-3p. LINC00636 was positively correlated with the expression of miR-450a-2-3p. The overexpression of miR-450a-2-3p suppressed the MAPK1 expression in CFs, thereby inhibiting the expression of α-SMA, COL1, and COL3 and preventing CF proliferation. In HUVECs, the miR-450a-2-3p overexpression upregulated the expression of VE-Cadherin (VE-Cad) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by inhibiting the mitogen-activated protein kinase 1 (MAPK1) expression, whereas the expression levels of vimentin, COL1, and COL3 decreased. These results indicate that LINC00636, which is present in human pericardial fluid, is an antifibrotic molecule that inhibits MAPK1 through the miR-450a-2-3p overexpression and improves cardiac fibrosis in patients with atrial fibrillation.

MiR-194-5p protects against myocardial ischemia/reperfusion injury via MAPK1/PTEN/AKT pathway.

BackgroundMicroRNA (miRNA), which participates in various physiological and pathological processes, is a highly conserved small RNA sequence. This study aimed to investigate the role of miR-194-5p in hypoxia/reoxygenation (H/R)-induced cardiomyocyte apoptosis and myocardial ischemia/reperfusion (I/R) injury.MethodsWe set up an H/R H9c2 cell model in vitro and an I/R mouse model in vivo. Then, cell vitality, apoptosis, and histopathological evaluation were conducted. Reactive oxygen species (ROS) generation and the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) were examined by 2’,7’-Dichlorodihydrofluorescein diacetate (H2DCFDA), and enzyme-linked immunosorbent assay (ELISA), respectively. The level of creatine kinase isoenzyme (CK-MB), cardiac troponin I (cTnI), myoglobin (Mb) is examined by ELISA. The expression of Caspase-3, cleaved-Caspase-3, Bax, Bcl-2, phosphatase and tensin homolog deleted on chromosome ten (PTEN), and protein kinase B (AKT) was analyzed by western blot.ResultsData showed the expression of miR-194-5p was decreased in H/R-induced H9c2 cells and I/R-induced mouse. Conversely, overexpression of miR-194-5p could improve cardiomyocyte damage in ischemic models in vivo and in vitro. Furthermore, mitogen-activated protein kinase 1 (MAPK1) was found as a direct target of miR-194-5p, which negatively regulated the expression of MAPK1. The up-regulation of MAPK1 inhibited the myocardial protection previously observed by miR-194-5p.ConclusionsOur study shows overexpression of miR-194-5p protects against H/R injury in vitro and cardiac I/R injury in vivo, which involves the inhibition of cardiac apoptosis and oxidative stress by targeting MAPK1 expression via PTEN/AKT pathway. These findings supply novel insights into potential therapeutic targets for cardiovascular diseases.

MicroRNA-320c inhibits articular chondrocytes proliferation and induces apoptosis by targeting mitogen-activated protein kinase 1 (MAPK1).

To clarify the interaction of microRNA-320c (miR-320c) and mitogen-activated protein kinase 1 (MAPK1), and to investigate the effects of miR-320c on articular chondroctye proliferation and apoptosis. Lentiviral expression vectors were constructed and dual luciferase assays containing MAPK1 3'-untranslated regions (3'-UTRs) were performed. Small hairpin RNA (shRNA) was utilized to modulate MAPK1 expression. The messenger RNA and protein expression levels were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting respectively. Cell Counting Kit-8 and flow cytometry were conducted to detect the proliferation and apoptosis of Human Chondrocyte-articular (HC-a) cells. Besides that, the influences of miR-320c and MAPK1 on MAPK pathway activation were also evaluated. Our data identified MAPK1 as a direct target gene of miR-320c, and miR-320c can negatively regulate MAPK1 expression by directly binding to MAPK1 3'-UTR in HC-a cells. Further functional study displayed that miR-320c overexpression and MAPK1 shRNA significantly suppressed the proliferation of HC-a cells and promoted cell apoptosis. Meanwhile, MAPK1 shRNA could attenuate miR-320c inhibitor promotive effects on HC-a cell proliferation and reverse its inhibitory effect on cell apoptosis. MAPK1 overexpression could rescue the inhibitory effect of miR-320c on HC-a cell proliferation, and weaken the accelerating effect of miR-320c on cell apoptosis. However, neither miR-320c or MAPK1 shRNA regulate the expression of c-JUN, JNK and c-Fos. miR-320c inhibits articular chondrocyte proliferation and induces apoptosis by targeting MAPK1, suggesting that miR-320c perhaps participates in the pathogenesis of osteoarthritis and acts as a potential target for the therapeutic treatment of osteoarthritis.

