JunD Expression Research Articles

B cell function in patients with systemic lupus erythematosus is regulated by the upregulation of JunD

PurposeSystemic lupus erythematosus (SLE) is largely caused by B cell dysfunction. JunD is an activator protein 1 family protein that has been linked to the regulation of apoptotic and proliferative activities. However, the precise mechanism(s) by which JunD functions remains to be fully elucidated. Accordingly, this study aimed to clarify the functional importance of JUND gene expression in SLE, with further analyses of the functional role that JunD plays as a regulator of B cell proliferation and immune function. MethodsReverse transcriptase quantitative polymerase chain reaction techniques were used to analyze JunD expression in B cells of patients with SLE and healthy subjects. Cell Counting Kit-8 (CCK-8) assays and flow cytometry methods were used to characterise proliferative activity, cell cycle progression, and apoptosis of B cells in which JunD was either knocked down or overexpressed. The immune status and autophagic activity of these cells were assessed using Western immunoblotting and enzyme-linked immunosorbent assay (ELISA). Additionally, a JunD knockdown mouse model was established, and the functional role of B cell JunD expression in the pathogenesis of SLE was assessed using Western immunoblotting, ELISA, and haematoxylin and eosin staining. ResultsB cells from patients with SLE exhibited upregulation of JunD, with overexpression facilitating in vitro cellular proliferation and modulation of the immune and autophagic status of these B cells. JunD knockdown was also sufficient to modulate in vivo immune function and the autophagic status of B cells. ConclusionJunD was upregulated in the B cells of patients with SLE, where it regulates proliferation, autophagy, and immunity.

LncRNA SNHG7 Functions as an Oncogene in Cervical Cancer by Sponging miR-485-5p to Modulate JUND Expression [Retraction

[This retracts the article DOI: 10.2147/OTT.S237802.].

Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial–mesenchymal transition in recurrent miscarriage

Research questionIs four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial–mesenchymal transition (EMT) in recurrent miscarriage? DesignVillus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. ResultsSignificantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. ConclusionsFHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

SINGLE‐CELL RNA‐SEQ OF CLASSIC HAIRY CELL LEUKEMIA REVEALS DISEASE DRIVERS LINKED TO INTRINSIC TREATMENT RESISTANCE AND IDENTIFIES DUSP1 AS POTENTIAL NEW THERAPEUTIC TARGET

Introduction: Standard treatment with purine analogs such as cladribine (2-CdA) achieves excellent remissions in classic hairy cell leukemia (HCL), but up to 25% of patients relapse early and there is a non-diminishing risk of late relapse in complete responders. Even when targeting the recurrent genetic driver mutation BRAF V600E, HCL cells frequently persist in the bone marrow suggesting cooperating biologic alterations facilitating disease survival and persistence. Methods: Single-cell RNA sequencing (scRNA-Seq) was performed in primary HCL cells derived from three long-term (>10 years progression-free survival, PFS) and three short-term responders after 2-CdA treatment (SR, <3 years PFS) and from up to three time points (HCL diagnosis, first, and second relapse). Functional studies were performed using the BRAF D594E-mutated HAIR-M cell line after validating BRAF D594E as an activating mutation with similar downstream signaling and inhibitory potential with BRAF inhibitors as compared to BRAF V600E in primary HCL cells. Results: scRNA-Seq revealed distinct HCL cell clusters characterized by marked overexpression of DUSP1, FOS and JUND in all six patients that were outgrowing at first and second relapse in all three SR. In these DUSP1hi/FOShi/JUNDhi HCL cells, PROGENy cancer pathway analysis demonstrated induced activation of MAPK pathways and gene ontology analysis found negative regulation of the pro-apoptotic p38-MAPK-cascade as potential underlying biologic alteration. BRAF D594E-mutated HAIR-M cells were selected as substitute for primary HCL cells in cell culture due to comparable changes upon exposure to BRAF inhibitors in BRAF kinase activity conformation as assessed by the modular kinase conformation biosensor platform (KinCon) and similar reduction in ERK phosphorylation levels in HEK293T cells transiently overexpressing either flag-tagged BRAF D594E or BRAF V600E. In HAIR-M, we confirm stable expression levels of DUSP1, FOS and JUND, which were highly increased when co-cultured with HS5 stromal cells. Whereas increased expression of FOS and JUND was reversible upon exposure to BRAF inhibitors, expression of DUSP1, a direct target of BRAF-MEK-ERK signaling, and phosphorylation of p38 remained unaffected. As DUSP1 preferentially dephosphorylates/inactivates p38, we studied exposure of the DUSP1-inhibitor BCI in co-culture and found re-phosphorylated p38. Conclusion: DUSP1hi/FOShi/JUNDhi HCL cells may represent a distinct subset of HCL cells highly dependent on environmental cues with an inherently more resistant phenotype to treatment with 2-CdA or BRAF-inhibitors. Inhibition of DUSP1 may represent a novel therapeutic approach by effectively truncating HCL cells from extracellular pro-survival stimuli. Finally, our results suggest that BRAF D594E-mutated HAIR-M cells may serve as a representative disease model for functional studies. Keywords: bioinformatics, computational and systems biology, indolent non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Temporal pattern of Fos and Jun families expression after mitogenic stimulation with FGF-2 in rat neural stem cells and fibroblasts.

