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While existing research has illuminated the environmental dangers and neurotoxic effects of MC-LR exposure, the molecular underpinnings of brain damage from environmentally-relevant MC-LR exposure remain elusive. Employing a comprehensive approach involving RNA sequencing, histopathological examination, and biochemical analyses, we discovered genes differentially expressed and enriched in the ferroptosis pathway. This finding was associated with mitochondrial structural impairment and downregulation of Gpx4 and Slc7a11 in mice brains subjected to low-dose MC-LR over 180 days. Mirroring these findings, we noted reduced cell viability and GSH/GSSH ratio, along with an increased ROS level, in HT-22, BV-2, and bEnd.3 cells following MC-LR exposure. Intriguingly, MC-LR also amplified phospho-Erk levels in both in vivo and in vitro settings, and the effects were mitigated by treatment with PD98059, an Erk inhibitor. Taken together, our findings implicate the activation of the Erk/MAPK signaling pathway in MC-LR-induced ferroptosis, shedding valuable light on the neurotoxic mechanisms of MC-LR. These insights could guide future strategies to prevent MC-induced neurodegenerative diseases.
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Células Endoteliais , Ferroptose , Camundongos , Animais , Encéfalo , Transdução de SinaisRESUMO
Neurologic deterioration after massive cerebral infarct should be identified at an early stage for medical and surgical treatments. We investigated the effect of hydrogen sulfide on the excitotoxity of PC12 cells exposed to oxygen-glucose deprivation (OGD) and its effect on the apoptosis of brain tissues in rats with middle cerebral artery occlusion (MCAO). Rats with MCAO were treated with SAM, a cystathionine beta-synthase (CBS) activator, or AOAA, a CBS inhibitor. Hydrogen sulfide content in the brain tissues of infarcted patients or rats with MCAO was decreased, whereas glutamate (GLU) content was increased. In addition, SAM reduced reactive oxygen species content, lactate dehydrogenase release, and apoptosis levels in the brain tissues of rats with MCAO. The PC12 cells that were exposed to OGD were also treated with 20 mM GLU and later treated with SAM or AOAA. In PC12 cells, SAM reduced the apoptosis caused by GLU after OGD. The protective effects of hydrogen sulfide was elicited through the sulfur-sulfhydrylation modification of NMDAR and the induction of ERK/MAPK signaling. Our results showed that hydrogen sulfide exerts a protective effect on the PC12 cells and the rats with MCAO, which might represent a possible therapeutic agent against massive cerebral infarct.
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Isquemia Encefálica , Sulfeto de Hidrogênio , Traumatismo por Reperfusão , Animais , Apoptose , Isquemia Encefálica/tratamento farmacológico , Cistationina beta-Sintase/farmacologia , Cistationina beta-Sintase/uso terapêutico , Glucose/farmacologia , Ácido Glutâmico , Humanos , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Oxigênio , Células PC12 , Ratos , Traumatismo por Reperfusão/tratamento farmacológicoRESUMO
OBJECTIVE: Disc degeneration is the common life-threatening disease characterized by flank pain. The gene expression of insulin-like growth factor binding protein 3 (IGFBP3) is increased in patients with disc degeneration, however, its mechanism is still unknown. This study aimed to investigate the influence of IGFBP3 gene silencing mediated inhibition of extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling on proliferation, apoptosis, autophagy, and cell senescence in rats nucleus pulposus (NP) cells. METHODS: The expression of IGFBP3 in disc NP of patients was assessed by real-time PCR (RT-PCR) and western blot. RT-PCR, transwell assay, immunohistochemical staining, SA-ß-Gal staining, and western blot were performed to explore the molecular mechanism of IGFBP3 in NP cell migration and invasion. RESULTS: In this study, IGFBP3 was highly expressed in disc NP of patients. With RT-PCR, transwell assay, immunohistochemical staining, SA-ß-Gal staining, and western blot, downregulated IGFBP3 could inhibit NP cells' migration and invasion by targeting the ERK/MAPK signaling pathway. CONCLUSION: Our findings revealed that the inhibition of the ERK/MAPK pathway was mediated by IGFBP3 silencing that had effects on proliferation, apoptosis, autophagy, and cell senescence. Furthermore, our findings suggested the underlying mechanism of disc degeneration.
