Jnk1 and downstream signalling hubs regulate anxiety-like behaviours in a zebrafish larvae phenotypic screen.

Current treatments for anxiety and depression show limited efficacy in many patients, indicating the need for further research into the underlying mechanisms. JNK1 has been shown to regulate anxiety- and depressive-like behaviours in mice, however the effectors downstream of JNK1 are not known. Here we compare the phosphoproteomes from wild-type and Jnk1-/- mouse brains and identify JNK1-regulated signalling hubs. We next employ a zebrafish (Danio rerio) larvae behavioural assay to identify an antidepressant- and anxiolytic-like (AA) phenotype based on 2759 measured stereotypic responses to clinically proven antidepressant and anxiolytic (AA) drugs. Employing machine learning, we classify an AA phenotype from extracted features measured during and after a startle battery in fish exposed to AA drugs. Using this classifier, we demonstrate that structurally independent JNK inhibitors replicate the AA phenotype with high accuracy, consistent with findings in mice. Furthermore, pharmacological targeting of JNK1-regulated signalling hubs identifies AKT, GSK-3, 14-3-3 ζ/ε and PKCε as downstream hubs that phenocopy clinically proven AA drugs. This study identifies AKT and related signalling molecules as mediators of JNK1-regulated antidepressant- and anxiolytic-like behaviours. Moreover, the assay shows promise for early phase screening of compounds with anti-stress-axis properties and for mode of action analysis.

Di-n-butyl phthalate induces toxicity in male fetal mouse testicular development by regulating the MAPK signaling pathway.

"White pollution" has a significant impact on male reproduction. Di-n-butyl phthalate (DBP) is one of the most important factors in this type of pollution. Currently, research from international sources has demonstrated the significant reproductive toxicity of DBP. However, most of these studies have focused mainly on hormones expression at the protein and mRNA levels and the specific molecular targets of DBP and its mechanisms of action remain unclear. In this study, we established a Sprague Dawley pregnant mouse model exposed to DBP, and all male offspring were immediately euthanized at birth and bilateral testes were collected. We found through transcriptome sequencing that cell apoptosis and MAPK signaling pathway are the main potential pathways for DBP induced reproductive toxicity. Molecular biology analyses revealed a significant increase in the protein levels of JNK1(MAPK8) and BAX, as well as a significant increase in the BAX/BCL2 ratio after DBP exposure. Therefore, we propose that DBP induces reproductive toxicity by regulating JNK1 expression to activate the MAPK signaling pathway and induce reproductive cell apoptosis. In conclusion, our study provides the first evidence that the MAPK signaling pathway is involved in DBP-induced reproductive toxicity and highlights the importance of JNK1 as a potential target of DBP in inducing reproductive toxicity.

Cold Stress Induces Apoptosis in Silver Pomfret via DUSP-JNK Pathway.

We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 ℃, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 ℃ detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 ℃, 23 ℃, 20 ℃, 17 ℃, and 14 ℃). The cell viability significantly declined from 14 to 20 ℃; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 ℃ and 14 ℃; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 ℃ and 14 ℃, and PIDD was up-regulated at 20 ℃, 17 ℃, and 14 ℃. DUSP3 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃, and PARP was down-regulated at 17 ℃ and 14 ℃. JNK2 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.

Spatio-temporal pattern of c-Jun N-terminal kinase isoforms in the cochleae of C57BL/6J mice with presbycusis.

The c-Jun N-terminal kinase (JNK) pathway is a vital component of the mitogen-activated protein kinase cascade, which regulates cell death and survival. The present study aimed to explore the Spatio-temporal changes in all JNK isoforms in the cochleae of C57/BL6J mice with age-related hearing loss. Changes in the three isoforms of JNKs in the cochleae of an animal model with presbycusis and the senescent HEI-OC1 cell line were tested by immunohistochemistry staining and western blotting. Our results demonstrated that all three JNK isoforms are distributed in the cochleae, and the expression patterns of JNK1, JNK2, and JNK3 differed in hair cells, spiral ganglion neurons, and stria vascularis, with great significance in the cochleae of adult C57BL/6J mice. The levels of JNK1, JNK2, and JNK3 showed various spatio-temporal changes in the aging mice. In a senescent hair cell model, changes in JNK1, JNK2, and JNK3 expression levels were similar to those observed in the cochleae. Our study is the first to show that JNK3 is highly expressed in the hair cells of C57BL/6J mice and further increases in conjunction with age-related hearing loss, suggesting that it may play a more critical role than previously believed in hair cell loss and spiral ganglion degeneration.

c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor that regulates early retinal development.