Intense stimulation of most living cells triggers the activation of immediate early genes, such as Fos and Jun families. These genes are important in cellular and biochemical processes, such as mitosis and cell death. The present study focused on determining the temporal expression pattern of Fos and Jun families in fibroblasts and neural stem cells of cerebellum, hippocampus, and subventricular zone (SVZ) of rats of different ages at 0, 0.5, 1, 3, and 6 h after stimulation with fibroblast growth factor (FGF)-2. In neonates, a similar expression pattern was observed in all cells analyzed, with lower expression in basal condition, peak expression at 0.5 h after stimulation, returning to baseline values between 1 and 3 h after stimulation. On the other hand, cells from adult animals only showed Fra1 and JunD expression after stimulation. In fibroblasts and hippocampus, Fra1 reached peak expression at 0.5 h after stimulation, while in the SVZ, peak level was observed at 6 h after stimulation. JunD in fibroblasts presented two peak expressions, at 0.5 and 6 h after stimulation. Between these periods, the expression observed was at a basal level. Nevertheless, JunD expression in SVZ and hippocampus was low and without significant changes after stimulation. Differences in mRNA expression in neonate and adult animals characterize the significant differences in neurogenesis and cell response to stimulation at different stages of development. Characterizing these differences might be important for the development of cell cultures, replacement therapy, and the understanding of the physiological response profile of different cell types.

JUND Promotes Tumorigenesis via Specifically Binding on Enhancers of Multiple Oncogenes in Cervical Cancer.

Enhancers are genomic regulatory elements located distally from the target gene, which play a critical role in determining cell identity and function. Dysregulation of enhancers has been frequently observed in various types of cancer, including cervical cancer. However, the identity of enhancers and their associated transcriptional regulators that are involved in cervical cancer remains unclear. With bioinformatics and 3D genomics, we revealed the enhancers in cervical cancer cell line and calculated which transcription factor (TF) is specifically binding on them based on TFs motif database. We knockdowned this TF and studied its function in cervical cancer cell line in vivo and in vitro. We found 14,826 activated enhancers and predicted that JUND (JunD Proto-Oncogene) is relatively enriched in the sequences of these enhancers. Well-known oncogene MYC and JUN were regulated by JUND through enhancers. To further explore the roles of JUND in cervical cancer, we analyzed the gene expression data of clinical cervical cancer samples and knock-downed JUND by CRISPR-Cas9 in Hela cell line. We found JUND is over-expressed in cervical cancer and the expression of JUND increased along with the cervical cancer progresses. Knockdown of JUND decreased the proliferation of Hela cells in vitro and in vivo and blocked cell cycle in G1-phase. Transcriptome sequencing analysis revealed the identification of 2,231 differentially expressed genes in response to the JUND knockdown treatment. This perturbation resulted in the modulation of several biological processes and pathways that have been previously linked to cancer. These findings provide evidence for the significant involvement of JUND in cervical cancer pathogenesis, thereby positioning JUND as a potential therapeutic target for the treatment of this disease.

