RESUMO
BACKGROUND: Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive. METHODS: In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining. RESULTS: USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells. CONCLUSIONS: These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.
Assuntos
Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Osteoblastos , Osteogênese , Osteoblastos/metabolismo , Osteoblastos/citologia , Diferenciação Celular/fisiologia , Diferenciação Celular/genética , Humanos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/genética , Proliferação de Células/fisiologia , Proliferação de Células/genética , Osteogênese/fisiologia , Osteogênese/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Linhagem Celular , Apoptose/genética , Apoptose/fisiologia , Ubiquitinação , Técnicas de Silenciamento de GenesRESUMO
Osteosarcoma is a highly metastatic, aggressive bone cancer that occurs in children and young adults worldwide. Circular RNAs (circRNAs) are crucial molecules for osteosarcoma progression. In this study, we aimed to investigate the impact of circMRPS35 overexpression and its interaction with FOXO1 via evaluating apoptosis, cell cycle, and bioinformatic analyses on the malignant development of osteosarcoma in MG63 and MNNG/HOS cells. We found that circMRPS35 overexpression reduced osteosarcoma cell viability and inhibited tumor growth in vivo. It increased the apoptosis rate and induced cell cycle arrest in osteosarcoma cells. We identified a potential interaction between circMRPS35 and FOXO1 with miR-105-5p using bioinformatics analysis. Overexpression of circMRPS35 decreased miR-105-5p expression, whereas miR-105-5p mimic treatment increased its expression. This mimic also suppressed the luciferase activity of circMRPS35 and FOXO1 and reduced FOXO1 expression. Overexpression of circMRPS35 elevated FOXO1 protein levels, but this effect was reversed by co-treatment with the miR-105-5p mimic. We demonstrated that inhibiting miR-105-5p decreased viability and induced apoptosis. Overexpression of FOXO1 or treatment with a miR-105-5p inhibitor could counteract the effects of circMRPS35 on viability and apoptosis in osteosarcoma cells. Therefore, we concluded that circMRPS35 suppressed the malignant progression of osteosarcoma via targeting the miR-105-5p/FOXO1 axis.
Assuntos
Apoptose , Neoplasias Ósseas , Proteína Forkhead Box O1 , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Osteossarcoma , RNA Circular , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , RNA Circular/genética , RNA Circular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Humanos , Linhagem Celular Tumoral , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Apoptose/genética , Camundongos , Progressão da Doença , Proliferação de Células/genética , Camundongos Nus , Sobrevivência Celular/genéticaRESUMO
BACKGROUND/AIMS: Accumulating evidences have shown an important role of circular RNAs (circRNAs) in the tumorigenesis of gastric cancer (GC). Nevertheless, whether circSNX27 plays a role in GC remains undetermined. MATERIALS AND METHODS: Relative expression of circRNAs and related microRNAs (miRNAs) in GC tissues and cells were tested by quantitative reverse transcription polymerase chain reaction. Specific short hairpin RNAs were designed to knockdown the expression of circSNX27 in GC cells. CCK-8, colony formation, flow cytometry, wound healing, and transwell assays were used to access the function of circSNX27 silencing on GC cells. The target miRNAs of circSNX27 were predicted by 2 databases, circBank and Circinteractome. Dualluciferase reporter assay was used to verify the interaction between circSNX27 and miR-638. RESULTS: circSNX27 was found to be upregulated in GC tissues and cell lines compared with normal controls. Silencing of circSNX27 repressed GC cell viability, proliferation, migration, and invasion. Moreover, circSNX27 silencing could accelerate GC cell apoptosis. Additionally, we found that circSXN27 decreased the expression of miR-638 by directly binding to it in GC cells. CONCLUSION: Our results indicated that circSXN27 facilitated GC progression by acting as a sponge of miR-638.
Assuntos
Apoptose , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , MicroRNAs , RNA Circular , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , Proliferação de Células/genética , Linhagem Celular Tumoral , Apoptose/genética , Movimento Celular/genética , Regulação para Cima , Sobrevivência Celular/genéticaRESUMO
BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).
