MAPK1 Expression Research Articles

Exploration of the Pathogenesis of Chronic Obstructive Pulmonary Disease Caused by Smoking-Based on Bioinformatics Analysis and In Vitro Experimental Evidence.

This study was aimed at investigating the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by smoking-based on bioinformatics analysis and in vitro experimental evidence. The GEO, GEO2R, TargetScan, miRDB, miRWalk, DAVID, and STRING databases were used for bioinformatics analysis. The mRNA expression and the protein levels were determined by real-time PCR and ELISA. After taking the intersection of the diversified results of the databases, four differentially expressed miRNAs (hsa-miR-146a, hsa-miR-708, hsa-miR-150, and hsa-miR-454) were screened out. Subsequently, a total of 57 target genes of the selected miRNAs were obtained. The results of DAVID analysis showed that the selected miRNAs participated in COPD pathogenesis through long-term potentiation, the TGF-β signaling pathway, the PI3K-Akt signaling pathway, etc. The results of STRING prediction showed that TP53, EP300, and MAPK1 were the key nodes of the PPI network. The results of the confirmatory experiment showed that, compared with the control group, the mRNA expression of ZEB1, MAPK1, EP300, and SP1 were up-regulated, while the expression of MYB was down-regulated and the protein levels of ZEB1, MAPK1, and EP300 were increased. Taken together, miRNAs (hsa-miR-146a, hsa-miR-708, hsa-miR-150, and hsa-miR-454) and their regulated target genes and downstream protein molecules (ZEB1, EP300, and MAPK1) may be closely related to the pathological process of COPD.

Network pharmacology-based research on the effect of Scutellaria baicalensis on osteosarcoma and the underlying mechanism.

To explore the anti-tumor effects of Scutellaria baicalensis on osteosarcoma and its mechanism. Network pharmacology and molecular docking techniques were applied to investigate the effect and mechanism of Scutellaria baicalensis on osteosarcoma (OS). We analyzed the protein-protein interaction (PPI) network for potential targets of Scutellaria baicalensis for treating osteosarcoma and identified hub targets. We used KM curves to screen for hub targets that could effectively prolong the survival time of OS patients. We systematically performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of the Scutellaria baicalensis potential targets and predicted the Scutellaria baicalensis molecular mechanism and function in treating osteosarcoma. Through molecular docking, the binding process between the hub targets, which could prolong the survival time of sarcoma patients, and Scutellaria baicalensis was simulated. PPI network analysis of potential therapeutic targets discriminated 12 hub targets. The KM curves of the hub targets showed that upregulation of RXRA, RELA, ESR1, TNF, IL6, IL1B, and RB1 expression, and downregulation of MAPK1, VEGFA, MAPK14, CDK1, and PPARG expression were effective in improving the 5-year survival rate of OS patients. GO and KEGG enrichment demonstrated that Scutellaria baicalensis regulated multiple signaling pathways of OS. Molecular docking results indicated that Scutellaria baicalensis could bind freely to the above hub target, which could prolong the survival time of sarcoma patients. Scutellaria baicalensis acted on osteosarcoma by regulating a signaling network formed by hub targets connecting multiple signaling pathways. Scutellaria baicalensis appears to have the potential to serve as a therapeutic drug for osteosarcoma and to prolong the survival of OS patients.

MiR-22 inhibits angiotensin II-induced aortic dissection and protects aortic vessel wall in mice by targeting MAPK14