BACKGROUND: The transcription factor orthodenticle homeobox 2 (OTX2) has critical functions in brain and eye development, and its mutations in humans are related to retinal diseases, such as ocular coloboma and microphthalmia. However, the regulatory mechanisms of OTX2 are poorly identified. OBJECTIVE: The identification of JNK1 as an OTX2 regulatory protein through the protein interaction and phosphorylation. METHODS: To identify the binding partner of OTX2, we performed co-immunoprecipitation and detected with a pooled antibody that targeted effective kinases. The protein interaction between JNK1 and OTX2 was identified with the co-immunoprecipitation and immunocytochemistry. In vivo and in vitro kinase assay of JNK1 was performed to detect the phosphorylation of OTX2 by JNK1. RESULTS: JNK1 directly interacted with OTX2 through the transactivation domain at the c-terminal region. The protein-protein interaction and co-localization between JNK1 and OTX2 were further validated in the developing P0 mouse retina. In addition, we confirmed that the inactivation of JNK1 K55N mutant significantly reduced the JNK1-mediated phosphorylation of OTX2 by performing an immune complex protein kinase assay. CONCLUSION: c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor through the protein-protein interaction.

Silencing of UCA1 attenuates the ox-LDL-induced injury of human umbilical vein endothelial cells via miR-873-5p/MAPK8 axis.

LncRNA UCA1 plays a vital role in cardiovascular diseases. Endothelial cell dysfunction is a prerequisite for atherosclerosis (AS) development. However, the pathophysiological role of UCA1 in endothelial cell dysfunction induced by ox-LDL remains obscure. Here, we observed that UCA1 was upregulated in the sera of patients with AS and ox-LDL-treated endothelial cells. UCA1 knockdown dramatically reduced the cell apoptosis induced by ox-LDL and the production of pro-inflammatory cytokines and ROS in endothelial cells. Mechanistically, we found that UCA1 directly targeted miR-873-5p. UCA1 knockdown increased, while UCA1 overexpression decreased the expression of miR-873-5p. Further, we found that mitogen-activated protein kinase 8 (MAPK8) was a downstream target gene of miR-873-5p. MAPK8 overexpression or miR-873-5p knockdown reduced the enhancement of ox-LDL-induced cell apoptosis, oxidative stress, and pro-inflammatory cytokine production conferred by UCA1 knockdown. In conclusion, UCA1 can protect Human Umbilical Vein Endothelial Cells from ox-LDL-induced injury via the miR-873-5p/MAPK8 axis.

Scaffold protein MAPK8IP2 expression is a robust prognostic factor in prostate cancer associated with AR signaling activity.

Mitogen-activated protein kinase-8-interacting protein 2 (MAPK8IP2) is a scaffold protein that modulates MAPK signal cascades. Although MAPK pathways were heavily implicated in prostate cancer progression, the regulation of MAPK8IP2 expression in prostate cancer is not yet reported. We assessed MAPK8IP2 gene expression in prostate cancer related to disease progression and patient survival outcomes. MAPK8IP2 expression was analyzed using multiple genome-wide gene expression datasets derived from The Cancer Genome Atlas (TCGA) RNA-sequence project and complementary DNA (cDNA) microarrays. Multivariable Cox regressions and log-rank tests were used to analyze the overall survival outcome and progression-free interval. MAPK8IP2 protein expression was evaluated using the immunohistochemistry approach. The quantitative PCR and Western blot methods analyzed androgen-stimulated MAPK8IP2 expression in LNCaP cells. In primary prostate cancer tissues, MAPK8IP2 mRNA expression levels were significantly higher than those in the case-matched benign prostatic tissues. Increased MAPK8IP2 expression was strongly correlated with late tumor stages, lymph node invasion, residual tumors after surgery, higher Gleason scores, and preoperational serum prostate-specific antigen (PSA) levels. MAPK8IP2 upregulation was significantly associated with worse overall survival outcomes and progression-free intervals. In castration-resistant prostate cancers, MAPK8IP2 expression strongly correlated with androgen receptor (AR) signaling activity. In cell culture-based experiments, MAPK8IP2 expression was stimulated by androgens in AR-positive prostate cancer cells. However, MAPK8IP2 expression was blocked by AR antagonists only in androgen-sensitive LNCaP but not castration-resistant C4-2B and 22RV1 cells. These results indicate that MAPK8IP2 is a robust prognostic factor and therapeutic biomarker for prostate cancer. The potential role of MAPK8IP2 in the castration-resistant progression is under further investigation.