Assuntos
Oócitos , Folículo Ovariano , Proteínas Proto-Oncogênicas c-kit , Animais , Feminino , Oócitos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Folículo Ovariano/metabolismo , Sobrevivência Celular/genética , Reprodução , Camundongos Knockout , Biomarcadores , Fator 9 de Diferenciação de Crescimento/metabolismo , Fator 9 de Diferenciação de Crescimento/genética , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/genéticaRESUMO
Few efficacious treatment options are available for patients with small cell lung carcinoma (SCLC), indicating the need to develop novel therapeutic approaches. In this study, we explored kinesin family member 11 (KIF11), a potential therapeutic target in SCLC. An analysis of publicly available data suggested that KIF11 mRNA expression levels are significantly higher in SCLC tissues than in normal lung tissues. When KIF11 was targeted by RNA interference or a small-molecule inhibitor (SB743921) in two SCLC cell lines, Lu-135 and NCI-H69, cell cycle progression was arrested at the G2/M phase with complete growth suppression. Further work suggested that the two cell lines were more significantly affected when both KIF11 and BCL2L1, an anti-apoptotic BCL2 family member, were inhibited. This dual inhibition resulted in markedly decreased cell viability. These findings collectively indicate that SCLC cells are critically dependent on KIF11 activity for survival and/or proliferation, as well as that KIF11 inhibition could be a new strategy for SCLC treatment.
Assuntos
Sobrevivência Celular , Cinesinas , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Cinesinas/metabolismo , Cinesinas/genética , Cinesinas/antagonistas & inibidores , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proliferação de Células , Proteína bcl-X/metabolismo , Proteína bcl-X/genética , Regulação Neoplásica da Expressão Gênica , Apoptose/genética , Benzamidas , QuinazolinasRESUMO
Latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer.
Assuntos
Progressão da Doença , Neoplasias da Próstata , Receptores Androgênicos , Masculino , Humanos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Linhagem Celular Tumoral , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Movimento Celular/genética , Movimento Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Janus Quinase 2/metabolismo , Janus Quinase 2/genética , Receptores de Peptídeos/metabolismo , Receptores de Peptídeos/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Transdução de Sinais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Processamento AlternativoRESUMO
Objectives: To investigate the effects and the related signaling pathway of miR-362-3p on OS. Methods: The bioinformatics analysis approaches were employed to investigate the target pathway of miR-362-3p. After the 143B and U2OS cells and nu/nu male mice were randomly divided into blank control (BC) group, normal control (NC) group, and overexpression group (OG), the CCK-8, EdU staining, wound healing assay, Transwell assay, and TUNEL staining were adopted to respectively determine the effects of overexpressed miR-362-3p on the cell viability, proliferation, migration, invasion, and apoptosis of 143B and U2OS cells in vitro, tumor area assay and hematoxylin and eosin staining were employed to respectively determine the effects of overexpressed miR-362-3p on the growth and pathological injury of OS tissue in vivo. The qRT-PCR, Western blot, and immunohistochemical staining were applied to respectively investigate the effects of overexpressed miR-362-3p on the IL6ST/JAK2/STAT3 pathway in OS in vivo and in vitro. Results: The bioinformatics analysis approaches combined qRT-PCR indicated that the IL6ST/JAK2/STAT3 is one of the target pathways of miR-362-3p. Compared with NC, the cell viability, proliferation, migration, and invasion of 143B and U2OS cells were dramatically (P < 0.01) inhibited but the apoptosis was prominently (P <0 .0001) promoted in OG. Compared with NC, the growth of OS tissue was significantly (P < 0.05) suppressed and the pathological injury of OS tissue was substantially aggravated in OG. The gene expression levels of IL6ST, JAK2, and STAT3 and the protein expression levels of IL6ST, JAK2, p-JAK2, STAT3, and p-STAT3 in 143B and U2OS cells were memorably (P < 0.0001) lower in OG than those in NC. In addition, the positively stained areas of proteins of IL6ST, JAK2, p-JAK2, STAT3, and p-STAT3 of OS tissue in OG were markedly (P < 0.01) reduced compared with those in NC. Conclusion: The overexpression of miR362-3p alleviates OS by inhibiting the IL6ST/JAK2/STAT3 pathway in vivo and in vitro.