Assuntos
Apoptose , Autofagia , Senescência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Sistema de Sinalização das MAP Quinases , Núcleo Pulposo/citologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Degeneração do Disco Intervertebral/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Ratos Sprague-Dawley , terc-Butil Hidroperóxido/farmacologiaRESUMO
We have extensively studied the phenotypic heterogeneity of patient-derived melanoma cells. Here, whole-exome sequencing revealed novel variants of genes associated with the MAPK, NOTCH, Hippo, cell-cycle, senescence, and ubiquitin-dependent pathways, which could contribute to the observed phenotypic diversity between cell lines. Focusing on mutations in the MAPK pathway-associated genes, we found BRAF (BRAFV600E ) and RAS subtypes, including NRASQ61R and the rare HRASQ61R variant, and additional alterations potentially leading to different ERK1/2 activity. Both RASQ61R cell lines harbored a MEK1P124S variant and exerted a low level of phospho-MEK1/2. Activity of the MAPK pathway was further attenuated in NRASQ61R /MEKP124S cells by trametinib, and this effect was also shown in HRASQ61R /MEKP124S melanoma cells. The observed variability in doubling time might be a consequence of diverse MAPK and PI3K/AKT pathway activities, but not exclusively, as a senescence program was also executed to different extent in distinct melanoma cell lines. Low percentages of senescent cells might result from mutations in CDKN2A, E2F3, and EZH2, and a high c-MYC expression. Vemurafenib and trametinib induced senescence concomitantly with c-MYC downregulation and irrespectively of CDKN2A mutation, but the EZH2S412C variant might limit senescence induction. Damaging alterations in Hippo pathway-associated genes were accompanied with variability in the phosphorylation level of YAP1/TAZ and CTGF expression. Our study also suggests opposite activity of NOTCH2F1209V and NOTCH2N2002S variants. Additionally, we found a novel FBXW7V418M variant that retained its function in melanoma cells. The obtained molecular data might be further exploited in genotype-phenotype relationship studies and in identifying novel biomarkers and therapies for melanomas.
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Biomarcadores Tumorais/genética , Senescência Celular , Sequenciamento do Exoma/métodos , Melanoma/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Apoptose , Ciclo Celular , Proliferação de Células , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Fenótipo , Fosforilação , Células Tumorais CultivadasRESUMO
This study is supposed to investigate the effect of FGF-23 on parathyroid hormone (PTH) secretion through ERK/MAPK signaling pathway in secondary hyperparathyroidism (SHPT) rat model. Thirty rats were equally served as the normal and SHPT groups. After transfection, parathyroid cells was assigned into blank, NC, pcDNA3.1-FGF-23, siRNA-FGF-23, U0126, and siRNA-FGF-23 + U0126 groups. The serum levels of Calcium (Ca), Phosphorus (P), alkaline phosphatase (ALP), and PTH were detected. HE and immunohistochemical (IHC) staining were used for the histopathological changes and the FGF-23, EKR1/2, and pEKR1/2 expressions. qRT-PCR and Western blotting were performed to determine the mRNA and protein expression of FGF-23, PTH, MAPK, EKR1/2, and Klotho. The proliferation, apoptosis, and cell cycle were all measured for parathyroid cells by CCK-8 assay, TUNEL staining and Flow cytometry. Compared with the normal group, the SHPT group showed increased serum levels PTH, P, ALP, and FGF-23 and mRNA and protein expressions of FGF-23 and PTH, whereas declined Ca and p-ERK1/2 expression, mRNA and protein expression of Klotho, cell apoptosis rate was reduced. Furthermore, compared to the blank and NC groups, the pcDNA3.1-FGF-23 and U0126 groups had a decreased mRNA expression of Klotho, protein expression of EKR1/2 and Klotho, and cell apoptosis rate was down-regulated, whereas the RNA and protein expressions of FGF-23 and PTH were up-regulated, and cell proliferation was elevated. The opposite results were observed in the siRNA-FGF-23 group. Our study demonstrated that FGF-23 could inhibit signaling transduction of ERK/MAPK pathway and accelerate the secretion of PTH in rats with SHPT.