Downregulation of the Long Noncoding RNA IALNCR Targeting MAPK8/JNK1 Promotes Apoptosis and Antagonizes Bovine Viral Diarrhea Virus Replication in Host Cells.

Bovine viral diarrhea virus (BVDV) is the causative agent of the bovine viral diarrhea-mucosal disease, which is a leading cause of economic losses in the cattle industry worldwide. To date, many underlying mechanisms involved in BVDV-host interactions remain unclear, especially the functions of long noncoding RNAs (lncRNAs). In our previous study, the lncRNA expression profiles of BVDV-infected Madin-Darby bovine kidney (MDBK) cells were obtained by RNA-seq, and a significantly downregulated lncRNA IALNCR targeting MAPK8/JNK1 (a key regulatory factor of apoptosis) was identified through the lncRNA-mRNA coexpression network analysis. In this study, the function of IALNCR in regulating apoptosis to affect BVDV replication was further explored. Our results showed that BVDV infection-induced downregulation of the lncRNA IALNCR in the host cells could suppress the expression of MAPK8/JNK1 at both the mRNA and protein levels, thereby indirectly promoting the activation of caspase-3, leading to cell-autonomous apoptosis to antagonize BVDV replication. This was further confirmed by the small interfering RNA (siRNA)-mediated knockdown of the lncRNA IALNCR. However, the overexpression of the lncRNA IALNCR inhibited apoptosis and promoted BVDV replication. In conclusion, our findings demonstrated that the lncRNA IALNCR plays an important role in regulating host antiviral innate immunity against BVDV infection. IMPORTANCE Bovine viral diarrhea-mucosal disease caused by BVDV is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BVDV-host interactions are complex. To date, most studies focused only on how BVDV escapes host innate immunity. By contrast, how the host cell regulates anti-BVDV innate immune responses is rarely reported. In this study, a significantly downregulated lncRNA, with a potential function of inhibiting apoptosis (inhibiting apoptosis long noncoding RNA, IALNCR), was obtained from the lncRNA expression profiles of BVDV-infected cells and was experimentally evaluated for its function in regulating apoptosis and affecting BVDV replication. We demonstrated that downregulation of BVDV infection-induced lncRNA IALNCR displayed antiviral function by positively regulating the MAPK8/JNK1 pathway to promote cell apoptosis. Our data provided evidence that host lncRNAs regulate the innate immune response to BVDV infection.

Tumor Necrosis Factor-α-Induced C-C Motif Chemokine Ligand 20 Expression through TNF Receptor 1-Dependent Activation of EGFR/p38 MAPK and JNK1/2/FoxO1 or the NF-κB Pathway in Human Cardiac Fibroblasts.

Tumor necrosis factor (TNF)-α is involved in the pathogenesis of cardiac injury, inflammation, and apoptosis. It is a crucial pro-inflammatory cytokine in many heart disorders, including chronic heart failure and ischemic heart disease, contributing to cardiac remodeling and dysfunction. The implication of TNF-α in inflammatory responses in the heart has been indicated to be mediated through the induction of C-C Motif Chemokine Ligand 20 (CCL20). However, the detailed mechanisms of TNF-α-induced CCL20 upregulation in human cardiac fibroblasts (HCFs) are not completely defined. We demonstrated that in HCFs, TNF-α induced CCL20 mRNA expression and promoter activity leading to an increase in the secretion of CCL20. TNF-α-mediated responses were attenuated by pretreatment with TNFR1 antibody, the inhibitor of epidermal growth factor receptor (EGFR) (AG1478), p38 mitogen-activated protein kinase (MAPK) (p38 inhibitor VIII, p38i VIII), c-Jun amino N-terminal kinase (JNK)1/2 (SP600125), nuclear factor kappaB (NF-κB) (helenalin), or forkhead box O (FoxO)1 (AS1841856) and transfection with siRNA of TNFR1, EGFR, p38α, JNK2, p65, or FoxO1. Moreover, TNF-α markedly induced EGFR, p38 MAPK, JNK1/2, FoxO1, and NF-κB p65 phosphorylation which was inhibited by their respective inhibitors in these cells. In addition, TNF-α-enhanced binding of FoxO1 or p65 to the CCL20 promoter was inhibited by p38i VIII, SP600125, and AS1841856, or helenalin, respectively. Accordingly, in HCFs, our findings are the first to clarify that TNF-α-induced CCL20 secretion is mediated through a TNFR1-dependent EGFR/p38 MAPK and JNK1/2/FoxO1 or NF-κB cascade. We demonstrated that TNFR1-derived EGFR transactivation is involved in the TNF-α-induced responses in these cells. Understanding the regulation of CCL20 expression by TNF-α on HCFs may provide a potential therapeutic strategy in cardiac inflammatory disorders.