Assuntos
Apoptose , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Janus Quinase 2 , MicroRNAs , Osteossarcoma , Fator de Transcrição STAT3 , Transdução de Sinais , MicroRNAs/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Janus Quinase 2/metabolismo , Janus Quinase 2/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Movimento Celular/genética , Apoptose/genética , Masculino , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor gp130 de Citocina/metabolismo , Receptor gp130 de Citocina/genética , Biologia Computacional/métodos , Modelos Animais de Doenças , Sobrevivência Celular/genéticaRESUMO
Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.
Assuntos
Homeostase , Regeneração , Telomerase , Telomerase/metabolismo , Telomerase/genética , Animais , Homeostase/genética , Homeostase/efeitos da radiação , Camundongos , Regeneração/efeitos da radiação , Regeneração/genética , Humanos , Glândulas Salivares/efeitos da radiação , Glândulas Salivares/metabolismo , Glândulas Salivares/citologia , Proliferação de Células/efeitos da radiação , Proliferação de Células/genética , Sobrevivência Celular/efeitos da radiação , Sobrevivência Celular/genética , Glândula Submandibular/efeitos da radiação , Glândula Submandibular/metabolismo , Células-Tronco/efeitos da radiação , Células-Tronco/metabolismo , Células-Tronco/citologia , Radioterapia/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Células CultivadasRESUMO
Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.
Assuntos
Adenosina , Homólogo AlkB 5 da RNA Desmetilase , Asma , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Asma/genética , Asma/metabolismo , Asma/patologia , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Proliferação de Células/genética , Metilação , Progressão da Doença , Linhagem Celular , Ferroptose/genética , Células Epiteliais/metabolismo , Regulação para Baixo , Brônquios/patologia , Brônquios/metabolismo , Técnicas de Silenciamento de Genes , Sobrevivência Celular/genéticaRESUMO
LncRNA is a group of transcripts with a length exceeding 200 nucleotides that contribute to tumour development. Our research group found that LINC00052 expression was repressed during the formation of breast cancer (BC) multicellular spheroids. Intriguingly, LINC00052 precise role in BC remains uncertain. We explored LINC00052 expression in BC patients` RNA samples (TCGA) in silico, as well as in an in-house patient cohort, and inferred its cellular and molecular mechanisms. In vitro studies evaluated LINC00052 relevance in BC cells viability, cell cycle and DNA damage. Results. Bioinformatic RNAseq analysis of BC patients showed that LINC00052 is overexpressed in samples from all BC molecular subtypes. A similar LINC00052 expression pattern was observed in an in-house patient cohort. In addition, higher LINC00052 levels are related to better BC patient´s overall survival. Remarkably, MCF-7 and ZR-75-1 cells treated with estradiol showed increased LINC00052 expression compared to control, while these changes were not observed in MDA-MB-231 cells. In parallel, bioinformatic analyses indicated that LINC00052 influences DNA damage and cell cycle. MCF-7 cells with low LINC00052 levels exhibited increased cellular protection against DNA damage and diminished growth capacity. Furthermore, in cisplatin-resistant MCF-7 cells, LINC00052 expression was downregulated. Conclusion. This work shows that LINC00052 expression is associated with better BC patient survival. Remarkably, LINC00052 expression can be regulated by Estradiol. Additionally, assays suggest that LINC00052 could modulate MCF-7 cells growth and DNA damage repair. Overall, this study highlights the need for further research to unravel LINC00052 molecular mechanisms and potential clinical applications in BC.
Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante , Feminino , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Biologia Computacional/métodos , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Células MCF-7 , Prognóstico , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: This study investigated the mechanism by which tazarotene-induced gene 1 (TIG1) inhibits melanoma cell growth. The main focus was to analyze downstream genes regulated by TIG1 in melanoma cells and its impact on cell growth. METHODS: The effects of TIG1 expression on cell viability and death were assessed using water-soluble tetrazolium 1 (WST-1) mitochondrial staining and lactate dehydrogenase release assays. RNA sequencing and Western blot analysis were employed to investigate the genes regulated by TIG1 in melanoma cells. Additionally, the correlation between TIG1 expression and its downstream genes was analyzed in a melanoma tissue array. RESULTS: TIG1 expression in melanoma cells was associated with decreased cell viability and increased cell death. RNA-sequencing (RNA-seq), quantitative reverse transcription PCR (reverse RT-QPCR), and immunoblots revealed that TIG1 expression induced the expression of Endoplasmic Reticulum (ER) stress response-related genes such as Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 (HERPUD1), Binding immunoglobulin protein (BIP), and DNA damage-inducible transcript 3 (DDIT3). Furthermore, analysis of the melanoma tissue array revealed a positive correlation between TIG1 expression and the expression of HERPUD1, BIP, and DDIT3. Additionally, attenuation of the ER stress response in melanoma cells weakened the impact of TIG1 on cell growth. CONCLUSIONS: TIG1 expression effectively hinders the growth of melanoma cells. TIG1 induces the upregulation of ER stress response-related genes, leading to an increase in caspase-3 activity and subsequent cell death. These findings suggest that the ability of retinoic acid to prevent melanoma formation may be associated with the anticancer effect of TIG1.
Assuntos
Sobrevivência Celular , Estresse do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Melanoma , Humanos , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Morte Celular/genética , Apoptose/genética , Apoptose/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/efeitos dos fármacos , Proteínas de MembranaRESUMO
Mortalin (encoded by HSPA9) is a mitochondrial chaperone often overexpressed in cancer through as-yet-unknown mechanisms. By searching different RNA-sequencing datasets, we found that ESRRA is a transcription factor highly correlated with HSPA9 in thyroid cancer, especially in follicular, but not C cell-originated, tumors. Consistent with this correlation, ESRRA depletion decreased mortalin expression only in follicular thyroid tumor cells. Further, ESRRA expression and activity were relatively high in thyroid tumors with oncocytic characteristics, wherein ESRRA and mortalin exhibited relatively high functional overlap. Mechanistically, ESRRA directly regulated HSPA9 transcription through a novel ESRRA-responsive element located upstream of the HSPA9 promoter. Physiologically, ESRRA depletion suppressed thyroid tumor cell survival via caspase-dependent apoptosis, which ectopic mortalin expression substantially abrogated. ESRRA depletion also effectively suppressed tumor growth and mortalin expression in the xenografts of oncocytic or ESRRA-overexpressing human thyroid tumor cells in mice. Notably, our Bioinformatics analyses of patient data revealed two ESRRA target gene clusters that contrast oncocytic-like and anaplastic features of follicular thyroid tumors. These findings suggest that ESRRA is a tumor-specific regulator of mortalin expression, the ESRRA-mortalin axis has higher significance in tumors with oncocytic characteristics, and ESRRA target gene networks can refine molecular classification of thyroid cancer.