Purpose: To study the effect of miR-22 on angiotensin II-induced aortic dissection in mice, and its protective effect on aortic vessel wall, as well the involvement of MAPK-14 in these processes.
 Methods: A mouse aortic dissection model was established via subcutaneous implantation of angiotensin II (1 µg/kg/min) micropump in the dorsal region. The mice (n = 30) were assigned in equal numbers to 5 groups (n = 6). All injections were given via the tail vein. The miR-22 expressions in aortas of mice in each group were determined with quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Western blot assay was used to determine the expressions of MAPK-14 protein, while H&E staining was used to measure the ratio of aortic thickness-to-diameter, and contents of collagen and elastic fibers.
 Results: The expression of miR-22 in aorta of mice in miR-22 overexpression group was significantly higher than that in overexpression control group, but significantly lower in miR-22 inhibition mouse than in inhibition control mouse (p < 0.05). There was significantly lower protein expression of MAPK-14 in mice aorta in miR-22 overexpression mice than in overexpression control mice, but significantly upregulated in miR-22 inhibition mice, relative to that in inhibition control mice (p < 0.05). In the miR-22 overexpression mice, the ratio of membrane thickness-to-diameter was higher than the corresponding value in miR-22 inhibition mice. There were significantly higher contents of aortic elastic and collagen fibers in miR-22 overexpression mice than in overexpression control and miR-22 inhibition groups (p < 0.05).
 Conclusion: Overexpression of miR-22 inhibits the up-regulation of expression of its target gene mapk14, increases thickness of aortic media and aortic elasticity in mice, and increase the content of collagen fibers, thereby exerting protective effect on aortic wall structure.

Histone lactylation-derived LINC01127 promotes the self-renewal of glioblastoma stem cells via the cis-regulating the MAP4K4 to activate JNK pathway

Gliomas are the most prevalent and aggressive brain tumors, exhibiting high proliferation, abnormal glycolysis, and poor prognosis. LncRNAs act as regulatory molecules and play crucial roles in the malignant behaviors of GBM cells, including cell self-renewal. However, the regulatory mechanisms involved are largely unknown. In this study, we performed bioinformatics analysis to explore NF-κB pathway-related lncRNAs. ECAR and qRT-PCR were used to measure the relationship between glycolytic activity and lncRNA expression. Assays such as RIP-PCR and ChIP-PCR were employed to reveal the regulatory mechanisms of the lncRNA. Neurosphere formation and limiting dilution assays were performed to evaluate the self-renewal capacity of GBM cells. In our study, we identified an NF-κB pathway-related lncRNA named LINC01127 in GBM, which was found to be associated with poor progression of GBM. Functionally, the NF-κB pathway promoted warburg effect, which, in turn, induced the lactylation of H3 histone and increased the expression of LINC01127. Consequently, this enhancement of LINC01127 expression led to the promotion of self-renewal in GBM cells. Furthermore, LINC01127 regulated MAP4K4 expression in cis by directly guiding POLR2A to the MAP4K4 promoter regions, thereby leading to JNK pathway activation, and ultimately modulating the self-renewal of GBM cells. Moreover, the activated JNK pathway promoted the phosphorylation of IκBα. Overall, targeting LINC01127-mediated axis impeded orthotopic tumor growth in GBM xenografts. Taken together these results revealed that warburg effect-induced histone lactylation drives NF-κB-related LINC01127 expression, thereby promoting the self-renewal of GBM cells through the MAP4K4/JNK/NF-κB axis, and providing substantial evidence that LINC01127 might provide a novel therapeutic strategy for GBM patients.

MAPK1 knockdown ameliorated immune and inflammatory abnormalities in a mouse model of refractory asthma

Purpose: To evaluate the potential molecular mechanisms involved in refractory asthma in an animal model, and the potential therapeutic effect of MAPK1 knockdown on the disease.Methods: Eighteen female Institute of Cancer Research (ICR) mice, aged 8 - 10 weeks, were randomly divided into three groups: control, asthma model and refractory asthma, with 6 mice in each group. The expression of MAPK1 was knocked down in mice using an adenoviral vector. Subsequently, the methylation levels of MAPK1 promoter in mouse lung tissue were determined using methylation assays. Hematoxylin and eosin (H&E) staining and Periodic Acid-Schiff (PAS) staining were used to determine inflammatory and histological changes in lung tissues. Levels of immune cells were determined using flow cytometry, while Western blotting was used to measure the protein expression levels of ERK1/2, JNK, MEK1/2 and p38.Results: Methylation assay results show that mean methylation level of cg11335969 locus was significantly reduced in the refractory asthma mouse model (p < 0.05). The levels of IgG1 and IgM in refractory asthmatic mice were reduced after MAPK1 knockdown. There was a significantly reduced degree of lung lesions in mice (p < 0.05), as was reflected in effectively decreased histopathological changes. Protein levels of ERK1/2, JNK, MEK1/2 and p38, and the levels of neutrophils, dendritic cells, and macrophages were significantly decreased (p < 0.05).Conclusion: There is hypermethylated modification of MAPK1 at cg11335969 site in refractory asthma mouse model. Knockdown of MAPK1 attenuates inflammation and tissue damage, and reverses abnormal immune cell numbers in refractory asthma mice. Thus, MAPK1 inhibition may be a novel strategy for ameliorating immune abnormalities in refractory asthma.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