JNK1 and JNK3: divergent functions in hippocampal metabolic-cognitive function.

BACKGROUND AND AIM: The appearance of alterations in normal metabolic activity has been increasingly considered a risk factor for the development of sporadic and late-onset neurodegenerative diseases. In this report, we induced chronic metabolic stress by feeding of a high-fat diet (HFD) in order to study its consequences in cognition. We also studied the effects of a loss of function of isoforms 1 and 3 of the c-Jun N-terminal Kinases (JNK), stress and cell death response elements. METHODS: Animals were fed either with conventional chow or with HFD, from their weaning until their sacrifice at 9 months. Before sacrifice, body weight, intraperitoneal glucose and insulin tolerance test (IP-GTT and IP­ITT) were performed to evaluate peripheral biometrics. Additionally, cognitive behavioral tests and analysis of spine density were performed to assess cognitive function. Molecular studies were carried out to confirm the effects of metabolic stressors in the hippocampus relative to cognitive loss. RESULTS: Our studies demonstrated that HFD in Jnk3-/- lead to synergetic responses. Loss of function of JNK3 led to increased body weight, especially when exposed to an HFD and they had significantly decreased response to insulin. These mice also showed increased stress in the endoplasmic reticulum and diminished cognitive capacity. However, loss of function of JNK1 promoted normal or heightened energetic metabolism and preserved cognitive function even when chronically metabolically stressed. CONCLUSIONS: Downregulation of JNK3 does not seem to be a suitable target for the modulation of energetic-cognitive dysregulations while loss of function of JNK1 seems to promote a good metabolic-cognitive profile, just like resistance to the negative effects of chronic feeding with HFD.

Expression profiles and functions of ferroptosis-related genes in the placental tissue samples of early- and late-onset preeclampsia patients.

BACKGROUND: The accumulation of reactive oxygen species (ROS) resulting from upregulated levels of oxidative stress is commonly implicated in preeclampsia (PE). Ferroptosis is a novel form of iron-dependent cell death instigated by lipid peroxidation that likely plays an important role in PE pathogenesis. This study aimed to investigate the expression profiles and functions of ferroptosis-related genes (FRGs) in early-onset preeclampsia (EOPE) and late-onset preeclampsia (LOPE). METHODS: Gene expression data and clinical information were downloaded from the Gene Expression Omnibus (GEO) database. The "limma" R package was used to screen differentially expressed genes. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analyses were conducted to investigate the bioinformatics functions and molecular interactions of significantly different FRGs. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to verify the expression of hub FRGs in PE. RESULTS: A total of 4215 differentially expressed genes (DEGs) were identified between EOPE and preterm cases while 556 DEGs were found between LOPE and term controls. Twenty significantly different FRGs were identified in EOPE subtypes, while only 3 FRGs were identified in LOPE subtypes. Functional enrichment analysis revealed that the differentially expressed FRGs were mainly involved in EOPE and enriched in hypoxia- and iron-related pathways, such as the response to hypoxia, iron homeostasis and iron ion binding process. PPI network analysis and verification by RT-qPCR resulted in the identification of the following five FRGs of interest: FTH1, HIF1A, FTL, MAPK8 and PLIN2. CONCLUSIONS: EOPE and LOPE have distinct underlying molecular mechanisms, and ferroptosis may be mainly implicated in the pathogenesis of EOPE. Further studies are necessary for deeper inquiry into placental ferroptosis and its role in the pathogenesis of EOPE.