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Fatores de Crescimento de Fibroblastos/metabolismo , Hiperparatireoidismo Secundário/enzimologia , Hiperparatireoidismo Secundário/patologia , Sistema de Sinalização das MAP Quinases , Glândulas Paratireoides/enzimologia , Glândulas Paratireoides/patologia , Hormônio Paratireóideo/metabolismo , Fosfatase Alcalina/sangue , Animais , Apoptose , Cálcio/sangue , Ciclo Celular , Proliferação de Células , Creatinina/sangue , Modelos Animais de Doenças , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Fatores de Crescimento de Fibroblastos/genética , Glucuronidase/sangue , Glucuronidase/genética , Hiperparatireoidismo Secundário/sangue , Proteínas Klotho , Masculino , Hormônio Paratireóideo/sangue , Fósforo/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate the role of the striatal extracellular signal-regulated kinase (Erk1/2) and its phosphorylation (p-Erk1/2) in aerobic training to alleviate the development of the L-DOPA induced dyskinesia (LID) in PD mice. METHODS: Forty-eight male C57BL/6â¯N mice were randomly divided into the 6-OHDA surgery group (6-OHDA, n=42) and the sham surgery group (Sham, n=6). A two-point injection of 6-OHDA into the right striatum was used to establish a lateralized injury PD model. PD mice were randomly divided into a PD control group (PD, n=13) and a PD exercise group (PDE, n=16), this is followed by 4 weeks of L-DOPA treatment, and PDE mice received concurrent running table training (18â¯m/min, 40â¯min/day, 5 times/week). AIM scores were performed weekly, and mice were assessed for motor function after 4 weeks using the rotarod, open field, and gait tests. Immunohistochemistry was used to test nigrostriatal TH expression, Western blot was used to determine Erk1/2 and p-Erk1/2 protein expression, and immunofluorescence double-labeling technique was used to detect Erk1/2 and p-Erk1/2 co-expression with prodynorphin (PDYN). RESULTS: (1) All AIM scores of PD and PDE mice increased significantly (P < 0.05) with the prolongation of L-DOPA treatment. Compared with PD, all AIM scores were significantly lower in PDE mice (P < 0.05). (2) After 4 weeks, the motor function of PD mice was significantly reduced compared with Sham (P < 0.05 or P < 0.01); compared with PD, the motor function of PDE mice was significantly increased (P < 0.05). (3) Compared with Sham, the expression of Erk1/2 protein, the number of positive cells of Erk1/2 and p-Erk1/2 and the number of positive cells co-expressed with PDYN were significantly increased in PD mice (P < 0.05); compared with PD, Erk1/2 protein expression was significantly decreased in PDE mice (P < 0.05), and the number of Erk1/2 and p-Erk1/2 positive cells was significantly reduced (P < 0.05). CONCLUSION: 4 weeks of aerobic exercise can effectively alleviate the development of L-DOPA-induced dyskinesia and improve motor function in PD mice. The related mechanism may be related to the inhibition of striatal Erk/MAPK signaling pathway overactivation by aerobic exercise, but this change did not occur selectively in D1-MSNs.
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Discinesia Induzida por Medicamentos , Exercício Físico , Doença de Parkinson , Animais , Masculino , Camundongos , Antiparkinsonianos/farmacologia , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Discinesia Induzida por Medicamentos/metabolismo , Levodopa , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos C57BL , Oxidopamina/farmacologia , Doença de Parkinson/terapia , Doença de Parkinson/metabolismo , HumanosRESUMO
Cognitive impairment is a major complication of cerebral ischemia-reperfusion (CIR) injury and has an important impact on the quality of life of patients. However, the precise mechanisms underlying cognitive impairment after CIR injury remain elusive. In the current study, we investigated the role of interleukin 17 A (IL-17A) on CIR injury-induced cognitive impairment in wild-type and IL-17A knockout mice using RNA sequencing analysis, neurological assessments, Golgi-Cox staining, dendritic spine analysis, immunofluorescence assay, and western blot analysis. RNA sequencing identified 195 CIR-induced differentially expressed genes (83 upregulated and 112 downregulated), highlighting several enriched biological processes (negative regulation of phosphorylation, transcription regulator complex, and receptor ligand activity) and signaling pathways (mitogen-activated protein kinase [MAPK], tumor necrosis factor, and IL-17 signaling pathways). We also injected adeno-associated virus into the bilateral hippocampal CA1 regions of CIR mice to upregulate or downregulate cyclic AMP response element-binding protein. IL-17A knockout activated the extracellular signal-regulated kinase (ERK)/MAPK signaling pathway and further improved synaptic plasticity, structure, and function in CIR mice. Together, our findings suggest that IL-17A deficiency alleviates CIR injury by activating the ERK/MAPK signaling pathway and enhancing hippocampal synaptic plasticity.
