C-jun mRNA Research Articles

P21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2

Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK.

Mode of action exploration for prostate epithelial cell injury caused by bisphenol A

Bisphenol A (BPA) is a typical food chemical contaminant with various detrimental effects, especially on reproductive system. Male prostate damage is also one of its major adverse health effects, of which mode of action (MOA) remains unclear. This study aims to explore the MOA for prostate toxicity of BPA using human normal prostate epithelial cell RWPE-1 for 28-day human-relevant-level exposure. A physiological based pharmacokinetic model was used to determine the concentration of BPA based on the actual oral exposure in China. The possible key events were identified by high-throughput transcriptome sequencing and validated by qPCR, Western blot and cell cycle assay, and the benchmark concentration analysis were conducted. The enriched KEGG pathways include the endocytosis, cell cycle, cellular senescence, MAPK and TNF signaling pathways. With increasing BPA concentrations, the increased mRNA and/or protein expressions of MAPKAPK2, c-JUN and c-fos in the MAPK signaling pathway, the increased mRNA expressions of CCND1 and CDKN1A, the decreased mRNA expression of CDC25C, the increased proportion of G0/G1 phase and S phase, as well as the decreased proportion of G2/M phase, were observed. The lowest value of benchmark concentration lower confidence limit (BMCL) was retrieved from G2/M phase ratio, with 110.580 and 175.862 nM for BMCL5 and BMCL10, respectively, much higher than the male gonad maximum concentration of 0.019 nM of BPA at the current exposure level of adult Chinese males. In conclusion, the MOA of BPA induced male prostatic toxicity at human-relevant levels may include: key event (KE)1-MAPK signaling pathway activation, KE2-disorder of cell cycle regulatory gene expression (increased expression of CCND1 and CDKN1A, decreased expression of CDC25C), and KE3-disturbance of cell cycle (increased proportion of G0/G1 and S phases, decreased proportion of G2/M phases). However, more studies are needed to validate and complete the MOA.

Topical metformin accelerates wound healing by promoting collagen synthesis and inhibiting apoptosis in a diabetic wound model.

The wound healing process, which is a pathophysiological process that includes various phases, is interrupted in diabetes due to hyperglycemia, and since deterioration occurs in these phases, a normal healing process is not observed. The aim of the current study is to investigate the proliferative and antiapoptotic effects of metformin on wound healing after topical application on diabetic and non-diabetic wounds. For this purpose, we applied metformin topically on the full-thickness excisional wound model we created in diabetic and nondiabetic groups. We investigated the effects of metformin on the apoptotic index by the Terminal deoxynucleotidyl transferase mediated dUTP Nick-End Labeling method and on collagen-I, collagen-III, p53, and c-jun expression levels by quantitative reverse transcription polymerase chain reaction technique in wound biopsy tissues. Our results showed that c-jun and p53 mRNA levels and apoptotic index increased with the effect of diabetes, while collagen synthesis was disrupted. As a result of the study, we showed that metformin increases cellular proliferation and has anti-apoptotic effects by increasing collagen-I/III expression and decreasing p53/c-jun level, especially in diabetic wounds and also in normal wounds. In conclusion, the topical effect of metformin on diabetic wounds reversed the adverse effects caused by diabetes, increasing the wound healing rate and improving the wound repair process.

The sodium borate relieves the hypertrophic damage induced during pregnancy, it improves contractibility, reduces oxidative stress and stimulates cell proliferation