Farnesoid X receptor agonist attenuates subchondral bone osteoclast fusion and osteochondral pathologies of osteoarthritis via suppressing JNK1/2/NFATc1 pathway.

Osteoarthritis (OA) is a prevalent degenerative disease of the joint, featured by articular cartilage destruction and subchondral bone marrow lesions. Articular cartilage and subchondral bone constitute an osteochondral unit that guarantees joint homeostasis. During OA initiation, activated osteoclasts in subchondral bone ultimately result in impaired capacities of the subchondral bone in response to mechanical stress, followed by the degradation of overlying articular cartilage. Thus, targeting osteoclasts could be a potential therapeutic option for treating OA. Here, we observed that farnesoid X receptor (FXR) expression and osteoclast fusion and activity in subchondral bone were concomitantly changed during early-stage OA in the OA mouse model established by anterior cruciate ligament transection (ACLT). Then, we explored the therapeutic effects of FXR agonist GW4064 on the osteochondral pathologies in ACLT mice. We showed that GW4064 obviously ameliorated subchondral bone deterioration, associated with reduction in tartrate-resistant acid phosphatase (TRAP) positive multinuclear osteoclast number, as well as articular cartilage degradation, which were blocked by the treatment with FXR antagonist Guggulsterone. Mechanistically, GW4064 impeded osteoclastogenesis through inhibiting subchondral bone osteoclast fusion via suppressing c-Jun N-terminal kinase (JNK) 1/2/nuclear factor of activated T-cells 1 (NFATc1) pathway. Taken together, our results present evidence for the protective effects of GW4064 against OA by blunting osteoclast-mediated aberrant subchondral bone loss and subsequent cartilage deterioration. Therefore, GW4064 demonstrates the potential as an alternative therapeutic option against OA for further drug development.

Virtual Screening and Molecular Docking to Study the Mechanism of Chinese Medicines in the Treatment of Coronavirus Infection.

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.

The urinary exosomes derived from premature infants attenuate cisplatin-induced acute kidney injury in mice via microRNA-30a-5p/ mitogen-activated protein kinase 8 (MAPK8).

Acute kidney injury (AKI) is a susceptible factor for chronic kidney disease (CKD). There is still a lack of effective prevention methods in clinical practice. This study investigated the protective effect of the urinary exosomes from premature infants on cisplatin-induced acute kidney injury. Here we isolated exosomes from the fresh urine of premature infants. A C57BL/6 mice model of cisplatin-induced acute kidney injury was given 100 ug urinary exosomes 24 hours after model establishment. The kidneys were collected for pathological examination and the evaluation of renal tubular damage and apoptosis. In the in vitro experiment, human renal cortex/proximal tubular cells (HK-2) were induced by cisplatin to assess the effect of the urine exosomes from premature infants. Exosome microRNA (miRNA) sequencing technology was applied to investigate the miRNAs enriched in exosomes and the dual-luciferase gene reporter system to examine the targeting relationship of the miRNA with target genes. The results indicated that the urinary exosomes could decrease the serum creatinine level and the apoptosis of renal tubular cells, and reduce mice mortality. In addition, miR-30a-5p was the most abundant miRNA in the exosomes. It protected HK-2 cells from cisplatin-induced apoptosis by targeting and down-regulating the mitogen-activated protein kinase 8 (MAPK8). Together, our findings identified that the urinary exosomes derived from premature infants alleviated cisplatin-induced acute kidney injury and inhibited the apoptosis of HK-2 via miR-30a-5p, which could target MAPK8. These findings implied that urinary exosomes from premature infants riched in miR-30a-5p might become a potential treatment for AKI.

c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1.

The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 can act as either tumor suppressors or pro-oncogenic kinases in human cancers. The isoform-specific roles for JNK1 and JNK2 in human pancreatic cancer are still unclear, the question which should be addressed in this project. Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 clones were established either expressing either JNK1 or -2 shRNA in a stable manner. Basal anchorage-dependent and -independent cell growth, single-cell movement, and invasion using the Boyden chamber assay were analyzed. Xenograft growth was assessed using an orthotopic mouse model. All seven tested pancreatic cancer cell lines expressed JNKs as did human pancreatic cancer samples determined by immunohistochemistry. Pharmacological, unspecific JNK inhibition (SP600125) reduced cell growth of all cell lines but PANC-1. Especially inhibition of JNK2 resulted in overall increased oncogenic potential with increased proliferation and invasion, associated with alterations in cytoskeleton structure. Specific inhibition of JNK1 revealed opposing functions. Overall, JNK1 and JNK2 can exert different functions in human pancreatic cancer and act as counter players for tumor invasion. Specifically modulating the activity of JNKs may be of potential therapeutic interest in the future.