Association of Promoter Methylation Patterns with Expression of MAPK14 in Tissue of Papillary Thyroid Cancer Patients.

Thyroid cancer is one of the most prevalent malignancies worldwide. Genetic and epigenetic alterations are one of the main causes of thyroid tumor that is responsible to the activation of oncogenes as well as the inactivation of tumor suppressor genes. This research aimed to investigate the relationship of promoter methylation patterns with the expression of P38α in Iranian patients with thyroid cancer. We collected 40 thyroid tumor samples and 40 adjacent normal thyroid samples from 40 Iranian patients with papillary thyroid cancer. The promoter methylation pattern of P38α gene was investigated by methylation-sensitive high-resolution melting (MS-HRM) method. Moreover, mRNA expression of P38α was investigated by Real-Time PCR method. Further validation of the obtained results was performed by the Cancer Genome Atlas (TCGA) dataset. The obtained results indicated that the expression of the P38α (MAPK-14) gene in the thyroid cancer sample was considerably higher than tumor margin sample. Also, P38α gene promoter methylation was higher in thyroid margin tissue as compared to tumor tissue. These results were additionally confirmed by TCGA analysis. The receiver operating characteristic (ROC) curve analysis showed a high accuracy of P38α gene expression as a diagnostic biomarker for thyroid malignancy. Our study demonstrated that the P38α expression level gene was associated with thyroid cancer pathogenesis among the Iranian population. We suggested that this gene expression might be used as a biomarker for diagnosis of thyroid tumor.

Map4k4 is up-regulated and modulates granulosa cell injury and oxidative stress in polycystic ovary syndrome via activating JNK/c-JUN pathway: An experimental study

The regulatory mechanism on granulosa cells (GCs) oxidative injury is becoming increasingly important in polycystic ovary syndrome (PCOS) studies. Serine/threonine kinase mitogen-activated protein 4 kinase 4 (Map4k4) is linked with oxidative injury and possibly associated with premature ovarian failure and ovarian dysgenesis. Herein, we investigated the function and mechanism of Map4k4 in a PCOS rat model. A microarray from GEO database identified Map4k4 was up-regulated in the ovarian of PCOS rats, and functional enrichments suggested that oxidative stress-associated changes are involved. We verified the raised Map4k4 expression in an established PCOS rat model and also in the isolated PCOS-GCs, which were consistent with the microarray data. Map4k4 knockdown in vivo contributed to regular estrous cycle, restrained steroid concentrations and ovarian injury in PCOS rats. Both Map4k4 silencing in vivo and in vitro attenuated the PCOS-related GC oxidative stress and apoptosis. Mechanically, Map4k4 activated the JNK/c-JUN signaling pathway. Importantly, a JNK agonist restored the suppressive effects of Map4k4 silencing on PCOS-induced granulosa cell injury and oxidative stress. Besides, Map4k4 may be a target gene of miR-185-5p. In conclusion, Map4k4, a potential target of miR-185-5p, is up-regulated and induces ovarian GC oxidative injury by activating JNK/c-JUN pathway in PCOS. The Map4k4/JNK/c-JUN mechanism may provide a new idea on the treatment of PCOS.