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Isquemia Encefálica , Traumatismo por Reperfusão , Humanos , Animais , Camundongos , Região CA1 Hipocampal/metabolismo , Interleucina-17/metabolismo , Qualidade de Vida , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Traumatismo por Reperfusão/metabolismoRESUMO
OBJECTIVE: To explore the role of zinc finger protein 36(ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. METHODS: ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. RESULTS: During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7(P < 0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P < 0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P < 0.05). CONCLUSIONS: ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.
Assuntos
Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais , Osteoblastos , Osteogênese , Animais , Camundongos , Fosfatase Alcalina/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Fator 1 de Resposta a Butirato/metabolismo , Fator 1 de Resposta a Butirato/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismoRESUMO
5-Fluorouracil (5-Fu) is the first-line chemotherapeutic option for colorectal cancer. However, its efficacy is inhibited by drug resistance. Cytokines play an important role in tumor drug resistance, even though their mechanisms are largely unknown. Using a cytokine array, we established that tissue inhibitor metalloproteinase 2 (TIMP-2) is highly expressed in 5-Fu resistant colorectal cancer patients. Analysis of samples from 84 patients showed that elevated TIMP-2 expression levels in colorectal patients were correlated with poor prognostic outcomes. In a 5-Fu-resistant patient-derived xenograft (PDX) model, TIMP-2 was also found to be highly expressed. We established an autocrine mechanism through which elevated TIMP-2 protein levels sustained colorectal cancer cell resistance to 5-Fu by constitutively activating the ERK/MAPK signaling pathway. Inhibition of TIMP-2 using an anti-TIMP-2 antibody or ERK/MAPK inhibition by U0126 suppressed TIMP-2 mediated 5-Fu-resistance in CRC patients. In conclusion, a novel TIMP-2-ERK/MAPK mediated 5-Fu resistance mechanism is involved in colorectal cancer. Therefore, targeting TIMP-2 or ERK/MAPK may provide a new strategy to overcome 5-Fu resistance in colorectal cancer chemotherapy.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fluoruracila/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Idoso , Animais , Butadienos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Experimentais/tratamento farmacológico , Nitrilas/farmacologia , Inibidor Tecidual de Metaloproteinase-2/genética , Transcriptoma , Adulto JovemRESUMO
OBJECTIVE: To explore the role of RN181 in the pathogenesis of oral squamous cell carcinoma (OSCC) cells via mediating ERK/MAPK signaling. METHODS: The expression of RN181 was detected in OSCC tissues and cells. CAL27 and SCC-15 cells were divided into Control, Empty, RN181, si-RN181, U0126 (an inhibitor of ERK/MAPK pathway) and si-RN181 + U0126 groups. MTT was used to determine cell proliferation, flow cytometry to determine cell cycle and apoptosis, Transwell assay and wound healing test to determine cell invasion and migration, respectively. Western blotting was used to measure the protein expression. Furthermore, a xenograft tumor model was established to observe the effect of RN181 on the in vivo growth of OSCC cells. RESULTS: RN181 was down-regulated in OSCC tissues and cells. As compared to the Control group, CAL27 and SCC-15 cells in the RN181 group and U0126 group presented with decreases in the proliferation, invasion and migration, but increases in the cell ratio at the G0/G1 phase and apoptosis, while the p-ERK 1/2/ERK 1/2 was down-regulated. Cells in the si-RN181 group manifested the opposite changes. U0126 could reverse the positive effect of si-RN181 on the growth of OSCC cells. In vivo experiment demonstrated that the tumor growth and weight were reduced in the RN181 group, with decreased Ki67 positive expression and elevated TUNEL positive cells. CONCLUSION: RN181 was down-regulated in OSCC, and it could inhibit the proliferation, invasion and migration, cause the G0/G1 arrest, while promote the apoptosis of OSCC cells via inhibiting ERK/MAPK pathway.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Bucais/enzimologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/enzimologia , Ubiquitina-Proteína Ligases/fisiologia , Adulto , Idoso , Animais , Apoptose , Butadienos/farmacologia , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Antígeno Ki-67/biossíntese , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Nitrilas/farmacologia , Transdução de Sinais , CicatrizaçãoRESUMO