IntroductionFetal and postnatal hypertrophy develop in response to such different exposures or illnesses the mother suffers during gestation as anti-infectious and physical agents, obesity, hypertension, diabetes, and even advanced maternal age. This gives rise to high comorbidities in the newborn; therefore, looking for alternatives that contribute to cardiac homeostasis is quite necessary to inhibit the overgrowth of myocytes. Boron-derivative compounds could play a key role in exerting a repairing effect on chronic cardiac damage induced during gestation. MethodologyThe cardiotoxic effect of 6.4, 12 and 100 mg/kg of sodium tetraborate administered by oral delivery route to healthy pregnant mice was assessed. After that, the use of the chemical compound was tested in the treatment of pregnant mice previously subjected to isoproterenol (fetal hypertrophy model) on the fifth day post coitus. Prior to the sacrifice of the pups of mice an electrocardiography (ECG) was done. Morphological and histological changes of heart were assessed in newborn pups. As a damage marker, the concentration of p38 nitrogen-activated protein kinases were evaluated by using Western Blot and the levels of malondialdehyde (MDA) as well as glutathione antioxidants (GSH) and glutathione peroxidase (GPx) were tested by spectrometry. Moreover, the mRNA expression for early response genes (c-jun, c-fos y c-myc), late response (GATA-4, Mef2c, NFAT) and heart damage (ANP and BNP) was measured by qPCR real time. ResultsThe supply of 6,4 and 12 mg/kg-sodium tetraborate favored ventricular remodeling with histological alterations. By comparison, 100 mg/kg of sodium tetraborate administered during the fetal stage did not alter neither the cardiac morphology of six-week old pups nor the p38/P-p38MAPK ratio remained the same and no oxidative stress was observed.When pregnant females treated with isoproterenol were treated with 100 mg/kg sodium tetraborate during the fetal stage, an improvement in contractility was detected in the pups with an actual reduction in myocardial fibrosis and oxidative stress, but cardiac mass increased. In addition, the expression levels of c-jun, c-myc, GATA-4, MEF2c and ANP mRNA declined in comparison with CTR. However, the hypertrophic damage mechanism was sustained by c-fos, NFAT and BNP expressions. ConclusionsThe set of results achieved suggests that high concentrations of sodium tetraborate have no cardiotoxic effects. Furthermore, sodium tetraborate mitigates hypertrophy induced during pregnancy, thereby improving contractibility, reducing oxidative stress and stimulating cell proliferation. Therefore, sodium tetraborate could be an excellent prophylactic treatment administered by delivery oral route during pregnancy when there is a risk of developing fetal left ventricular hypertrophy (LVH).

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients.A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Siponimod ameliorates experimental autoimmune neuritis

BackgroundGuillain–Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are human autoimmune peripheral neuropathy. Besides humoral immunity, cellular immunity is also believed to contribute to these pathologies, especially CIDP. Sphingosine-1-phosphate receptor 1 (S1PR1) regulates the maturation, migration, and trafficking of lymphocytes. As of date, the therapeutic effect of sphingosine-1-phosphate receptor (S1PR) agonists on patients with GBS or CIDP remains unclear.MethodsTo evaluate the effect of siponimod, an agonist of S1PR1 and S1PR5, on experimental autoimmune neuritis (EAN), an animal model of autoimmune peripheral neuropathy, was used. Lewis rats were immunized with 125 μg of synthetic peptide from bovine P2 protein. Rats in the siponimod group were orally administered 1.0 mg/kg siponimod and those in the EAN group were administrated the vehicle on days 5–27 post-immunization (p.i.) daily. The symptom severity was recorded daily. The changes in the expression of cytokines and transcription factors in the lymph nodes and cauda equina (CE) which correlate with the pathogenesis of EAN and recovery of injured nerve were measured using reverse transcription quantitative PCR. Histological study of CE was also performed.ResultsFlaccid paralysis developed on day 11 p.i. in both groups. Siponimod relieved the symptom severity and decreased the expression of interferon-gamma and IL-10 mRNAs in lymph nodes and CE compared with that in the EAN group. The expression of Jun proto-oncogene (c-Jun) mRNA increased from the peak to the recovery phase and that of Sonic hedgehog signaling molecule (Shh) and Glial cell line-derived neurotrophic factor (Gdnf) increased prior to increase in c-Jun with no difference observed between the two groups. Histologically, siponimod also reduced demyelinating lesions and inflammatory cell invasion in CE.ConclusionsSiponimod has a potential to ameliorate EAN. Shh and Gdnf, as well as C-Jun played a significant role during the recovery of injured nerves.