Active ingredients Isorhamnetin of Croci Srigma inhibit stomach adenocarcinomas progression by MAPK/mTOR signaling pathway

Gastric cancer (GC) remains the third leading cause of cancer-related mortality in the world, and ninety-five percent of GC are stomach adenocarcinomas (STAD). The active ingredients of Croci Stigma, such as Isorhamnetin, Crocin, Crocetin and Kaempferol, all have antitumor activity. However, their chemical and pharmacological profiles remain to be elusive. In this study, network pharmacology was used to characterize the action mechanism of Croci Stigma. All compounds were obtained from the traditional Chinese medicine systems pharmacology (TCMSP) database, and active ingredients were selected by their oral bioavailability and drug-likeness index. The targets of Croci Stigma active ingredients were obtained from the traditional Chinese medicine integrated database (TCMID), whereas the related genes of STAD were obtained from DisGeNET platform. Cytoscape was used to undertake visual analyses of the Drug Ingredients–Gene Symbols–Disease (I–G–D) network, and 2 core genes including MAPK14, ERBB3 were obtained, which are the predicted targets of isorhamnetin (IH) and quercetin, respectively. Data analysis from TCGA platform showed that MAPK14 and ERBB3 all upregulated in STAD patients, but only the effect of MAPK14 expression on STAD patients’ survival was significant. Molecular docking showed that IH might affect the function of MAPK14 protein, and then the underlying action mechanisms of IH on STAD were experimentally validated using human gastric cancer cell line, HGC-27 cells. The results showed that IH can inhibit cell proliferation, migration, clonal formation, and arrest cell cycle, but promote the apoptosis of HGC-27 cells. qRT-PCR data demonstrated that IH downregulated the MAPK14 mRNA expression and EMT related genes. WB results showed that IH regulates MAPK/mTOR signaling pathway. These findings suggest that IH has the therapeutic potential for the treatment of STAD.

Arteriovenous malformation Map2k1 mutation affects vasculogenesis

Somatic activating MAP2K1 mutations in endothelial cells (ECs) cause extracranial arteriovenous malformation (AVM). We previously reported the generation of a mouse line allowing inducible expression of constitutively active MAP2K1 (p.K57N) from the Rosa locus (R26GT-Map2k1-GFP/+) and showed, using Tg-Cdh5CreER, that EC expression of mutant MAP2K1 is sufficient for the development of vascular malformations in the brain, ear, and intestines. To gain further insight into the mechanism by which mutant MAP2K1 drives AVM development, we induced MAP2K1 (p.K57N) expression in ECs of postnatal-day-1 pups (P1) and investigated the changes in gene expression in P9 brain ECs by RNA-seq. We found that over-expression of MAP2K1 altered the transcript abundance of > 1600 genes. Several genes had > 20-fold changes between MAP2K1 expressing and wild-type ECs; the highest were Col15a1 (39-fold) and Itgb3 (24-fold). Increased expression of COL15A1 in R26GT-Map2k1-GFP/+; Tg-Cdh5CreER+/− brain ECs was validated by immunostaining. Ontology showed that differentially expressed genes were involved in processes important for vasculogenesis (e.g., cell migration, adhesion, extracellular matrix organization, tube formation, angiogenesis). Understanding how these genes and pathways contribute to AVM formation will help identify targets for therapeutic intervention.

O-104 The Impact of application of CRISPR /dCas9 systems for increasing the expression of FSH receptor in human granulosa cells