Background: Chronic heart failure (CHF) is a major public health problem with high mortality and morbidity worldwide. Shexiang Tongxin Dropping Pill (STDP) is a widely used traditional Chinese medicine preparation for coronary heart disease and growing evidence proves that STDP exerts beneficial effects on CHF in the clinic. However, the molecular mechanism of the therapeutic effects of STDP on CHF remains largely unknown. Objective: This study aimed to elucidate the mechanism of action of STDP against CHF by integrating network pharmacology analysis and whole-transcriptome sequencing. Methods: First, the mouse model of CHF was established by the transverse aortic constriction (TAC) surgery, and the efficacy of STDP against CHF was evaluated by assessing the alterations in cardiac function, myocardial fibrosis, and cardiomyocyte hypertrophy with echocardiography, Masson's trichrome staining, and wheat germ agglutinin staining. Next, a CHF disease network was constructed by integrating cardiovascular disease-related genes and the transcriptome sequencing data, which was used to explore the underlying mechanism of action of STDP. Then, the key targets involved in the effects of STDP on CHF were determined by network analysis algorithms, and pathway enrichment analysis was performed to these key genes. Finally, important targets in critical pathway were verified in vivo. Results: STDP administration obviously improved cardiac function, relieved cardiomyocyte hypertrophy, and ameliorated myocardial fibrosis in CHF mice. Moreover, STDP significantly reversed the imbalanced genes that belong to the disease network of CHF in mice with TAC, and the number of genes with the reverse effect was 395. Pathway analysis of the crucial genes with recovery efficiency revealed that pathways related to fibrosis and energy metabolism were highly enriched, while TGF-ß pathway and ERK/MAPK pathway were predicted to be significantly affected. Consistently, validation experiments confirmed that inhibiting ERK/MAPK and TGF-ß signaling pathways via reduction of the phosphorylation level of Smad3 and ERK1/2 is the important mechanism of STDP against CHF. Conclusion: Our data demonstrated that STDP can recover the imbalanced CHF network disturbed by the modeling of TAC through the multi-target and multi-pathway manner in mice, and the mechanisms are mainly related to inhibition of ERK/MAPK and TGF-ß signaling pathways.
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PURPOSE: The aim of this study was to explore the regulatory role and mechanism of long noncoding RNA LINC00152 in gastric cancer (GC) cells. METHODS: LINC00152 expression in GC tissues and cells was detected by reverse transcription-polymerase chain reaction (qRT-PCR). MKN45 and MGC-803 cells were selected and assigned into different groups after transfection with si-LINC00152, activated ERK/MAPK signaling pathway (SA), or negative control. Cell proliferation, apoptosis, cycle, migration and invasion were assessed by CCK-8, flow cytometry, Transwell assay and Scratch test, respectively. Western blot analysis was conducted to detect the expression of E-cadherin, N-cadherin and ERK/MAPK signaling pathway protein. RESULTS: Compared with the normal tissues, higher expression of LINC00152 was found in GC tissues and LINC00152 was remarkably correlative with clinical stage and lymphatic metastasis. LINC00152 expression in GC cells was higher than that in GES-1 cells. Compared with the NC group, the cell proliferation rate, cells in G2/M phase, migration and invasion abilities as well as the expression of N-cadherin and p-ERK-1/2 were significantly decreased, and the expression of E-cadherin, cells in G0/G1 phase and cell apoptosis rate were significantly increased in the si-LINC00152-1 group. ERK/MAPK signaling pathway activator SA could reverse the biological role of LINC00152 in GC cells. CONCLUSION: These results demonstrated that the interference of LINC00152 expression may inhibit the invasion and migration of GC cells by inhibiting the ERK/MAPK signaling pathway.