Long noncoding RNA plasmacytoma variant translocation 1 promotes progression of colorectal cancer by sponging microRNA-152-3p and regulating E2F3/MAPK8 signaling.

The purpose of this study was to investigate the pathogenesis of colorectal cancer (CRC) and the effects of the long noncoding RNA plasmacytoma variant translocation 1 (PVT1) on CRC progression. Bioinformatics analysis verified PVT1 expression in tumor and normal tissues. Quantitative PCR and western blotting were used to measure mRNA and protein levels, respectively. The MTT, Transwell, colony formation, and in vivo assays were used to assess the effects of PVT1 on proliferation, migration, and invasion by CRC cells. Both PVT1 and microRNA (miR)-152-3p were shown to be colocalized in CRC cells using FISH assay. The target genes of miR-152-3p were predicted and verified by bioinformatics analysis, luciferase assay, and RNA pull-down assay. The ChIP assay revealed that E2F3 binds with the promoter of MAPK8. We found that PVT1 was overexpressed in CRC specimens, and its expression was higher in CRC cells than normal intestinal cells. Overexpression of PVT1 enhanced the proliferation, migration, and invasion of CRC cells, whereas PVT1 knockdown inhibited these processes. MicroRNA-152-3p was a target of PVT1, and E2F3 was a target of miR-152-3p. Rescue experiments confirmed the interaction between miR-152-3p and PVT1 and between miR-152-3p and E2F3. Luciferase and ChIP assay results confirmed that E2F3 modulates the transcriptional activation of MAPK8. Long noncoding RNA PVT1 activated E2F3 signaling by sponging miR-152-3p. The PVT1/miR-152-3p/E2F3/MAPK8 axis promoted CRC progression.

c-Jun N-terminal kinase (JNK) signaling contributes to cystic burden in polycystic kidney disease.

Polycystic kidney disease is an inherited degenerative disease in which the uriniferous tubules are replaced by expanding fluid-filled cysts that ultimately destroy organ function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form, afflicting approximately 1 in 1,000 people. It primarily is caused by mutations in the transmembrane proteins polycystin-1 (Pkd1) and polycystin-2 (Pkd2). The most proximal effects of Pkd mutations leading to cyst formation are not known, but pro-proliferative signaling must be involved for the tubule epithelial cells to increase in number over time. The c-Jun N-terminal kinase (JNK) pathway promotes proliferation and is activated in acute and chronic kidney diseases. Using a mouse model of cystic kidney disease caused by Pkd2 loss, we observe JNK activation in cystic kidneys and observe increased nuclear phospho c-Jun in cystic epithelium. Genetic removal of Jnk1 and Jnk2 suppresses the nuclear accumulation of phospho c-Jun, reduces proliferation and reduces the severity of cystic disease. While Jnk1 and Jnk2 are thought to have largely overlapping functions, we find that Jnk1 loss is nearly as effective as the double loss of Jnk1 and Jnk2. Jnk pathway inhibitors are in development for neurodegeneration, cancer, and fibrotic diseases. Our work suggests that the JNK pathway should be explored as a therapeutic target for ADPKD.

Knockdown of transient receptor potential melastatin 2 reduces renal fibrosis and inflammation by blocking transforming growth factor-ß1-activated JNK1 activation in diabetic mice.