The pivotal regulatory factor circBRWD1 inhibits arsenic exposure-induced lung cancer occurrence by binding mRNA and regulating its stability

Multiple studies have indicated that circular RNAs (circRNAs) play a regulatory role in different stages of tumors by interacting with various molecules. With continuous in-depth research on the biological functions of circRNAs, increasing evidence has shown that circRNAs play important roles in carcinogenesis caused by environmental pollutants. However, the function and mechanism of circRNAs in arsenic exposure-induced lung cancer occurrence have not been reported. In this study, RNA sequencing and qPCR assays revealed that the expression of circBRWD1 was decreased in BEAS-2B-As cells and multiple lung cancer cell lines. Silencing circBRWD1 promoted cell viability and proliferation, inhibited cell apoptosis, and accelerated the G0/G1 phase transition in BEAS-2B-As cells; however, these functions were abrogated by circBRWD1 overexpression. Mechanistically, under arsenic exposure, expression of decreased circBRWD1 led to enhanced stability of the mRNA to which it directly binds (c-JUN, c-MYC, and CDK6 mRNA), increasing its expression. This mechanism promotes the malignant transformation of lung cells and ultimately leads to lung cancer. Our findings thus reveal the molecular mechanism of arsenic carcinogenesis.

Trehalose decreases mRNA and protein expressions of c-Jun and JunB in human cervical cancer HeLa cells.

Increasing evidence suggests that trehalose, a non-reducing disaccharide, ameliorates disease phenotypes by activating autophagy in animal models of various human diseases, including neurodegenerative diseases. Multiple in vitro studies suggest that activation of transcription factor EB, a master regulator of lysosomal biogenesis and autophagy genes, is a major contributor to trehalose-induced autophagy at later stages of exposure. However, underlying causes of trehalose-induced autophagy possibly occur at the early stage of the exposure period. In this study, we investigated the effects of short-term exposure of HeLa cells to trehalose on several signal transduction pathways to elucidate the initial events involved in its beneficial effects. Phospho-protein array analysis revealed that trehalose decreases levels of phosphorylated c-Jun, a component of the transcription factor activator protein-1, after 6h. Trehalose also rapidly reduced mRNA expression levels of c-Jun and JunB, a member of the Jun family, within 1h, resulting in a subsequent decrease in their protein levels. Future studies, exploring the interplay between decreased c-Jun and JunB protein levels and beneficial effects of trehalose, may provide novel insights into the mechanisms of trehalose action.

Intracellular miRNA-Triggered Surface-Enhanced Raman Scattering Imaging and Dual Gene-Silencing Therapy of Cancer Cell.

Development of theranostic nanosystems integrating cascaded surface-enhanced Raman scattering (SERS) imaging and gene silencing therapy for accurate cancer diagnosis and treatment is still a big challenge and rarely reported. Herein, a novel Au nanoparticles (AuNPs)-based theranostic nanosystem containing AuNP-Ys and AuNP-Ds for highly sensitive and specific cancer diagnosis and treatment was proposed for cascaded SERS imaging of intracellular cancer-related miR-106a and miR-106a-triggered DNAzyme-based dual gene-silencing therapy of cancer cells. The AuNP-Ys were prepared by modifying the AuNPs with specially designed Y-motifs, and the AuNP-Ds were obtained by colabeling Raman molecules and dsDNA linkers on AuNPs. When identifying the intracellular cancer-related miRNAs, the Y-motifs and dsDNA linkers undergoes miRNA-triggered ATP-driven conformational transitions and releases the miRNA for recycling, which results in the formation of AuNP network nanostructures to generate significantly enhanced SERS signals for sensitive identification of the cancer cells as well as the amplification and specific activation of DNAzymes to catalyze the Mg2+-assisted cleavage of the Survivin and c-Jun mRNAs for effective dual gene-silencing therapy of cancer cells. The AuNP-based theranostic nanosystem achieves the synergism of target-triggered SERS imaging and DNAzyme-based dual gene-silencing therapy with enhanced specificity, sensitivity, and curative effect, which can be a powerful tool for accurate diagnosis and efficient treatment of cancers.