JUND facilitates proliferation and angiogenesis of esophageal squamous cell carcinoma cell via MAPRE2 up-regulation

ObjectiveEsophageal squamous cell carcinoma (ESCC) is a globally aggressive malignant tumor. This study aimed to investigate the mechanism of JUND in ESCC development via MAPRE2. MethodsESCC cells (KYSE-450 and ECA109) were transfected with small interfering RNA (si)-JUND, si-MAPRE2, si-JUND, or pcDNA3.1-MAPRE2. JUND and MAPRE2 expression in ESCC cells was detected with quantitative real-time polymerase chain reaction and western blot. Cell counting kit-8 and 5-ethynyl-2′-deoxyuridine assays were used to determine ESCC cell proliferation. Dual-luciferase reporter gene and chromatin immunoprecipitation assays were performed to assess binding between JUND and MAPRE2. Human umbilical vein endothelial cells (HUVECs) were co-cultured with ESCC cell supernatants. Angiogenesis was assessed with an in vitro angiogenesis assay. Western blot was conducted to evaluate the expression of angiogenic proteins [vascular endothelial growth factor A (VEGFA), matrix metallopeptidase 9 (MMP-9), and angiopoietin-2 (ang2)]. ResultsThe levels of expression of JUND and MAPRE2 were high in ESCC cells. Mechanistically, JUND bound to MAPRE2 promoter and increased MAPRE2 transcription. Downregulation of JUND or MAPRE2 inhibited KYSE-450 and ECA109 cell proliferation and reduced the levels of expression of VEGFA, MMP-9, and ang2 and tube formation in HUVECs co-cultured with ESCC cell supernatants. MAPRE2 upregulation counteracted the inhibitory effects of JUND silencing on cell proliferative and angiogenic capabilities in ESCC. ConclusionsJUND promoted MAPRE2 transcription, thereby facilitating cell proliferative and angiogenic abilities in ESCC.

Molecular mechanism of di-n-butyl phthalate promotion of bladder cancer development

PurposeTo determine whether di-n-butyl phthalate (DBP) promotes the occurrence of bladder cancer (BCa) and explore the action of DBP acts on BCa cells at the cellular and molecular levels. MethodsMTT and Transwell assays were used to investigate the tumorigenic actions of DBP on BCa cells. Second-generation sequencing was used to identify differences in gene expression before and after DBP treatment. Differential gene expression was verified by q-PCR and analyzed using bioinformatics. Cells were transfected to overexpress genes of interest and proliferation and migration were measured using MTT and Transwell assays, respectively. ResultsDBP treatment stimulated both proliferation and invasion in BCa cells. Second-generation sequencing identified differences in the expression of FOSB, JUND, ATP6V1C2, and RHOQ before and after DBP treatment. FOSB expression was confirmed by q-PCR and bioinformatic analyses. FOSB overexpression increased both proliferation and invasion in BCa cells. ConclusionDBP promoted BCa tumorigenesis by inducing changes in gene expression.

Aryl hydrocarbon receptor–targeted therapy for CD4+ T cell–mediated idiopathic pneumonia syndrome in mice