Abstract Study question What are the molecular and physiological effects on human granulosa cells, when CRISPR/dCas9 epigenome edition technology is efficiently used to increase FSH receptor (FSHR) activity? Summary answer The significant increase in expression of FSHR maintained by dCAS9 systems has effects on several fundamental cellular events involving steroidogenic, life-sustaining,cell death related signaling sub-pathways. What is known already Manipulation of the expression of FSHR can result in biological events that include fundamental activities of FSH as regulation of folliculogenesis, production of steroidogenic hormones. FSHR can be activated in vitro by FSH analogues, and signaling networks activate ERK1/2 pathway through p38 mitogen-activated protein kinases. CRISPR/dCAS9 can be used to activate or inhibit gene action without silencing the gene. In spite of being used in many systems, interference of FSHR expression by epigenome edition is very limited as well as the use of in vitro tools to increase FSHR expression for investigating IVM, folliculogenesis, PCOS, FSH hyper/hyposensitivity, cancer biology. Study design, size, duration In-vitro cultured HGrC1 (human granulosa cell line) cells were treated by follitropin-alfa (Gonal-f). The experimental groups were designed in triplicates with cells i. without any genome edition and cells that have i. non-targeting gRNA, ii.dCAS9 activated FSHR, iii. dCAS9 activated FSHR and FSH treatment for 5 min, 15 min, or 1 hr; iv. only FSH treatment for 5 min, 15 min or 1 hr. FSHR related gene and protein expressions and estrogen production were evaluated. Participants/materials, setting, methods For human FSHR gene, two separate gRNAs were designed. Annealing, digestion, ligation, transformation, colony formation, plasmid isolation, Sanger sequencing analysis, MOI were accomplished for dCAS9/SAM system. HGrC1 (immortalized human granulosa cell line) cells were treated with 75 IU/ml Gonal-f in related groups. After treatment, in triplicates, gene/protein expression for MTOR, FSHR, AKT, MAPK8, MAPK1, TP 53, CASP 3, ESR1, CYP 19A1 were evaluated by qPCR, WB and IF. Estradiol was measured by chemiluminescence immunoassay. Main results and the role of chance FSHR has increased significantly in dCas9, dac9 + 5min Gonal-f, dCas9 + 15min Gonal-f, dCas9 + 1h Gonal-f; mostly in 5 min compared to groups without dCas9. The system plasmids did not show any non-specific effects.MAPK1, MAPK8, ESR1, MTOR, AKT, P53 and CASP3 gene expressions showed significant increase in dcas9 system groups compared to control and NT, whereas in all time-dependent treatments, a significant increase was seen in groups that have dcas9+Gonal-f compared to the ones treated with only Gonal-f. As time dependent manner has been investigated, expressions of MAPK1, MAPK8 and ESR1 were highest at min 5, expressions of CASP and AKT were highest at min 15 and the expression of MTOR was highest at hour 1, whereas FSHR and p53were strongly expressed at all time points (p &amp;lt; 0.05 for all mentioned results). Gene expression profiles were also confirmed for P-38 MAPK, AKT, FSHR by WB. IF stainings have revealed higher expression of AKT and MAPK in both dCas9 and dcas9+Gonal-f 5 min compared to the control group, however the intensity of the staining was not satisfactory enough. Interestingly, estrodiol(E2) levels were found to be higher in the 5 minutes only Gonal-f treatment and dcas9 with Gonal-f treatment, while the other groups were similar to control. Limitations, reasons for caution -This study has been conducted in cell line, rather than primary granulosa cells which are not easy targets for epigenome edition. -No testosterone derivatives were used as substrate for aromatase. This has been reflected in the results as significant increase in FSHR and related genes but non-significant increase in E2 production. Wider implications of the findings -dcas9/SAM system can be used efficiently increase other FSH-receptors, which is a potent way to reveal molecular sub-pathways in a temporal manner;eg. in male reproductive system or in other cells of female RS. -FSH related conditions as PCOS can be examined in vitro. -FHSR unresponsive conditions can be modeled in vitro. Trial registration number not applicable

Triterpene acid from Antrodia camphorata alleviates inflammation in acute liver injury.

This study aimed to investigate the role and mechanism of Anctin A, the Antrodia camphorata terpene component, in resisting liver injury. Network pharmacology analysis revealed that MAPK3 was the major action target of Antcin A. Furthermore, experimental research suggested that Antcin A suppressed mouse liver injury, reduced the inflammatory factor levels, and enhanced the anti-oxidative capacity. Meanwhile, it suppressed the expression of MAPK3 and the downstream NF-κB signal, while it did not significantly affect the expression of MAPK1. Based on network pharmacology method, this study discovers that the anti-liver injury effect of Antcin A is mainly related to MAPK3, and that Antcin A can suppress the activation of MAPK3 and its downstream NF-κB to inhibit mouse ALI.