Assuntos
Sobrevivência Celular , Receptor ERRalfa Relacionado ao Estrogênio , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Choque Térmico HSP70 , Receptores de Estrogênio , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Animais , Camundongos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Estrogênio/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Apoptose/genética , Regiões Promotoras Genéticas/genética , Proteínas MitocondriaisRESUMO
DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1, DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1 , DNA Helicases , Replicação do DNA , DNA de Cadeia Simples , Exodesoxirribonucleases , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Replicação do DNA/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Sobrevivência Celular/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Dano ao DNARESUMO
Cancer stem cells (CSCs), which are distinct subpopulations of tumor cells, have a substantially higher tumor-initiating capacity and are closely related to poor clinical outcomes. Damage to organelles can trigger CSC pool exhaustion; however, the underlying mechanisms are poorly understood. ZER6 is a zinc-finger protein with two isoforms possessing different amino termini: p52-ZER6 and p71-ZER6. Since their discovery, almost no study reported on their biological and pathological functions. Herein, we found that p52-ZER6 was crucial for CSC population maintenance; p52-ZER6-knocking down almost abolished the tumor initiation capability. Through transcriptomic analyses together with in vitro and in vivo studies, we identified insulin like growth factor 1 receptor (IGF1R) as the transcriptional target of p52-ZER6 that mediated p52-ZER6 regulation of CSC by promoting pro-survival mitophagy. Moreover, this regulation of mitophagy-mediated CSC population maintenance is specific to p52-ZER6, as p71-ZER6 failed to exert the same effect, most possibly due to the presence of the HUB1 domain at its N-terminus. These results provide a new perspective on the regulatory pathway of pro-survival mitophagy in tumor cells and the molecular mechanism underlying p52-ZER6 oncogenic activity, suggesting that targeting p52-ZER6/IGF1R axis to induce CSC pool exhaustion may be a promising anti-tumor therapeutic strategy.
Assuntos
Mitofagia , Células-Tronco Neoplásicas , Receptor IGF Tipo 1 , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Humanos , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 1/genética , Mitofagia/genética , Animais , Camundongos , Progressão da Doença , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Sobrevivência Celular/genéticaRESUMO
To explore the effects of ß-Sitosterol upon hepatocellular carcinoma cell proliferation, apoptosis, migration, invasion, and epithelial-mesenchymal transition (EMT), and to investigate the underlying mechanism using network pharmacology. Human hepatocellular carcinoma cell lines (Huh-7 and HCCLM3) were expose to gradient concentrations of ß-Sitosterol (5 µg/mL, 10 µg/mL, and 20 µg/mL). Cell viability and proliferation were assessed using MTT, CCK-8, colony formation, and EdU assays.Flow cytometry was employed to evaluate cell cycle and apoptosis. Scratch and Transwell assays were performed, respectively, to detect cell migration and invasion. The levels of apoptosis-associated proteins (BAX, BCL2, and cleaved caspase3) as well as EMT-associated proteins (E-cadherin, N-cadherin, Snail, and Vimentin) were detected in Huh-7 and HCCLM3 cell lines using Western blot analysis. The drug target gene for ß-Sitosterol was screened via PubChem and subsequently evaluated for expression in the GSE112790 dataset. In addition, the expression level of glycogen synthase kinase 3 beta (GSK3B) within the Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database was analyzed, along with its correlation to the survival outcomes of patients with hepatocellular carcinoma. The diagnostic efficiency of GSK3B was assessed by analyzing the ROC curve. Subsequently, Huh-7 and HCCLM3 cell lines were transfected with the overexpression vector of GSK3B and then treated with ß-Sitosterol to further validate the association between GSK3B and ß-Sitosterol. GSK3B demonstrated a significantly elevated expression in patients with hepatocellular carcinoma, which could predict hepatocellular carcinoma patients' impaired prognosis based on GEO dataset and TCGA database. GSK3B inhibitor (CHIR-98014) notably inhibited cell proliferation and invasion, promoted cell apoptosis and cell cycle arrest at G0/G1 phase in hepatocellular carcinoma cells. ß-Sitosterol treatment further promoted the efffects of GSK3B inhibitor on hepatocellular carcinoma cells. GSK3B overexpression has been found to enhance the proliferative and invasive capabilities of hepatocellular carcinoma cells. Furthermore it has been observed that GSK3B overexpression, it has been obsear can partially reverse the inhibitory effect of ß-Sitosterol upon hepatocellular. ß-Sitosterol suppressed hepatocellular carcinoma cell proliferation and invasion, and enhanced apoptosis via inhibiting GSK3B expression.