AbstractWe previously demonstrated that interferon γ (IFN-γ) derived from donor T cells co-opts the indoleamine 2,3-dioxygenase 1 (IDO1) → aryl hydrocarbon receptor (AHR) axis to suppress idiopathic pneumonia syndrome (IPS). Here we report that the dysregulated expression of AP-1 family genes in Ahr−/− lung epithelial cells exacerbated IPS in allogeneic bone marrow transplantation settings. AHR repressed transcription of Jund by preventing STAT1 from binding to its promoter. As a consequence, decreased interleukin-6 impaired the differentiation of CD4+ T cells toward Th17 cells. IFN-γ– and IDO1-independent induction of Ahr expression indicated that the AHR agonist might be a better therapeutic target for IPS than the IDO1 activator. We developed a novel synthetic AHR agonist (referred to here as PB502) that potently inhibits Jund expression. PB502 was highly effective at inducing AHR activation and ameliorating IPS. Notably, PB502 was by far superior to the endogenous AHR ligand, L-kynurenine, in promoting the differentiation of both mouse and human FoxP3+ regulatory CD4+ T cells. Our results suggest that the IDO1-AHR axis in lung epithelial cells is associated with IPS repression. A specific AHR agonist may exhibit therapeutic activity against inflammatory and autoimmune diseases by promoting regulatory T-cell differentiation.

Sodium/glucose cotransporter 2 (SGLT2) inhibitors improve cardiac function by reducing JunD expression in human diabetic hearts

BackgroundThe pathogenesis of experimental diabetic cardiomyopathy may involve the activator protein 1 (AP-1) member, JunD. Using non-diabetic heart transplant (HTX) in recipients with diabetes, we examined the effects of the diabetic milieu (hyperglycemia and insulin resistance) on cardiac JunD expression over 12 months. Because sodium/glucose cotransporter-2 inhibitors (SGLT2i) significantly reverse high glucose-induced AP-1 binding in the proximal tubular cell, we investigated JunD expression in a subgroup of type 2 diabetic recipients receiving SGLT2i treatment. MethodsWe evaluated 77 first HTX recipients (40 and 37 patients with and without diabetes, respectively). Among the recipients with diabetes, 17 (45.9%) were receiving SGLT2i treatment. HTX recipients underwent standard clinical evaluation (metabolic status, echocardiography, coronary computed tomography angiography, and endomyocardial biopsy). In the biopsy samples, we evaluated JunD, insulin receptor substrates 1 and 2 (IRS1 and IRS2), peroxisome proliferator-activated receptor-γ (PPAR-γ), and ceramide levels using real-time polymerase chain reaction and immunofluorescence. The biopsy evaluations in this study were performed at 1–4 weeks (basal), 5–12 weeks (intermediate), and up to 48 weeks (final, end of 12-month follow-up) after HTX. ResultsThere was a significant early and progressive increase in the cardiac expression of JunD/PPAR-γ and ceramide levels, along with a significant decrease in IRS1 and IRS2 in recipients with diabetes but not in those without diabetes. These molecular changes were blunted in patients with diabetes receiving SGLT2i treatment. ConclusionEarly pathogenesis in human diabetic cardiomyopathy is associated with JunD/PPAR-γ overexpression and lipid accumulation following HTX in recipients with diabetes. Remarkably, this phenomenon was reduced by concomitant therapy with SGLT2i, which acted directly on diabetic hearts.

The antipsychotic drug pimozide is effective against human T-cell leukemia virus type 1-infected T cells

Patients with adult T-cell leukemia (ATL), caused by the human T-cell leukemia virus type 1 (HTLV-1), exhibit poor prognosis owing to drug resistance. Pimozide is a dopamine D2 receptor antagonist and antipsychotic shown to exhibit anticancer activity. Herein, we investigated whether pimozide exerts anti-ATL effects and explored the mechanisms underlying these effects. Pimozide inhibited cell growth and survival in HTLV-1-infected T cells but not in the uninfected T cells. The dopamine D2 receptor subfamily mRNA expression levels in HTLV-1-infected T cells were high. Pimozide induced G1 cell cycle arrest concomitant with the upregulation of p21/p27/p53, and suppression of cyclin D2/E, cyclin-dependent kinase 2/4/6 and c-Myc expression, and pRb phosphorylation. Pimozide also induced apoptosis by activating caspases, upregulating pro-apoptotic proteins and downregulating anti-apoptotic proteins. Additionally, it promoted reactive oxygen species (ROS) generation and increased the expression of the endoplasmic reticulum stress marker activating transcription factor 4 and the DNA damage-inducible protein GADD45α and the phosphorylation of the DNA damage marker H2AX. Furthermore, pimozide-induced cytotoxicity was partially inhibited by a ROS scavenger, and pan-caspase and necroptosis inhibitors, indicating the involvement of caspase-dependent and -independent lethal pathways. The activities of the nuclear factor-κB, Akt, STAT3/5 and AP-1 signaling pathways were inhibited via the dephosphorylation of IκBα, IκB kinase α/β, Akt and STAT3/5, in addition to reduced JunB and JunD expression in HTLV-1-infected T cells. Pimozide also exhibited potent anti-ATL activity in the xenograft mouse model. These findings demonstrated the efficacy of pimozide as a potential therapeutic agent for ATL.