LncRNA PVT1 inhibits endothelial cells apoptosis in coronary heart disease through regulating MAPK1 expression via miR-532-3p

Background: Coronary atherosclerotic heart disease (CAD) is an inflammatory vascular disease caused by atherosclerosis. Long non-coding RNAs are involved in the pathophysiological process of coronary heart disease. Here we investigated the regulatory effects of lncRNA PVT1 (PVT1) in human coronary artery endothelial cells (HCAECs). Methods: qRT-PCR and western blot were performed to detect gene and protein expressions. CCK-8, flow cytometry and wound healing assays were used to determine cell viability, apoptosis and migration of HCAECs. The binding relationship among miR-532-3p, PVT1 and MAPK1 was verified by dual luciferase reporter assay. Results: Overexpression of PVT1 markedly reduced cell apoptosis and increased cell proliferation and migration. However, miR-532-3p upregulation suppressed cell proliferation and migration and promoted apoptosis of HCAECs. PVT1 suppressed the expression of miR-532-3p via directly targeting miR-532-3p. And miR-532-3p overexpression abolished the effect of PVT1 upregulation on proliferation and apoptosis in HCAECs. Furthermore, MAPK1 acted as a target gene of miR-532-3p and miR-532-3p inhibited MAPK1 expression. Conclusion: PVT1 promoted MAPK1 expression by targeting miR-532-3p, thus inhibiting HCAECs apoptosis and promoting cell proliferation, suggesting PVT1 might have great potential as a therapeutic target for CAD.

MiR-92a-1-5p enriched prostate cancer extracellular vesicles regulate osteoclast function via MAPK1 and FoxO1

BackgroundWe have previously reported that extracellular vesicles (EVs) derived from osteoblastic, osteoclastic and mixed prostate cancer cells promote osteoclast differentiation and inhibit osteoblast differentiation via transferring miR-92a-1-5p. In the present study, we focused on engineering miR-92a-1-5p into EVs and determining any therapeutic roles and mechanisms of the engineered EVs.MethodsA stable prostate cancer cell line (MDA PCa 2b) overexpressing miR-92a-1-5p was constructed by lentivirus, and EVs were isolated by ultracentrifugation. The overexpression of miR-92a-1-5p in both cells and EVs was tested using qPCR. Osteoclast function was evaluated by Trap staining, mRNA expression of osteoclastic markers ctsk and trap, immunolabeling of CTSK and TRAP and microCT using either in vitro and in vivo assays. Target gene of miR-92a-1-5p was proved by a dual-luciferase reporter assay system. siRNAs were designed and used for transient expression in order to determine the role of downstream genes on osteoclast differentiation.ResultsStable overexpression cells of miRNA-92a-5p was associated with EVs upregulating this microRNA, as confirmed by qPCR. Further, miR-92a-1-5p enriched EVs promote osteoclast differentiation in vitro by reducing MAPK1 and FoxO1 expression, associated with increased osteoclast function as shown by TRAP staining and mRNA expression of osteoclast functional genes. siRNA targeting MAPK1 or FoxO1 resulted in similar increase in osteoclast function. In vivo, the miR-92a-1-5p enriched EVs given via i.v. injection promote osteolysis, which was associated with reduction of MAPK1 and FoxO1 expression in bone marrow.ConclusionThese experiments suggest that miR-92a-1-5p enriched EVs regulate osteoclast function via reduction of MAPK1 and FoxO1.Graphical

Abstract 5822: YAP inhibition via targeting MAP3K3-dependent lysosomal degradation pathway overcomes BRAF and CDK4/6 inhibitor resistance