Effect of Doxycycline on Intrinsic Apoptosis of Myeloma Cell Line H929 and Its Mechanism

To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity. Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively. DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05). DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.

Baicalin enhances the thermotolerance of mouse blastocysts by activating the ERK1/2 signaling pathway and preventing mitochondrial dysfunction

Heat stress causes oxidative damage and induces excessive cell apoptosis and thus affects the development and/or even causes the death of preimplantation embryos. The effects of baicalin on the developmental competence of heat-stressed mouse embryos were investigated in this experiment. Two-cell embryos were cultured in the presence of baicalin and subjected to heat stress (42 °C for 1 h) at their blastocyst stage followed by continuous culture at 37 °C until examination. The results showed that heat stress (H group) increased reactive oxygen species (ROS) production, apoptosis and even embryo death, along with reductions in both mitochondrial activity and membrane potential (ΔΨm). Both heat stress (H group) and inhibition of the ERK1/2 signaling pathway (U group) led to significantly reduced expression levels of the genes c-fos, AP-1 and ERK2, and the phosphorylation of ERK1/2 and c-Fos, along with significantly increased c-Jun mRNA expression and phosphorylation levels. These negative effects of heat stress on the ERK1/2 signaling pathway were neutralized by baicalin treatment. To explore the signal transduction mechanism of baicalin in improving embryonic tolerance to heat stress, mitochondrial quality and apoptosis rate in the mouse blastocysts were also examined. Baicalin was found to up-regulate the expression of mtDNA and TFAM mRNA, increased mitochondria activity and ΔΨm, and improved the cellular mitochondria quality of mouse blastocysts undergoing heat stress. Moreover, baicalin decreased Bax transcript abundance in blastocyst, along with an increase in the blastocyst hatching rate, which were negatively affected by heat stress. Our findings suggest that baicalin improves the developmental capacity and quality of heat-stressed mouse embryos via a mechanism whereby mitochondrial quality is improved by activating the ERK1/2 signaling pathway and inducing anti-cellular apoptosis.

Stem cells from human exfoliated deciduous teeth relieve pain via downregulation of c-Jun in a rat model of trigeminal neuralgia.

Stem cells from human exfoliated deciduous teeth (SHED) have excellent immunomodulatory and neuroprotective abilities. It is possible that systemic SHED transplantation could ameliorate trigeminal neuralgia. The phosphorylation of c-Jun contributes to the development of hyperalgesia and allodynia. The present study aimed to evaluate whether systemic SHED transplantation could lead to analgesic effects by regulating peripheral c-Jun in the trigeminal ganglia (TG) in a rat model of trigeminal neuralgia. Chronic constriction injury of the infraorbital nerve (CCI-ION) was performed to establish a rat pain model. SHED were obtained from discarded exfoliated deciduous teeth from children and transplanted by a single infusion through the tail vein. SHED were labelled with the PKH26 red fluorescent cell linker mini kit for tract distribution. The mechanical threshold was determined using von Frey filaments. The mRNA levels of c-Jun in the ipsilateral TG were quantified. The phosphorylation of c-Jun in the ipsilateral TG was assessed by immunohistochemistry and Western blotting. PKH26-labelled SHED were distributed to both sides of TG, lung, liver and spleen. Systemic SHED transplantation significantly elevated the mechanical thresholds in CCI-ION rats and blocked the upregulation of c-Jun mRNA levels in the TG caused by nerve ligation. The activation of c-Jun in the TG was blocked by SHED transplantation. These findings demonstrate that systemic SHED administration reverts trigeminal neuralgia via downregulation of c-Jun in the TG.