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Patients with Alzheimer's disease often undergo anxiety and depression. Our previous studies have shown that α7nAChR protects against Aß-induced neurotoxicity via downregulation of p38 and JNK MAPKs, but the role of α7nAChR on Aß-induced anxiety and depressive-like behaviors and the effect of α7nAChR on the regulation of MAPKs pathways remain unknown. To examine the effects of α7nAChR and MAPKs pathways on Aß-induced anxiety and depression-like behaviors and to explore their relationships between them, elevated plus maze, open field and forced swim tests were performed. Protein levels of 5-HT1A receptor, 5-HT2C receptor, α7nAChR, t-ERK1/2 and p-ERK1/2 in the amygdala were analyzed by western blotting and immunostaining. Our study found out that Aß oligomers induced anxiety and depression-like behaviors in C56BL/6 mice with open field, elevated plus maze and forced swim tests. However, activation of α7nAChR or inhibition of ERK pathways showed significant antidepressant and anxiolytic-like effects on Aß-injected mice. Moreover, Aß significantly decreased the level of 5-HT1A receptor but increased the level of 5-HT2C receptor in the basolateral amygdala. Treatment with α7nAChR agonist PNU282987 or ERK inhibitor U0126 reversed Aß-induced 5-HT1A and 5-HT2C receptor changes. Moreover, activation of α7nAChR inhibited ERK pathway in the amygdala of Aß1-42-injected mice. Our study provides a new insight into the mechanism of α7nAChR in Aß-induced depression and anxiety-related symptoms through the regulation of ERK1/2 pathway and the potential association with serotonin receptors. Together, our data suggests that α7nAChR is protective against Aß-induced anxiety and depression-like behaviors in mice.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Ansiedade/metabolismo , Benzamidas/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/efeitos dos fármacos , Animais , Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/tratamento farmacológico , Transtornos de Ansiedade/metabolismo , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Regulação para Cima/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) are suitable cell sources for dental pulp regeneration, but the mechanism of BMMSCs differentiation into odontogenic lineage remains unknown. The aim of the present study was to reveal the role of magnesium transporter protein 1 (MagT1) and MAPK pathways in the odontogenic differentiation of BMMSCs. METHODS: The RNA sequencing (RNA-seq) was performed to explore the altered transcriptome of BMMSCs undergoing odontogenic differentiation induced by tooth germ cell-condition medium (TGC-CM). Pathway analysis was conducted to explore enriched pathways of the differential expression signature. Automated western blot, real-time PCR, shRNA lentivirus, and flow cytometry were used to detect the function of MagTl and MAPK pathway in odontogenic differentiation of BMMSCs. RESULTS: RNA-seq identified 622 differentially expressed genes associated with odontogenic differentiation of BMMSCs induced by TGC-CM, some of which were responsible for MAPK pathway. Consistently, we verified that TGC-CM induced odontogenic differentiation of BMMSCs through activating ERK/MAPK pathway, and the inactivation of ERK/MAPK pathway inhibited the odontogenic differentiation induced by TGC-CM. We also found MagT1 protein was significantly increased during odontogenic differentiation of BMMSCs induced by TGC-CMM, in accordance, MagT1 knockdown significantly decreased the extent of mineralized nodules and the protein levels of alkaline phosphatase (ALP), dentin matrix protein 1 (DMP-1), and dentin sialophosphoprotein (DSP). Flow cytometry showed that intracellular Mg2+ was significantly reduced in MagT1-knockdown BMMSCs, indicating the suppression of MagT1 inhibited odontogenic differentiation of BMMSCs by decreasing intracellular Mg2+. Finally, we performed RNA-seq to explore the altered transcriptome of MagT1-knockdown BMMSCs undergoing odontogenic differentiation and identified 281 differentially expressed genes, some of which were involved in MAPK pathway. Consistently, automated western blot analysis found the ERK/MAPK pathway was inhibited in MagT1-knockdown BMMSCs during odontogenic differentiation, indicating that suppression of MagT1 inhibited odontogenic differentiation of BMMSCs via ERK/MAPK pathway. CONCLUSIONS: This study identified the significant alteration of transcriptome in BMMSCs undergoing odontogenic differentiation induced by TGC-CM. We clarified the pivotal role of MagT1 and ERK/MAPK pathway in odontogenic differentiation of BMMSCs, and suppression of MagT1 inhibited the odontogenic differentiation of BMMSCs by decreasing the intracellular Mg2+ and inactivating ERK/MAPK pathway.