BACKGROUND: Diabetic nephropathy is a major complication of diabetes. We explore the protective effect of TRPM2 knockdown on the progression of diabetic nephropathy. METHODS: A type 2 diabetes animal model was established in C57BL/6N mice by long-term high-fat diet (HFD) feeding combined with a single injection of 100 mg/kg streptozotocin (STZ). Genetic knockdown of TRPM2 in mouse kidneys was accomplished by the intravenous injection via the tail vein of adeno-associated virus type 2 carrying TRPM2 shRNA. RESULTS: Mice with HFD/STZ-induced diabetes exhibited kidney dysfunction, as demonstrated by increased blood creatinine and urea nitrogen levels, accompanied by glomerulus derangement, tubule damage and extracellular matrix deposition in the interstitium. The protein expression of TRPM2, transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor, α-smooth muscles actin, fibronectin, collagen I and collagen III, and the mRNA expression and contents of inflammatory factors, including interleukin-1ß, interleukin-6, interferon-α, tumour necrosis factor -α and monocyte chemotactic protein -1, were significantly elevated in the renal tissues of the HFD/STZ-induced diabetes group compared to those of the two control groups. Furthermore, fluorescent staining of TRPM2 was markedly increased in the renal tubular epithelial cells from diabetic mice. Knockdown of TRPM2 significantly attenuated HFD/STZ-induced renal inflammatory responses and fibrosis, which was accompanied by activation of TGF-ß1-activated c-Jun N-terminal protein kinase-1 (JNK1) signalling. JNK1 inactivation reversed hyperglycaemia-induced fibrosis and inflammation in HK-2 cells. CONCLUSION: TRPM2 silencing significantly attenuated fibrosis and inflammation in the kidneys of mice with HFD/STZ-induced diabetes, which was largely achieved via the inhibition of TGF-ß1-activated JNK1 activation.

Oct4 downregulation-induced inflammation increases the migration and invasion rate of oral squamous cell carcinoma.

Inflammatory changes are involved in tumor cell proliferation, migration, and invasion. Tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) play important roles in inflammatory regulation during tumor development. Oct4 acts as a transcription factor that modulates inflammatory changes in mesenchymal stem cells. In this study, we explored the role of Oct4 in the invasion and migration of oral squamous cell carcinoma (OSCC) cells. LPS and TNF-α were used to treat the OSCC cell lines HN4 and CAL27 to induce inflammation. The generation of inflammatory cytokines, including TNF-α, interleukin (IL)-1ß, and IL-6, was evaluated by enzyme-linked immunosorbent assay and real-time quantitative PCR. Western blot analysis was employed to detect the expression and phosphorylation of JNK1, p65, and STAT3, which are key modulators of inflammation. Wound scratch healing and transwell invasion assays were further used to determine the role of inflammation in the invasion and migration of OSCC cells. Robust inflammation was observed in HN4 and CAL27 cells treated with LPS and TNF-α. A marked increase in JNK1, p65, and STAT3 phosphorylation levels in OSCC cells was also detected after LPS and TNF-α treatment. The migration and invasion of HN4 and CAL27 cells were significantly boosted by stimulation with LPS and TNF-α. Furthermore, Oct4 mRNA and protein levels were significantly upregulated by stimulation with LPS and TNF-α. Silencing of Oct4 led to reduced inflammation and decreased levels of phosphorylated JNK1, p65, and STAT3 and impaired invasion and migration in LPS- and TNF-α-stimulated OSCC cells. Overall, LPS- and TNF-α-induced inflammation suppressed the migration and invasion of OSCC cells by upregulating Oct4 expression.

Genetics plays a limited role in predicting chronic obstructive pulmonary disease treatment response and exacerbation.

BACKGROUND: Combination treatments, targeting multiple disease processes, benefit subjects with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). However, predicting treatment response and exacerbation risk remain challenging. OBJECTIVE: To identify genetic associations with AECOPD risk and response to combination therapy (fluticasone furoate, umeclidinium bromide and vilanterol). METHODS: The genetic basis of AECOPD disease was investigated in 19,841 subjects from 23 clinical studies and 2 disease cohorts to identify exacerbation disease targets. AECOPD pharmacogenetic effects were examined in 8439 moderate to severe COPD patients with exacerbation rate, lung function and quality of life endpoints; results were followed up in an additional 2201 subjects. RESULTS: We did not identify significant associations in the AECOPD disease analysis. In the AECOPD pharmacogenetics analysis, rs56195836 (MAPK8) was significantly associated with moderate to severe exacerbation rate in subjects on fluticasone furoate with baseline blood eosinophils ≥150 cells/µl (P = 1.8 × 10-8). Post-hoc, one variant was associated with on-treatment moderate to severe exacerbation rate stratifying by exacerbation history. AZU1 rs1962343 was significantly associated in subjects with frequent moderate exacerbation history when treated with fluticasone furoate/vilanterol (P = 1.1 × 10-8). Neither of these signals was supported in independent follow-up. CONCLUSION: Common genetic variants do not play major roles in AECOPD disease nor predict response to triple therapy or its components in moderate to very severe COPD.