The Dual Effects of Silibinin on Human Pancreatic Cells

Objective: Silibinin is a flavonoid with antihepatotoxic properties, and exhibits pleiotropic anticancer effects. However, the molecular mechanisms responsible for its anticancer actions in pancreatic cancer cells, and the effects on such cells and normal pancreatic cells, remain unclear. The objective of this study was to determine the effect of silibinin on human pancreatic cancer cells and normal ductal cells. Methods: Human pancreatic cancer cells (MIA PaCa-2 and PANC-1) and normal ductal cells (hTERT-HPNE) were cultured with 0-400 μM silibinin for 48 h. Thereafter, the proliferation, invasion, apoptosis, and signaling pathways of the pancreatic cells were evaluated. Results: Silibinin significantly inhibited the proliferation, invasion, and spheroid formation of human pancreatic cancer cells in vitro in a dosedependent manner (p&lt;0.05). It also induced apoptosis in a dose-dependent manner. Western blot analysis showed that silibinin downregulated extracellular signaling-regulated kinase (ERK) and serine/threonine protein kinase (AKT) in human pancreatic cancer cells. It also upregulated microtubule associated protein 1 light chain 3 β (LC3B) and cleaved caspase-3 via c-Jun N-terminal kinases (JNK) signaling. On the other hand, silibinin increased the mRNA and protein levels of c-Jun, Twist-related protein 1, and Snail. It also decreased exogenous p53 levels, but increased endogenous c-Jun protein levels in human pancreatic cancer cells. However, silibinin did not affect cell viability and endogenous c-Jun levels in pancreatic normal ductal cells. It increased exogenous p53 levels, but decreased stemness-related gene expression in pancreatic normal ductal cells. Silibinin increased Ki-67 levels in pancreatic cancer cells, but decreased them in pancreatic normal ductal cells. Conclusion: Silibinin not only exerted anticancer effects by inhibiting AKTERK and JNK signaling, but also upregulated cancer stemness-related genes in human pancreatic cancer cells. These results suggest that silibinin should be used as a therapeutic agent for human pancreatic cancer with caution.

Methionine- and Choline-Deficient Diet–Induced Nonalcoholic Steatohepatitis Is Associated with Increased Intestinal Inflammation

Inflammation drives the pathogenesis of nonalcoholic steatohepatitis (NASH). The current study examined changes in intestinal inflammation during NASH. In male C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in severe hepatic steatosis and inflammation relative to feeding a chow diet (CD). MCD-fed mice exhibited characteristics of mucosal and submucosal inflammatory responses compared with CD-fed mice. Moreover, intestinal phosphorylation states of c-Jun N-terminal protein kinase p46 and mRNA levels of IL-1B, IL-6, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 were significantly higher and intestinal mRNA levels of IL-4 and IL-13 were significantly lower in MCD-fed mice compared with those in CD mice. Surprisingly, upon treatment with MCD-mimicking media, the proinflammatory responses in cultured intestinal epithelial CMT-93 cells did not differ significantly from those in CMT-93 cells treated with control media. In contrast, in RAW264.7 macrophages, MCD-mimicking media significantly increased the phosphorylation states of c-Jun N-terminal protein kinase p46 and mitogen-activated protein kinases p38 and mRNA levels of IL-1B, IL-6, IL-10, and tumor necrosis factor alpha under either basal or lipopolysaccharide-stimulated conditions. Collectively, these results suggest that increased intestinal inflammation is associated with NASH phenotype. Thus, elevated proinflammatory responses in macrophages likely contribute to, in large part, increased intestinal inflammation in NASH.

Speckle-type POZ protein up-regulates c-Jun protein expression and promotes proliferation and invasion of renal carcinoma cells