Assuntos
Apoptose , Carcinoma Hepatocelular , Proliferação de Células , Transição Epitelial-Mesenquimal , Glicogênio Sintase Quinase 3 beta , Neoplasias Hepáticas , Sitosteroides , Humanos , Sitosteroides/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Expressão Gênica/genética , Expressão Gênica/efeitos dos fármacos , Fenótipo , Invasividade Neoplásica/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Farmacologia em Rede , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
BACKGROUND: TRAIL protein on binding to its cognate death receptors (DR) can induce apoptosis specifically in breast tumor cells sparing normal cells. However, TRAIL also binds to decoy receptors (DCR) thereby inhibiting the apoptotic pathways thus causing TRAIL resistance. Also, one of the barriers due to which TRAIL-based therapy could not become FDA-approved might be because of resistance to therapy. Therefore, in the current study we wanted to explore the role of transcription factors in TRAIL resistance with respect to breast cancer. METHODS: Microarray data from TRAIL-sensitive (TS) and TRAIL-resistant (TR) MDA-MB-231 cells were reanalyzed followed by validation of the candidate genes using quantitative PCR (qPCR), immunoblotting and immunofluorescence technique. Overexpression of the candidate gene was performed in MDA-MB-231 and MCF7 cells followed by cell viability assay and immunoblotting for cleaved caspase-3. Additionally, immunoblotting for DCR2 was carried out. TCGA breast cancer patient survival was used for Kaplan-Meier (KM) plot. RESULTS: Validation of the candidate gene i.e. ELF3 using qPCR and immunoblotting revealed it to be downregulated in TR cells compared to TS cells. ELF3 overexpression in MDA-MB-231 and MCF7 cells caused reversal of TRAIL resistance as observed using cell viability assay and cleaved caspase-3 immunoblotting. ELF3 overexpression also resulted in DCR2 downregulation in the MDA-MB-231 and MCF7 cells. Furthermore, KM analysis found high ELF3 and low DCR2 expression to show better patient survival in the presence of TRAIL. CONCLUSION: Our study shows ELF3 to be an important factor that can influence TRAIL-mediated apoptosis in breast cancer. Also, ELF3 and DCR2 expression status should be taken into consideration while designing strategies for successful TRAIL-based therapy.
Assuntos
Apoptose , Neoplasias da Mama , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Transcrição , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Linhagem Celular Tumoral , Apoptose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células MCF-7 , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Membro 10c de Receptores do Fator de Necrose Tumoral/genética , Membro 10c de Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Proto-Oncogênicas c-etsRESUMO
Colorectal cancer is one of the most common malignant cancers. Pseudogenes have been identified as oncogenes or tumor suppressor genes in the development of various cancers. However, the function of pseudogene CSPG4P12 in colorectal cancer remains unclear. Therefore, the aim of this study was to investigate the potential role of CSPG4P12 in colorectal cancer and explore the possible underlying mechanism. The difference of CSPG4P12 expression between colorectal cancer tissues and adjacent normal tissues was analyzed using the online Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database. Cell viability and colony formation assays were conducted to evaluate cell viability. Transwell and wound healing assays were performed to assess cell migration and invasion capacities. Western blot was used to measure the expression levels of epithelial-mesenchymal transition-related proteins. Colorectal cancer tissues had lower CSPG4P12 expression than adjacent normal tissues. The overexpression of CSPG4P12 inhibited cell proliferation, invasion, and migration in colorectal cancer cells. Overexpressed CSPG4P12 promoted the expression of E-cadherin, whereas it inhibited the expression of vimentin, N-cadherin, and MMP9. These findings suggested that CSPG4P12 inhibits colorectal cancer development and may serve as a new potential target for colorectal cancer.