MiR-673/menin/JunD axis modulates hyperglycemia-induced oxidative stress and inflammation in the diabetic heart

Abstract Background Hyperglycemia-induced reactive oxygen species generation in diabetic heart contributes to myocardial dysfunction. JunD, a member of the activated protein 1 (AP-1) family of transcription factors, is emerging as a major gatekeeper against oxidative stress. Previous studies have shown that downregulation of AP-1 transcription factor JunD is involved in vascular aging and heart failure. However, the role of JunD in diabetes-induced myocardial dysfunction is unknown. Purpose The present study was designed to investigate whether hyperglycemia-driven epigenetic regulation of JunD contributes to oxidative stress, inflammation and myocardial dysfunction in the diabetic heart. Methods Diabetes (DB) was induced in C57BL/6 wild-type (WT) mice by streptozotocin. After four weeks of DB, left ventricular (LV) function was assessed by standard and 2D speckle-tracking echocardiography in both groups (n=10). Then, the animals were euthanized and LV specimens were collected to determine JunD mRNA and protein expression as well as superoxide anion production by ESR spectroscopy. Chromatin modifications of JunD gene promoter were assessed by chromatin immunoprecipitation. Isolated DNA was analyzed for promoter methylation following Methylminer kit. Cardiac biopsies were collected from age-matched patients with and without diabetes. Results DB mice showed LV dysfunction with reduced ejection fraction and fractional shortening. JunD mRNA and protein expression were reduced in the myocardium of DB as compared to control mice. JunD downregulation was associated with oxidative stress, increased NF-kB binding activity and expression of inflammatory mediators. Accordingly, expression of free radical scavenger superoxide dismutase 1 and aldehyde dehydrogenase 2 was reduced, whereas nicotinamide adenine dinucleotide phosphate oxidase subunits NOX2 and NOX4 were upregulated in DB. A reduction of JunD mRNA and protein expression was confirmed in LV specimens obtained from patients with diabetes. The downregulation of JunD was epigenetically regulated by promoter hypermethylation and histone modifications. Post-translational repression by tumor suppressor menin also contributed to JunD downregulation. Indeed, menin was significantly upregulated in DB hearts and co-immunoprecipitation experiments confirmed the binding of menin to JunD. Furthermore, rat ventricular myocytes exposed to high glucose (HG) showed increased menin expression. We found that miR-673 targeting menin was downregulated in hearts of DB mice. Reprogramming miR-673 in HG-treated myocytes was able to restore both menin and JunD expression to control levels. Conclusions Our findings show that downregulation of AP-1 transcription factor JunD contributes to diabetes-induced myocardial dysfunction and miR-673/menin/JunD represents a novel molecular axis involved in hyperglycemia-induced ROS-driven cardiac damage. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): European Society of Cardiology (ESC) Research Grant 2017

Characterization of grass carp FosB, Fosl2, JunD transcription factors in response to GCRV infection