Abstract The Hippo-yes-associated protein (YAP) signaling pathway activation underlies the resistance to a wide range of molecularly targeted anti-cancer agents. However, strategies to block YAP activity in cancer by promoting YAP degradation have been largely unsuccessful despite rigorous investigations into canonical Hippo-YAP regulatory mechanisms. Here, we report a novel mechanism of YAP stability regulation by MAP3K3 independently of LATS-mediated proteasomal degradation. We observed that MAP3K3 depletion significantly reduced YAP protein levels in both normal and malignant cells, and mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at its serine 405 residue. MAP3K3-mediated serine 405 YAP phosphorylation polarizes YAP interaction with BTRC while hindering its interaction with FBXW7, collectively preventing both proteasomal degradation and the p62-mediated lysosomal degradation of YAP. We investigated the role of this mechanism in overcoming anti-cancer resistance and observed robust YAP activation in CDK4/6 inhibitor-resistant luminal breast cancer cells. Genetic and pharmacological MAP3K3 inhibition suppressed YAP-induced drug resistance. Moreover, increased MAP3K3 expression supported the pro-survival activity of YAP in BRAF inhibitor-resistant melanoma cells, and MAP3K3 depletion downregulated YAP-dependent cell cycle gene signatures. Accordingly, the combination of vemurafenib and ponatinib was effective in reducing the survival of BRAF inhibitor-resistant melanoma cells. Collectively, our study identified a novel phosphorylation-mediated YAP stability regulation mechanism and underscored MAP3K3 inhibition as a promising strategy to overcome BRAF and CDK4/6 inhibitor resistance by directly promoting YAP degradation. Citation Format: Won-Ji Ryu, Sanghyun Park, Tae Yeong Kim, Yumi Hwang, Hyun Ju Han, Jeong Dong Lee, Gun Min Kim, Joohyuk Sohn, Sang Kyum Kim, Joon Kim, Min Hwan Kim. YAP inhibition via targeting MAP3K3-dependent lysosomal degradation pathway overcomes BRAF and CDK4/6 inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5822.

Seaweed Sargassum aquifolium extract ameliorates cardiotoxicity induced by doxorubicin in rats

Doxorubicin (DOX) is a potent anticancer drug with adverse cardiotoxic effects. Alginates are multifunctional biopolymers and polyelectrolytes derived from the cell walls of brown seaweeds. They are nontoxic, biocompatible, and biodegradable, and hence, utilized in several biomedical and pharmaceutical applications. Here, we investigated the potential cardioprotective effect of thermally treated sodium alginate (TTSA), which was extracted and purified from the seaweed Sargassum aquifolium, in treating acute DOX cardiotoxicity and apoptotic pathways in rats. UV–visible spectroscopy, Fourier-transform infrared, and nuclear magnetic resonance (1H–NMR) spectroscopy techniques were used to characterize TTSA. CK-MB and AST levels in sera samples were determined. The expression levels of Erk-2 (MAPK-1) and iNOS genes were investigated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of Erk-2, anti-apoptotic p53, and caspase-3 were analyzed using western blotting and ELISA. For the in vivo studies, sixty rats were randomly divided equally into six groups and treated with DOX, followed by TTSA. We revealed that treatment with TTSA, which has low molecular weight and enhanced antioxidant properties, improved DOX-mediated cardiac dysfunction and alleviated DOX-induced myocardial apoptosis. Furthermore, TTSA exhibited a cardioprotective effect against DOX-induced cardiac toxicity, indicated by the increased expression of MAPK-1 (Erk2) and iNOS genes, which are implicated in the adaptive responses regulating DOX-induced myocardial damage. Moreover, TTSA significantly (p < 0.05) suppressed caspase-3 and upregulated anti-apoptotic protein p53 expression. TTSA also rebalanced the cardiomyocyte redox potential by significantly (p < 0.05) increasing the levels of endogenous antioxidant enzymes, including catalase and superoxide dismutase. Our findings suggest that TTSA, particularly at a dose of 400 mg/kg b.w., is a potential prophylactic supplement for treating acute DOX-linked cardiotoxicity.

LncRNA FOXP4-AS1 silencing inhibits metastasis and epithelial-mesenchymal transition in nasopharyngeal carcinoma via miR-136-5p/MAPK1.