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Proteínas de Transporte de Cátions/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Diferenciação Celular/fisiologia , Meios de Cultivo Condicionados , Sistema de Sinalização das MAP Quinases , Odontogênese , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
In the present study, we aimed to identify specific lncRNAs and miRNAs, as well as mRNAs, involved in bile duct carcinoma (BDC) and to further explore the way in which lncRNA UCA1 regulates cell metastasis ability. Differentially expressed RNAs were screened out from the TCGA database. In in vitro experiments, qRT-PCR was used to measure lncRNA UCA1, miR-122 and CLIC1 expression. We performed a dual luciferase assay to validate the target relationships among UCA1, CLIC1 and miR-122. The cell migration ability was measured by a wound healing assay, and Transwell assays were applied to detect cell invasive ability. Western blot analysis was employed to detect the expression of related proteins in the MAPK signaling pathway. According to the bioinformatics analysis, lncRNA UCA1 and CLIC1 were both significantly upregulated in BDC, while the expression of miR-122 declined compared with the normal group. The target relationship among UCA1, CLIC1 and miR-122 was verified. UCA1 promoted BDC cell migration and invasiveness, while miR-122 inhibited their progression. CLIC1 served as the downstream target gene of miR-122 and had opposite effects. The ERK/MAPK signaling pathway was activated after upregulating UCA1. LncRNA-UCA1 promoted the metastasis of BDC cells by regulating the expression of miR-122 and its downstream gene mRNA CLIC1 and promoted the activation of the ERK/MAPK pathway, which expanded the horizons of targeted therapy of cholangiocarcinoma.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Canais de Cloreto/genética , Colangiocarcinoma/patologia , MicroRNAs/genética , RNA Longo não Codificante/fisiologia , Neoplasias dos Ductos Biliares/genética , Ductos Biliares Intra-Hepáticos/patologia , Estudos de Casos e Controles , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Análise em Microsséries , Metástase Neoplásica , RNA Longo não Codificante/genética , Regulação para Cima/genéticaRESUMO
Central nervous system diseases remain the most challenging pathologies, with limited or even no therapeutic possibilities and a poor prognosis. This study aimed to investigate the differentiation properties of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) transfected with recombinant adenovirus expressing enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) and the possible mechanisms involved. Cells were isolated, and MSC immunophenotypes were confirmed. The resulting differentiated cells treated with Adv-EGFP-CT-1 and cultured in neural induction medium (NIM) expressed higher levels of Nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) markers than cells in other treatments. Expression of glycoprotein 130/leukemia inhibitory factor receptor ß (gp130/LiFRß), Raf-1, phosphorylated Raf-1 (p-Raf-1), extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) increased gradually within 72 h after transfection with Adv-EGFP-CT-1 and NIM culture. Additionally, inhibition of extracellular signal-regulated kinase kinase (MEK) abrogated expression of p-ERK1/2, Nestin, GFAP and NeuN. Thus, the ERK1/2 pathway may contribute to CT1-stimulated neural differentiation of hUCB-MSCs.
RESUMO
Disuse osteoporosis (DOP) is a common complication of the lack of mechanical loading. The precise mechanism underlying DOP remains unknown, although epigenetic modifications may be a major cause. Recently, cumulative research has revealed that DNA methyltransferase (DNMT) proteins can catalyze the conversion of cytosine to 5-methylcytosine (5mC), altering the epigenetic state of DNA. Here, we report that DNMT1 expression and lncRNA-H19 methylation are upregulated in the femoral tissues of DOP rats, accompanied with inhibited Erk signaling pathway. Overexpression of DNMT1 in UMR-106 cells mimics 5mC enrichment in the H19 promoter, inhibition of Erk signaling and impairment of osteogenesis, which can be rescued by 5'-aza-deoxycytidine (5'-Aza) treatment. Moreover, local intramedullary injection of Dnmt1 siRNA (siDNMT1) in Sprague-Dawley (SD) rats abrogated disuse lncRNA-H19 (H19) downregulation, Erk signaling inhibition, histopathological changes, and bone microstructure declines in the distal femur in vivo. Therefore, our data identify for the first time a new signaling cascade in DOP: mechanical unloading causes upregulation of DNMT1 and hypermethylation of H19 promoter, which subsequently leads to downregulation of lncRNA-H19 and inhibition of the ERK signaling, suggesting a new potential therapeutic target.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/fisiologia , Epigênese Genética , Osteoporose/etiologia , Osteoporose/genética , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Regulação para Baixo , Fêmur/ultraestrutura , Elevação dos Membros Posteriores , Sistema de Sinalização das MAP Quinases , Osteoporose/patologia , Osteoporose/terapia , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