To investigate the effect of speckle-type POZ protein (SPOP) on proliferation, apoptosis, migration and invasion of renal cell carcinoma (RCC) and explore the potential mechanisms. Renal carcinoma cell lines (786-O, A704, and Caki-2) cultured in vitro were transfected with a SPOP-overexpressing plasmid, and the changes in proliferation of the cells were detected using colony formation and MTT assay; TUNEL assay was used to assess apoptosis of the cells. The changes in migration and invasion abilities of the cells were examined using wound healing assay and Transwell assay. The mRNA and protein levels of SPOP and c-Jun in the transfected cells were measured using real-time PCR and Western blotting. SPOP over-expression obviously promoted the proliferation, migration and invasion of 786-O, A704 and Caki-2 cells (P < 0.05). Compared with the control cells, 786-o and Caki-2 cells over-expressing SPOP exhibited significantly lowered apoptosis rates (P < 0.05). The results of real-time PCR demonstrated that the transfected cells did not show obvious changes in the mRNA level of c-Jun, but the protein expressions of SPOP and c-jun increased significantly as shown by Western blotting (P < 0.05). SPOP can promote proliferation, migration, and invasion and suppress apoptosis of renal carcinoma cells possibly by promoting the expression of c-Jun.

Expression of corticotropin releasing hormone in olive flounder (Paralichthys olivaceus) and its transcriptional regulation by c-Fos and the methylation of promoter

Corticotropin-releasing hormone (CRH) has been implicated in multiple physiological processes, such as circadian rhythms, food intake and anxiety-like behavior. In telelost fishes, CRH is predominantly expressed in the caudal neurosecretory system (CNSS), a much low level in hypothalamus and gonads, while undetectable levels in other tissues. However, the mechanisms governing this tissue-specific expression remain unknown. In this study, firstly, we investigated the expression pattern of CRH mRNA in different tissues of olive flounder (Paralichthys olivaceus). Secondly, we found that flounder exhibited low locomotor activity in the daytime, concomitant with the highest CRH mRNA expression in CNSS at noon. Thirdly, we examined whether epigenetic mechanisms through DNA methylation are involved in tissue-specific expression of CRH mRNA. Promoter methylation of the CRH gene was assessed through bisulphate sequencing methods. In the proximal promoter, almost no methylation was detected in the muscle, hypothamus and CNSS. However, different methylation was detected in the distal promoter of CRH. The methylation was 63% in CNSS, while that in hypothalamus and muscle was 85% and 96%, respectively. Lastly, bioinformatics analysis revealed that a conserved AP-1 binding site (5-TCACTGA-3) was located in the proximal promoter of CRH gene. In vivo experiment, lipopolysaccharide (LPS) intraperitoneally injection in the flounder up-regulated c-Fos and CRH mRNA in CNSS (P<0.01). In CNSS tissue culture experiment, forskolin treatment significantly induced the expression of c-Fos, c-Jun and CRH mRNA (P<0.01). Collectively, our data provide the evidence that distal promoter methylation and c-Fos signal pathway are involved in transcriptional regulation of CRH expression in flounder.

Apoptosis in Cancer Cells Is Induced by Alternative Splicing of hnRNPA2/B1 Through Splicing of Bcl-x, a Mechanism that Can Be Stimulated by an Extract of the South African Medicinal Plant, Cotyledon orbiculata.

Alternative splicing is deregulated in cancer and alternatively spliced products can be linked to cancer hallmarks. Targeting alternative splicing could offer novel effective cancer treatments. We investigated the effects of the crude extract of a South African medicinal plant, Cotyledon orbiculata, on cell survival of colon (HCT116) and esophageal (OE33 and KYSE70) cancer cell lines. Using RNASeq, we discovered that the extract interfered with mRNA regulatory pathways. The extract caused hnRNPA2B1 to splice from the hnRNPB1 to the hnRNPA2 isoform, resulting in a switch in the BCL2L1 gene from Bcl-xL to Bcl-xS causing activation of caspase-3-cleavage and apoptosis. Similar splicing effects were induced by the known anti-cancer splicing modulator pladienolide B. Knockdown of hnRNPB1 using siRNA resulted in decreased cell viability and increased caspase-3-cleavage, and over-expression of hnRNPB1 prevented the effect of C. orbiculata extract on apoptosis and cell survival. The effect of the hnRNPA2/B1 splicing switch by the C. orbiculata extract increased hnRNPA2B1 binding to Bcl-xl/s, BCL2, MDM2, cMYC, CD44, CDK6, and cJUN mRNA. These findings suggest that apoptosis in HCT116, OE33, and KYSE cancer cells is controlled by switched splicing of hnRNPA2B1 and BCL2L1, providing evidence that hnRNPB1 regulates apoptosis. Inhibiting this splicing could have therapeutic potential for colon and esophageal cancers. Targeting hnRNPA2B1 splicing in colon cancer regulates splicing of BCL2L1 to induce apoptosis. This approach could be a useful therapeutic strategy to induce apoptosis and restrain cancer cell proliferation and tumor progression. Here, we found that the extract of Cotyledon orbiculata, a South African medicinal plant, had an anti-proliferative effect in cancer cells, mediated by apoptosis induced by alternative splicing of hnRNPA2B1 and BCL2L1.