Nasopharyngeal carcinoma (NPC) is a malignant tumor caused by nasopharyngeal epithelium. Long non-coding RNAs (lncRNAs) and microRNAs have been identified as vital regulators in many tumors, including NPC. This study aimed to explain the biological roles and relevant mechanisms of lncRNA FOXP4-AS1 (FOXP4-AS1) in NPC. The levels of lncRNA FOXP4-AS1, miR-136-5p and MAPK1 in C666-1 and NP69 cells were analyzed by quantitative reverse transcription PCR (qRT-PCR). C666-1 cells viability, migration and invasion were evaluated by MTT and Transwell assay, respectively. The target gene of miR-136-5p predicted by TargetScan was further verified using dual luciferase reporter assay. Moreover, qRT-PCR and Western blot were adopted to assess epithelial-mesenchymal transition (EMT)-related gene expression, including E-cadherin and N-cadherin. We found that lncRNA FOXP4-AS1 was upregulated, while miR-136-5p was low-expressed in C666-1 cells, as opposed to NP69. Knockdown of FOXP4-AS1 notably suppressed C666-1 cell growth, inhibited cell migration and invasion. We also observed that E-cadherin expression was fortified and N-cadherin level was decreased in C666-1 cells after FOXP4-AS1-siRNA transfection. However, all these findings were eliminated in C666-1 cells after miR-136-5p inhibitor treatment. We also found miR-136-5p directly targeted MAPK1 and correlated inversely with MAPK1 expression in C666-1 cells. Further investigation suggested that MAPK1-plasmid reversed the effects of miR-136-5p mimic on cells viability, migration, invasion and EMT. To conclude, our data revealed that lncRNA FOXP4-AS1 knockdown alleviated metastasis and EMT in NPC via miR-136-5p/MAPK1, indicating that lncRNA FOXP4-AS1 may be a valuable therapeutic target for NPC diagnosis and treatment.

MAP4K4 promotes ovarian cancer metastasis through diminishing ADAM10-dependent N-cadherin cleavage

Peritoneal metastasis is a key feature of advanced ovarian cancer, but the critical protein required for ovarian cancer metastasis and progression is yet to be defined. Thus, an unbiased high throughput and in-depth study is warranted to unmask the mechanism. Transcriptomic sequencing of paired primary ovarian tumors and metastases unveiled that MAP4K4, a serine/threonine kinase belongs to the Ste20 family of kinases, was highly expressed in metastatic sites. Increased MAP4K4 expression in metastasis was further validated in other independent patients, with higher MAP4K4 expression associated with poorer survival, higher level of CA125 and more advanced FIGO stage. Down regulation of MAP4K4 inhibited cancer cell adhesion, migration, and invasion. Notably, MAP4K4 was found to stabilize N-cadherin. Further results showed that MAP4K4 mediated phosphorylation of ADAM10 at Ser436 results in suppression of N-cadherin cleavage by ADAM10, leading to N-cadherin stabilization. Pharmacologic inhibition of MAP4K4 abrogated peritoneal metastases. Overall, our data reveal MAP4K4 as a significant promoter in ovarian cancer metastasis. Targeting MAP4K4 may be a potential therapeutic approach for ovarian cancer patients.

Exosomal miR-370-3p increases the permeability of blood-brain barrier in ischemia/reperfusion stroke of brain by targeting MPK1.

Ischemia/reperfusion (I/R) damage induced by stroke poses a serious hazard to human life, while mechanism of blood-brain barrier (BBB) dysfunction is still unknown. To imitate stroke induced ischemia conditions in vivo, the rat model of cerebral I/R damage was created by middle cerebral artery occlusion (MCAO). In vitro, the rat microvascular endothelial cell line bEND.3 was subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Evans blue was used to evaluate the permeability of the blood-brain barrier (BBB). To evaluate gene expression at the mRNA and protein levels, researchers used real-time PCR and western blotting. Infarct volume and BBB permeability were considerably higher in cerebral (I/R) animals than in the Sham group. Exosomal miR-370-3p expression was shown to be higher in the brains of I/R injured rats and OGD/R treatment bEND.3. The BBB permeability was considerably increased when miR-370-3p was downregulated in OGD/R pretreated bEND.3. miR-370-3p regulates MAPK1 expression by targeting it. In bEND.3, OGD/R therapy increased BBB permeability substantially. OGD/R was inhibited by miR-370-3p mimic transfection, while miR-370-3p mimic was abolished by co-transfection with MAPK1 overexpression lentivirus. In cerebral I/R damage, exosomal miR-370-3p targets MAPK1 and aggregates BBB permeability.

Retracted: Long non-coding RNA HOTAIR regulates proliferation, migration and invasion of human cervical cancer cells by modulating expression of MAPK1.

[This retracts the article DOI: 10.5114/aoms.2019.83512.].