Endoplasmic reticulum (ER) stress, together with unfolded protein response (UPR), can remove unfolded proteins and promote survival. However, severe and prolonged ER stress leads to cell death, tissue injury, and many serious diseases. Therefore, it is essential to identify drugs that can attenuate ER stress for ER-related disease treatment. A great deal of research shows that selenoprotein S (SelS) is a sensitive and ideal marker of ER stress. Here, we used a firefly luciferase reporter driven by SelS gene promoter to screen natural compounds that can attenuate ER stress. Then we identified compound 20(S)-25-methoxyl-dammarane-3ß,12ß,20-triol (25-OCH3-PPD) could inhibit the promoter activity of SelS, further results showed that 25-OCH3-PPD effectively inhibited tunicamycin (TM) induced up-regulation of SelS expression in both mRNA and protein levels. Moreover, 25-OCH3-PPD significantly inhibited glucose-regulated protein 78 (GRP78; the major ER stress marker) expression in TM-induced ER stress in HepG2 and HEK293T cells, suggesting that 25-OCH3-PPD could attenuate ER stress in these cells. Mechanism studies showed that 25-OCH3-PPD significantly activated ERK/MAPK signaling pathway, and the inhibition of ERK/MAPK by U0126 dramatically abolished the inhibitory effect of 25-OCH3-PPD on ER stress, suggesting that 25-OCH3-PPD attenuated ER stress at least partially through activation of ERK/MAPK signaling pathway. Taken together, our studies indicate that 25-OCH3-PPD is a novel small molecular compound reducing ER stress, and a potential drug for treating diseases associated with ER stress.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Triterpenos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Selenoproteínas/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The past three decades have witnessed an enormous progress in the elucidation of the ERK/MAPK signaling pathway and its involvement in various cellular processes. Because of its importance and complex wiring, the ERK pathway has been an intensive subject for mathematical modeling, which facilitates the unraveling of key dynamic properties and behaviors of the pathway. Recently, however, it became evident that the pathway does not act in isolation but closely interacts with many other pathways to coordinate various cellular outcomes under different pathophysiological contexts. This has led to an increasing number of integrated, large-scale models that link the ERK pathway to other functionally important pathways. In this chapter, we first discuss the essential steps in model development and notable models of the ERK pathway. We then use three examples of integrated, multipathway models to investigate how crosstalk of ERK signaling with other pathways regulates cell-fate decision-making in various physiological and disease contexts. Specifically, we focus on ERK interactions with the phosphoinositide-3 kinase (PI3K), c-Jun N-terminal kinase (JNK), and ß-adrenergic receptor (ß-AR) signaling pathways. We conclude that integrated modeling in combination with wet-lab experimentation have been and will be instrumental in gaining an in-depth understanding of ERK signaling in multiple biological contexts.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Modelos Biológicos , Transdução de Sinais , Algoritmos , Animais , Apoptose , Biomarcadores , Proliferação de Células , Sobrevivência Celular , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the effects of microRNA-7 (miR-7) on the proliferation, migration and invasion of non-small cell lung cancer NSCLC) cells by targeting FAK through ERK/MAPK signaling pathway. METHODS: NSCLC tissues and adjacent normal tissues were obtained from 160 NSCLC patients after operation. NSCLC cell lines (A549, H1299 and H1355) and a normal human fetal lung fibroblast cell line (MRC-5) were obtained. NSCLC cells were assigned into miR-7 inhibitors, miR-7 mimics, blank, miR-7 mimics control, miR-7 inhibitors control, FAK siRNA and miR-7 inhibitors + FAK siRNA groups. The expressions of miR-7 and FAK mRNA in tissues and cell lines were detected by qRT-PCR and Western-Blotting. Cell proliferation, migration and invasion were detected by MTT assay, wound scratch assay and Transwell assay. RESULTS: Compared with adjacent normal tissues, miR-7 expression was down-regulated, but the mRNA and protein expressions of FAK, ERK and MAPK were up-regulated. Compared with the blank and mimics control groups, miR-7 significantly increased but FAK, ERK and MAPK expressions decreased in miR-7 mimics and FAK siRNA groups. Cell proliferation, migration and invasion were inhibited in the miR-7 mimics and FAK siRNA groups, while opposite regarding miR-7 inhibitors group. CONCLUSION: The miR-7 can inhibit the activation of ERK/MAPK signaling pathway by down-regulating FAK expression, thereby suppressing the proliferation, migration and invasion of NSCLC cells. The miR-7 and its target gene FAK may be novel targets for the diagnosis and treatment of NSCLC.