Knockdown of circPUM1 impedes cell growth, metastasis and glycolysis of papillary thyroid cancer via enhancing MAPK1 expression by serving as the sponge of miR-21-5p.

Circular RNAs (circRNAs) are a crucial class of regulatory RNAs in cancer procession, including papillary thyroid cancer (PTC). Circ-Pumilio 1 (circPUM1) is a novel circRNA with the oncogenic function in ovarian cancer and lung cancer. However, the role of circPUM1 in PTC is undiscovered. This study was performed to investigate the biological function and molecular mechanism of circPUM1 in PTC. CircPUM1 and microRNA-21-5p (miR-21-5p) levels were analyzed via quantitative real-time polymerase chain reaction (qRT-PCR). Cellular viability and metastasis were measured using Cell Counting Kit 8 (CCK-8) and transwell migration/invasion assay. Glycolysis was evaluated by glucose uptake and lactate production. Associated proteins were examined applying with western blot. Dual-luciferase reporter assay and RNA pull-down assay were used to analyze the interaction between circPUM1 or mitogen-activated protein kinase 1 (MAPK1) and miR-21-5p. Moreover, the role of circPUM1 in vivo was explored by xenograft tumor experiment. Significantly, circPUM1 was upregulated in PTC tissue samples and cells. Cell growth, metastasis and glycolytic process of PTC cells were all inhibited after downregulation of circPUM1. Besides, circPUM1 could sponge miR-21-5p and MAPK1 was a target gene of miR-21-5p. Furthermore, we found that the anti-cancer effect of circPUM1 knockdown on PTC was partly ascribed to MAPK1 downregulation by upregulating miR-21-5p. Silencing circPUM1 also impeded tumorigenesis of PTC in vivo via miR-21-5p/MAPK1 axis. These findings suggested that circPUM1 knockdown inhibited MAPK1 expression by targeting miR-21-5p, consequently leading to the repressive effect on PTC progression. CircPUM1 might be a promising target to improve the diagnosis and treatment of PTC.

Mechanism of Salviae Miltiorrhizae Radix et Rhizoma in the Treatment of Knee Osteoarthritis Based on Network Pharmacology

ObjectiveThe molecular mechanism of Salviae Miltiorrhizae Radix et Rhizoma (SMRR) in the treatment of knee osteoarthritis (KOA) was analyzed based on network pharmacology.MethodsActive components and potential targets of SMRR were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. KOA targets were obtained from the OMIM, DisGeNET, DrugBank, PharmGKB, and GeneCards Databases. The potential targets of SMRR in the treatment of KOA were identified by the Venn diagram. A protein-protein interaction network was generated with the STRING database. Visualization of the interactions in a potential pharmacodynamic component-target network was accomplished with Cytoscape software. The Database for Annotation, Visualization, and Integrated Discovery database and R software were used for Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation analyses of common targets. Molecular docking of the potential leading components, as determined by efficacy with the core target molecules, was performed with Discovery Studio.ResultsFifty-seven potential pharmacodynamic components and 58 potential targets of SMRR in the treatment of KOA were found. Bioinformatics analyses showed that the interleukin (IL)-17, hypoxia-inducible factor-1 (HIF-1), and tumor necrosis factor (TNF) signaling pathways, as well as the advanced glycation end product-receptor for advanced glycation end product signaling pathway in cases of diabetic complications, are related to the molecular mechanism of SMRR in the treatment of KOA. Molecular docking results showed that luteolin, tanshinone IIA, cryptotanshinone, and other components of SMRR had a strong affinity for MYC, signal transducer and activator of transcription 3, caspase-3 (CASP3), JUN, cyclin D1, prostaglandin endoperoxide synthase 2 (PTGS2), epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), protein kinase B, vascular endothelial growth factor A, and other targets.ConclusionSMRR indirectly regulates IL-17, HIF-1, TNF, and other signal transduction pathways by regulating the expression of proteins, including PTGS2, MAPK1, EGFR, and CASP3, thus playing a role in promoting chondrocyte proliferation, improving microcirculation, eliminating free radicals, and inhibiting inflammatory factors.