Activating protein-1 (AP-1) composed of Jun and Fos family proteins, which are frequently activated by pathogens to induce cellular inflammation and immune responses. In this study, by using RNA sequencing (RNA-seq) technology, we found that FosB, Fosl2, and JunD expression was co-induced 12 and 20 h in grass carp (Ctenopharyngodon idella) kidney cell line (CIK cells) after grass carp reovirus (GCRV) infection. Full-length cDNA of the three genes were cloned and phylogenetic analysis indicated that the corresponding proteins shared high homology with those in higher vertebrates. The tissue distribution analysis of the three genes showed they were ubiquitously expressed in various tissues of healthy grass carp. In infection assay, FosB, Fosl2, and JunD were up-regulated and co-expressed 8 h after GCRV infection in CIK cells. Protein interaction and co-expression network analysis revealed that FosB, Fosl2, and JunD showed similar functions and were co-expressed in mammals and fish. Taken together, these results indicated that the co-expression of the AP-1 family proteins in grass carp uniformly responds to GCRV infection. These findings provided insights into the further study of AP-1 transcription factors function during grass carp reovirus infection.

Transcriptomic responses to hypoxia in endometrial and decidual stromal cells.

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types, hypoxia-upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC, hypoxia restored an ESF-like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types, hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia-upregulated genes in ESF and DSC had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia-upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry, we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall, the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.

JUND-dependent up-regulation of HMOX1 is associated with cisplatin resistance in muscle-invasive bladder cancer.

The standard-of-care for metastatic muscle-invasive bladder cancer (MIBC) is platinum-based chemotherapy regimens. Acquired resistance that occurs frequently through unidentified mechanisms, however, remains the major obstacle for implementing therapeutic effectiveness. Here, using data mining and analysis on clinical samples, we show that expression of JUND, a core component of activator protein-1 family, was significantly induced in cisplatin (CDDP)-resistant MIBC. Accumulation of nuclear JUND was associated with low post-chemotherapy survival in MIBC patients. In both genetically engineered cell models and murine xenograft models, we provided evidence that bladder cancer (BC) cells with excessive JUND expression were less responsive to CDDP treatment. This CDDP resistance was further demonstrated to be mediated, at least in part, by transactivation of HMOX1 [the gene encoding heme oxygenase-1 (HO-1)], one of the most important antioxidant signalling pathways of cell adaptation to stress. One mutation within the HMOX1 promoter successfully abolished oxidative stress-enhanced and JUND-driven HMOX1 promoter activation, suggesting that this unique site synergized for maximal HO-1 induction in CDDP-challenged BC cells. Overall, our data highlight an indispensible role of JUND, both as a target as a modifier of the oxidative stress signalling, in conferring an adaptive response during the pathogenesis of CDDP resistance in MIBC.

LncRNA SNHG7 Functions as an Oncogene in Cervical Cancer by Sponging miR-485-5p to Modulate JUND Expression.

BackgroundLong non-coding RNA (LncRNA) SNHG7 is involved in the development of multiple cancers. However, its role in cervical cancer (CC) has not been elucidated. This study aimed to explore the function of SNHG7 in CC progression and the underlying mechanisms.Materials and MethodsThe expression levels of SNHG7 and miR-485-5p in CC tissues and cell lines were measured by qPCR. Functional experiments including CCK-8 assay, wound healing assay, transwell assay, flow cytometry, Western blot, luciferases reporter assay and immunoprecipitation (RIP) were performed to explore the SNHG7/miR-485-5p/JUND pathway. Additionally, in vivo study was carried out by establishing tumor xenograft models.ResultsWe found that SNHG7 was markedly enhanced in CC tissues and cell lines, and associated with poor clinical characteristics. In vitro, knockdown of SNHG7 inhibited CC cell proliferation, migration and invasion, as well as aggravated cell apoptosis. As to mechanism investigation, rescue experiments revealed that miR-485-5p inhibitor could partially reverse the effects on CC cells induced by SNHG7 knockdown. SNHG7 upregulated JUND expression via miR-485-5p. Moreover, tumor xenograft models were established to confirm the findings in vivo.ConclusionSNHG7 promoted CC progression through miR-485-5p/JUND axis. The SNHG7/miR-485-5p/JUND pathway might provide a novel therapeutic target for CC treatment.

Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance

BET bromodomain inhibitors (BETi), such as JQ1, have been demonstrated to effectively kill multiple types of cancer cells. However, the underlying mechanisms for BETi resistance remain largely unknown. Our evidences show that JQ1 treatment evicts BRD4 from the FOXD3-localized MIR548D1 gene promoter, leading to repression of miR-548d-3p. The loss of miRNA restores JunD expression and subsequent JunD-dependent transcription of RPS6KA2 gene. ERK1/2/5 kinases phosphorylate RSK3 (RPS6KA2), resulting in the enrichment of activated RSK3 and blockade of JQ1 killing effect. Dual inhibition of MEKs/ERKs or single EGFR inhibition are able to mimic the effect of JunD/RSK3-knockdown to reverse BETi resistance. Collectively, our study indicates that loss of BRD4/FOXD3/miR-548d-3p axis enhances JunD/RSK3 signalling and determines BET inhibition resistance, which can be reversed by targeting EGFR-MEK1/2/5-ERK1/2/5 signalling.

Escalated Oxycodone Self-Administration and Punishment: Differential Expression of Opioid Receptors and Immediate Early Genes in the Rat Dorsal Striatum and Prefrontal Cortex.

Opioid use disorder (OUD) is characterized by compulsive drug taking despite adverse life consequences. Here, we sought to identify neurobiological consequences associated with the behavioral effects of contingent footshocks administered after escalation of oxycodone self-administration. To reach these goals, Sprague-Dawley rats were trained to self-administer oxycodone for 4 weeks and were then exposed to contingent electric footshocks. This paradigm helped to dichotomize rats into two distinct behavioral phenotypes: (1) those that reduce lever pressing (shock-sensitive) and (2) others that continue lever pressing (shock-resistant) for oxycodone during contingent punishment. The rats were euthanized at 2-h after the last oxycodone plus footshock session. The dorsal striata and prefrontal cortices were dissected for use in western blot and RT-qPCR analyses. All oxycodone self-administration rats showed significant decreased expression of Mu and Kappa opioid receptor proteins only in the dorsal striatum. However, expression of Delta opioid receptor protein was decreased in both brain regions. RT-qPCR analyses documented significant decreases in the expression of c-fos, fosB, fra2, junB, egr1, and egr2 mRNAs in the dorsal striatum (but not in PFC) of the shock-sensitive rats. In the PFC, junD expression was reduced in both phenotypes. However, egr3 mRNA expression was increased in the PFC of only shock-resistant rats. These results reveal that, similar to psychostimulants and alcohol, footshocks can dichotomize rats that escalated their intake of oxycodone into two distinct behavioral phenotypes. These animals also show significant differences in the mRNA expression of immediate early genes, mainly, in the dorsal striatum. The increases in PFC egr3 expression in the shock-resistant rats suggest that Egr3 might be involved in the persistence of oxycodone-associated memory under aversive conditions. This punishment-driven model may help to identify neurobiological substrates of persistent oxycodone taking and abstinence in the presence of adverse consequences.

In vivo analysis of the presence of heme oxygenase-1, transcription factor Jun-D and CD90+/CD73+/CD105+/CD45- cells in the pulp of bleached teeth.