Assuntos
Movimento Celular , Proliferação de Células , Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Pseudogenes , Humanos , Transição Epitelial-Mesenquimal/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Pseudogenes/genética , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Sobrevivência Celular/genética , Invasividade Neoplásica/genéticaRESUMO
BACKGROUND: MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. METHODS AND RESULTS: HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. CONCLUSIONS: microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.
Assuntos
Fosfatases de Especificidade Dupla , Inflamação , Lipopolissacarídeos , MicroRNAs , Ligamento Periodontal , Células-Tronco , Proteínas Quinases p38 Ativadas por Mitógeno , Humanos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Ligamento Periodontal/metabolismo , Ligamento Periodontal/citologia , Periodontite/genética , Periodontite/metabolismo , Periodontite/patologia , Transdução de Sinais/genética , Células-Tronco/metabolismoRESUMO
Objective: Ubiquitin-specific peptidase 39 (USP39) plays a carcinogenic role in many cancers, but little research has been conducted examining whether it is involved in head and neck squamous cell carcinoma (HNSCC). Therefore, this study explored the functional role of USP39 in HNSCC. Method: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify differentially expressed proteins (DEPs) between the HNSCC tumor and adjacent healthy tissues. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to assess the functional enrichment of DEPs. Immunohistochemistry was used to detect protein expression. The viability and migration of two HNSCC cell lines, namely CAL27 and SCC25, were detected using the cell counting kit-8 assay and a wound healing assay, respectively. Quantitative real-time PCR was used to detect the expression level of signal transducer and activator of transcription 1 (STAT1) mRNA. Results: LC-MS/MS results identified 590 DEPs between HNSCC and adjacent tissues collected from 4 patients. Through GO and KEGG pathway analyses, 34 different proteins were found to be enriched in the spliceosome pathway. The expression levels of USP39 and STAT1 were significantly higher in HNSCC tumor tissue than in adjacent healthy tissue as assessed by LC-MS/MS analysis, and the increased expression of USP39 and STAT1 protein was confirmed by immunohistochemistry in clinical samples collected from 7 additional patients with HNSCC. Knockdown of USP39 or STAT1 inhibited the viability and migration of CAL27 and SCC25 cells. In addition, USP39 knockdown inhibited the expression of STAT1 mRNA in these cells. Conclusion: Our findings indicated that USP39 knockdown may inhibit HNSCC viability and migration by suppressing STAT1 expression. The results of this study suggest that USP39 may be a potential new target for HNSCC clinical therapy or a new biomarker for HNSCC.
Assuntos
Movimento Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço , Fator de Transcrição STAT1 , Carcinoma de Células Escamosas de Cabeça e Pescoço , Proteases Específicas de Ubiquitina , Humanos , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Movimento Celular/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Linhagem Celular Tumoral , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Sobrevivência Celular/genética , Espectrometria de Massas em Tandem , Proliferação de Células , Cromatografia Líquida , Feminino , Masculino , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proteômica/métodosRESUMO
BACKGROUND: This study aimed to identify a candidate gene associated with paclitaxel (PTX) resistance and to evaluate functionally its biological role in the PTX-resistant head and neck squamous cell carcinoma (HNSCC) cell lines and clinical specimens. METHODS: Microarray data series containing samples of different types of cancers resistant to PTX were analyzed and then a candidate gene associated with PTX resistance was identified using various bioinformatics tools. After the suppression of the target gene expression, changes in cell viability and colony-forming ability were evaluated in PTX-resistant FaDu and SCC-9 cell lines. RESULTS: Bioinformatics analyses of upregulated genes in PTX-resistant cancer cells indicated that OAS3 was associated with PTX resistance. The downregulation of OAS3 expression significantly reduced the viability and colony-forming capacity of PTX-resistant SCC-9 cells by inducing apoptosis and cell cycle arrest at G0/G1 phase. CONCLUSIONS: The therapeutic targeting of OAS3 may resensitize PTX-resistant HNSCC cells with high OAS3 expression to PTX treatment.