DNMT1-mediated methylation inhibits microRNA-214-3p and promotes hair follicle stem cell differentiate into adipogenic lineages

BackgroundDysfunction of the DNA methylation was associated with stem cell reprogramming. Moreover, DNA methyltransferase 1 (DNMT1) deficiency was involved in the differentiation of hair follicle stem cell (HFSc), but the molecular mechanisms remain unknown.MethodsHFSc from human scalp tissues were isolated and cultured. The oil red O staining was used to observe the adipogenesis. The interaction relationship between microRNA (miR)-214-3p and mitogen-activated protein kinase 1 (MAPK1) was accessed by dual-luciferase reporter gene assay. The methylation level of miR-214-3p promoter was detected by methylation-specific PCR and the enrichment of DNMT1 in miR-214-3p promoter by chromatin immunoprecipitation assay. A mouse model of trauma was established to observe the skin regeneration at 0, 6, and 14 days.ResultsExpression of DNMT1 and MAPK1 was increased in the HFSc, while the expression of miR-214-3p was reduced. Moreover, DNMT1 inhibited the expression of miR-214-3p by promoting the promoter methylation of miR-214-3p. Overexpression of DNMT1 could reduce the expression of miR-214-3p, but increase the expression of MAPK1 and the extent of extracellular signal regulated kinase (ERK)1/2 phosphorylation, leading to enhanced adipogenic differentiation. Importantly, DNMT1 promoted skin regeneration in vivo. Conversely, overexpression of miR-214-3p could reverse the effects of DNMT1 on adipogenesis of HFSc.ConclusionDNMT1 promotes adipogenesis of HFSc by mediating miR-214-3p/MAPK1/p-ERK1/2 axis. This study may provide novel biomarkers for the potential application in stem cell therapy.

LINC01436 Inhibited miR-585-3p Expression and Upregulated MAPK1 Expression to Promote Gastric Cancer Progression

Gastric cancer (GC) is a prevalent type of digestion system malignancies. Dysregulation of long non-coding RNAs (lncRNAs) has been proven to be prognostic factors and biological regulators in human cancers. The current study aimed to explore the role of long intergenic non-protein coding RNA 1436 (LINC01436) and its underlying mechanism in the progression of GC. RT-qPCR was conducted to measure RNA expression. Western blot was used for exploration of protein level. CCK-8, caspase-3 activity, and transwell assays were applied to evaluate the proliferative, apoptotic, and migratory abilities of GC cells, respectively. Mechanical experiments were used to probe the molecular interplay between genes. High LINC01436 level suggested low overall survival in GC patients, and LINC01436 was highly expressed in GC tissues and cells. Besides, LINC01436 knockdown hampered cell proliferation and migration, while facilitated cell apoptosis. Mechanistically, LINC01436 upregulated mitogen-activated protein kinase 1 (MAPK1) expression by competitively binding with miR-585-3p and inhibiting miR-585-3p expression. Furthermore, LINC01436 negatively regulated miR-585-3p expression by enhancing the zeste 2 polycomb repressive complex 2 subunit (EZH2)-induced trimethylation of histone H3 at lysine 27 (H3K27me3) on miR-585-3p promoter. Final rescue assays revealed that overexpression of MAPK1 could rescue the suppressive influence of LINC01436 depletion on GC progression. LINC01436 epigenetically silences miR-585-3p and acts as miR-585-3p to upregulate MAPK1 expression and promote GC progression.