To investigate hydrogen peroxide (H2 O2 )-induced responsiveness in pulp cells using heme oxygenase-1 (HO-1) immunolabelling, Jun-D immunolabelling to study the effects of H2 O2 on odontoblastic differentiation and CD90+/CD73+/CD105+/CD45- cell counting for in vivo identification of mesenchymal stem cells in the pulp. The maxillary molars of 50 rats were treated with a bleaching gel (35% H2 O2 , 1×30min) or placebo gel (control groups). At 2, 3, 7, 15 and 30days after the treatment (n=10), inflammation in pulp tissue was analysed by haematoxylin-eosin staining, HO-1- and Jun-D-immunolabelled cells were counted in each third of the pulp chamber, and the number of CD90+/CD73+/CD105+/CD45- cells was quantified by immunofluorescence. The results were assessed using the Paired t-test or Wilcoxon signed-rank test (P<0.05). Significant H2 O2 -induced inflammation was noted at 2 and 3days (P<0.05), with tertiary dentine formation occurring from 7days. The bleached specimens had greater HO-1 immunolabelling in the middle and cervical thirds of the coronal pulp at 2 and 3days, in all thirds at 7days, and in the occlusal third at 15days (P<0.05), and significant nuclear Jun-D immunolabelling in the cervical third at 2 and 3days and in the occlusal and middle thirds at 7days (P<0.05). Bleached and control groups had low numbers of CD90+/CD73+/CD105+/CD45- cells in the pulp at all periods (P>0.05). Pulp cells responded to oxidative stress by expressing HO-1 during the post-bleaching inflammation phase until the beginning of the repair phase. Jun-D expression occurred during the reduction of inflammation and the beginning of tertiary dentine production. The presence of oxidative stress did not influence the number of CD90+/CD73+/CD105+/CD45- cells identified in vivo in the dental pulp.

Abstract 877: Novel role of truncated HMGA2 in regulating reactive oxygen species in prostate cancer cells

Abstract High mobility group A (HMGA2), a non-histone protein, is up-regulated in several cancers. Due to chromosomal rearrangement of HMGA2 gene, full-length or wild-type HMGA2 can be truncated leading to loss of the C-terminus and the 3’UTR. Both wild-type and truncated isoforms have been detected in uterine leiomyoma patients. However, the functional role of truncated HMGA2 has not been investigated. We hypothesize that truncated HMGA2 plays a role in prostate cancer progression. We analysed expression of wild-type vs truncated HMGA2 in a panel of prostate cancer cell lines using real-time PCR and western blot analyses. We utilized LNCaP cells stably overexpressing wild-type HMGA2 or truncated HMGA2 in LNCaP cells to detect the basal reactive oxygen species (ROS) levels using DCFDA dye. We analysed Jun-D expression, a putative downstream effector of HMGA2, by western blot analysis, and utilized siRNA to knockdown Jun-D. We performed migration and cell viability assays following Jun-D knockdown. Additionally, we performed proteomic analysis following immunoprecipitation with HMGA2 antibody in nuclear extracts from LNCaP cells overexpressing wild-type HMGA2 WT or cytoplasmic extracts from cells overexpressing truncated HMGA2. Finally, we utilized tissue microarray to analyse expression of HMGA2 by immunohistochemisty. Our results showed that prostate cancer cell lines expressed varying amounts of wild-type and truncated HMGA2. LNCaP cells overexpressing truncated HMGA2 exhibited increased nuclear expression of Jun-D, as well as increased ROS compared to LNCaP cells overexpressing wild-type HMGA2 or empty vector Neo control. Knockdown Jun-D in LNCaP cells overexpressing truncated HMGA2 abrogated ROS induction and cell migration with no effect on cell proliferation. Additionally, truncated HMGA2 interacted with a number of proteins, including Ras GTPase-activating protein-binding protein 1(G3BP1), a cytoplasmic stress granule protein that responds to oxidative stress. Truncated HMGA2 is mainly localized to the cytoplasm, and interestingly we also observed cytoplasmic HMGA expression in prostatic tissue with chronic inflammation. Therefore, truncated HMGA2 may promote prostate cancer progression via Jun-D-mediated ROS. GRANT SUPPORT: NIH 1P20MD002285 and NIH/NCRR/RCMI G12RR003062-22 Citation Format: Ohuod Hawsawi, Veronica Henderson, Liza Burton, Jodi Dougan, Ana Cecillia Millena, Vanessa Adams, Elshaddai Z. White, Guang Di Wang, Qiang Zhang, Cimona Hinton, Shafiq Khan, Valerie Odero-Marah. Novel role of truncated HMGA2 in regulating reactive oxygen species in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 877.