Neuromedin B receptor mediates neuromedin B-induced COX-2 and IL-6 expression in human primary myometrial cells

The precise mechanisms that lead to parturition remain unclear. In our initial complementary DNA (cDNA) microarray experiment, we found that the neuromedin B receptor (NMBR) was differentially expressed in the...

Concentrated growth factor inhibits UVA-induced photoaging in human dermal fibroblasts via the MAPK/AP-1 pathway.

Ultraviolet (UV) radiation-induced photoaging is one of the contributors to skin aging. UV light triggers oxidative stress, producing a large number of matrix metalloproteinases (MMPs) and degrading the extracellular matrix in skin cells, thereby causing a series of photoaging symptoms. Concentrated growth factor (CGF) is a leukocyte- and platelet-rich fibrin biomaterial that plays a protective role in the occurrence and development of skin photoaging. In the present study, we investigated the underlying mechanism of CGF in the UVA-induced photoaging of human dermal fibroblasts (HDFs). A primary culture of HDFs was isolated from normal human facial skin. The cells were treated with CGF following UVA radiation. Proliferation of cells was detected using MTT assay, followed by measurement of reactive oxygen species (ROS) using immunofluorescence assay and flow cytometry. The mRNA and protein expression levels of P38, c-Jun, and MMP-1 were detected using real-time polymerase chain reaction and Western blot, respectively. CGF was found to improve cell viability by inhibiting the production of ROS and reducing oxidative damage. In addition, there was lower expression of p38 and c-Jun at the mRNA and protein levels following CGF treatment, thus resulting in the inhibition of MMP-1 expression. Our results suggest that CGF could protect HDFs against UVA-induced photoaging by blocking the P38 mitogen-activated protein kinase/activated protein-1 (P38MAPK/AP-1) signaling pathway. These findings provide a new clinical strategy for the prevention of skin photoaging.

Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver.

Growth hormone (GH) activates multiple signal transduction pathways. To investigate these pathways, we identified novel genes whose transcription was induced by GH in the liver of hypophysectomized (HPX) rats using the suppression subtractive hybridization technique. We found that regulator of calcineurin 1 (Rcan1) mRNA was upregulated by GH administration. RCAN1 regulates the activity of calcineurin, a Ca/calmodulin-dependent phosphatase. Rcan1 encodes two major transcripts, Rcan1-1 and Rcan1-4, resulting from differential promoter use and first exon choice. We found that a single injection of GH increased the levels of Rcan1-4 mRNA and RCAN1-4 protein transiently, but did not increase Rcan1-1 mRNA in HPX rat liver. Then the molecular mechanism of GH to induce Rcan1-4 transcription was examined in rat hepatoma H4IIE cells. Experiments using inhibitors suggested that c-JUN N-terminal kinase was required for the induction of Rcan1-4 mRNA by GH. GH increased the levels of phosphorylated c-JUN protein and c-Jun mRNA in HPX rat liver. The luciferase and electrophoretic mobility shift assays showed that c-JUN upregulated Rcan1-4 mRNA by binding to the cAMP-responsive element in the upstream of Rcan1 exon 4. These results indicate that GH activates c-JUN to affect the activity of calcineurin by the induction of Rcan1-4 in rat liver.