NEAT1/miR-140-3p/MAPK1 mediates the viability and survival of coronary endothelial cells and affects coronary atherosclerotic heart disease.

Studies have shown that long non-coding RNAs (lncRNA) play critical roles in coronary atherosclerotic heart disease (CAD). However, the function of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in CAD is unclear. In this study, we aimed to investigate the functions of lncRNA NEAT1 in CAD. RT-PCR and western blot analysis were carried out to examine the expressions of related RNAs. Colony formation assay, cell proliferation assay, apoptosis assay, and dual-luciferase reporter assay were conducted to investigate the abilities of colony migration, cell proliferation, apoptosis, and targeting. The results showed that NEAT1 was up-regulated in CAD blood samples and in human coronary endothelial cells (HCAECs). Transfection of pcNEAT1 significantly inhibited the survival rate of HCAECs and induced apoptosis of HCAECs. MiR-140-3p was down-regulated in HCAECs. NEAT1 directly targeted miR-140-3p, and the expression of miR-140-3p was inversely correlated with the expression of NEAT1 in CAD patients. In addition, co-transfection of NEAT1 with miR-140-3p mimic reversed the effect of pcNEAT1 on cell viability and apoptosis. mitogen-activated protein kinase 1 (MAPK1) was proved to be a target gene of miR-140-3p, and the miR-140-3p mimic was shown to reduce the expression of MAPK1 in HCAECs. pcNEAT1 significantly increased the expression level of MAPK1, while shNEAT1 significantly reduced the expression level of MAPK1. Our results revealed that lncRNA NEAT1 increased cell viability and inhibited CAD cell apoptosis possibly by activating the miR-140-3p/MAPK1 pathway, and lncRNA NEAT1 might serve as a potential therapeutic target for CAD.

P53 and H3K4me2 activate N6-methylated LncPGCAT-1 to regulate primordial germ cell formation via MAPK signaling.

Long noncoding RNAs (lncRNAs) participate in the formation of primordial germ cells (PGCs); however, the identity of the key lncRNAs and the molecular mechanisms responsible for the formation of PGCs remain unknown. Here, we identify a key candidate lncRNA (lncRNA PGC transcript-1, LncPGCAT-1) via RNA sequencing of embryonic stem cells, PGCs, and Spermatogonial stem cells (SSCs). Functional experiments confirmed that LncPGCAT-1 positively regulated the formation of PGCs by elevating the expression of Cvh and C-kit while downregulating the pluripotency(Nanog) in vitro and in vivo; PAS staining of genital ridges in vivo also showed that interference with LncPGCAT-1 can significantly reduce the number of PGCs in genital ridges, while overexpression of LncPGCAT-1 had the opposite result. The result of luciferase reporter assay combined with CHIP-qPCR showed that the expression of LncPGCAT-1 was promoted by the transcription factor P53 and high levels of H3K4me2. Mechanistically, the luciferase reporter assay confirmed that mitogen-activated protein kinase 1 (MAPK1) was the target gene of LncPGCAT-1 and gga-mir-1591. In the ceRNA system, high levels of N6 methylation of LncPGCAT-1 enhanced the adsorption capacity of LncPGCAT-1 for gga-mir-1591. Adsorption of gga-mir-1591 activated the MAPK1/ERK signaling cascade by relieving the gga-mir-1591-dependent inhibition of MAPK1 expression. Moreover, LncPGCAT-1 interacted with interleukin enhancer binding factor 3 (ILF3) to regulate the ubiquitination of P53 and phosphorylation of JNK. Interaction with ILF3 resulted in positive self-feedback regulation of LncPGCAT-1 and activation of JNK signaling, ultimately promoting PGC formation. Altogether, the study expands our knowledge of the function and molecular mechanisms of lncRNAs in PGC development.