RESUMO
Nickel pollution is a recognized factor contributing to lung cancer. Understanding the molecular mechanisms of its carcinogenic effects is crucial for lung cancer prevention and treatment. Our previous research identified the downregulation of a long noncoding RNA, maternally expressed gene 3 (MEG3), as a key factor in transforming human bronchial epithelial cells (HBECs) into malignant cells following nickel exposure. In our study, we found that deletion of MEG3 also reduced the expression of RhoGDIß. Notably, artificially increasing RhoGDIß levels counteracted the malignant transformation caused by MEG3 deletion in HBECs. This indicates that the reduction in RhoGDIß contributes to the transformation of HBECs due to MEG3 deletion. Further exploration revealed that MEG3 downregulation led to enhanced c-Jun activity, which in turn promoted miR-200c transcription. High levels of miR-200c subsequently increased the translation of AUF1 protein, stabilizing SOX2 messenger RNA (mRNA). This stabilization affected the regulation of miR-137, SP-1 protein translation, and the suppression of RhoGDIß mRNA transcription and protein expression, leading to cell transformation. Our study underscores the co-regulation of RhoGDIß expression by long noncoding RNA MEG3, multiple microRNAs (miR-200c and miR-137), and RNA-regulated transcription factors (c-Jun, SOX2, and SP1). This intricate network of molecular events sheds light on the nature of lung tumorigenesis. These novel findings pave the way for developing targeted strategies for the prevention and treatment of human lung cancer based on the MEG3/RhoGDIß pathway.
Assuntos
Neoplasias Pulmonares , MicroRNAs , RNA Longo não Codificante , Humanos , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Regulação para Baixo , Células Epiteliais/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Níquel , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/antagonistas & inibidores , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/genética , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro , Fatores de Transcrição SOXB1/genética , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismoRESUMO
BACKGROUND: Increasing evidences has indicated that primary and acquired resistance of ovarian cancer (OC) to platinum is mediated by multiple molecular and cellular factors. Understanding these mechanisms could promote the therapeutic efficiency for patients with OC. METHODS: Here, we screened the expression pattern of circRNAs in samples derived from platinum-resistant and platinum-sensitive OC patients using RNA-sequencing (RNA-seq). The expression of hsa_circ_0010467 was validated by Sanger sequencing, RT-qPCR, and fluorescence in situ hybridization (FISH) assays. Overexpression and knockdown experiments were performed to explore the function of hsa_circ_0010467. The effects of hsa_circ_0010467 on enhancing platinum treatment were validated in OC cells, mouse model and patient-derived organoid (PDO). RNA pull-down, RNA immunoprecipitation (RIP), and dual-luciferase reporter assays were performed to investigate the interaction between hsa_circ_0010467 and proteins. RESULTS: Increased expression of hsa_circ_0010467 is observed in platinum-resistant OC cells, tissues and serum exosomes, which is positively correlated with advanced tumor stage and poor prognosis of OC patients. Hsa_circ_0010467 is found to maintain the platinum resistance via inducing tumor cell stemness, and silencing hsa_circ_0010467 substantially increases the efficacy of platinum treatment on inhibiting OC cell proliferation. Further investigation reveals that hsa_circ_0010467 acts as a miR-637 sponge to mediate the repressive effect of miR-637 on leukemia inhibitory factor (LIF) and activates the LIF/STAT3 signaling pathway. We further discover that AUF1 could promote the biogenesis of hsa_circ_0010467 in OC. CONCLUSION: Our study uncovers the mechanism that hsa_circ_0010467 mediates the platinum resistance of OC through AUF1/hsa_circ_0010467/miR-637/LIF/STAT3 axis, and provides potential targets for the treatment of platinum-resistant OC patients.
Assuntos
Ribonucleoproteína Nuclear Heterogênea D0 , MicroRNAs , Neoplasias Ovarianas , RNA Circular , Animais , Feminino , Humanos , Camundongos , Hibridização in Situ Fluorescente , Fator Inibidor de Leucemia , MicroRNAs/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , RNA Circular/genética , Fator de Transcrição STAT3/genética , Ribonucleoproteína Nuclear Heterogênea D0/genéticaRESUMO
Mammalian target of rapamycin (mTOR), which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA-binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. We determined that AUF1 is required for phosphorylation of Akt at Thr308, Thr450, and Ser473 and that AUF1 also mediates phosphorylation of the mTORC2-modulated metabolic enzyme glutamine fructose-6-phosphate amidotransferase 1 at Ser243. In addition, AUF1 immunoprecipitation followed by quantitative RT-PCR revealed that the mRNAs of Akt, glutamine fructose-6-phosphate amidotransferase 1, and the mTORC2 component SIN1 associate with AUF1. Furthermore, expression of the p40 and p45, but not the p37 or p42, isoforms of AUF1 specifically mediate Akt phosphorylation. In the absence of AUF1, subcellular fractionation indicated that Akt fails to localize to the membrane. However, ectopic expression of a membrane-targeted allele of Akt is sufficient to allow Akt-Ser473 phosphorylation despite AUF1 depletion. Finally, conditions that enhance mTORC2 signaling, such as acute glutamine withdrawal, augment AUF1 phosphorylation, whereas mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in promoting membrane localization of Akt to facilitate its phosphorylation on this cellular compartment. Targeting AUF1 could have therapeutic benefit for cancers with upregulated mTORC2/Akt signaling.
Assuntos
Ribonucleoproteína Nuclear Heterogênea D0 , Proteínas Proto-Oncogênicas c-akt , Proteínas de Ligação a RNA , Proliferação de Células , Glutamina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Humanos , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Membrana Celular/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismoRESUMO
Heterogeneous Ribonucleoprotein D (hnRNPD) is an RNA binding protein involved in post-transcriptional regulation of multiple mediators of carcinogenesis. We previously demonstrated a strong association of hnRNPD over expression with poor outcome in Oral Squamous Cell Carcinoma (OSCC). However, hitherto the precise molecular mechanism of its overexpression in oral cancer was not clear. Therefore, in an attempt to elucidate the transcriptional regulation of hnRNPD expression, we cloned 1406 bp of 5' flanking region of human hnRNPD gene along with 257 bp of its first exon upstream to promoterless luciferase reporter gene in pGL3-Basic. Transfection of the resulting construct in SCC-4 cells yielded 1271 fold higher luciferase activity over parent vector. By promoter deletion analysis, we identified a canonical TATA box containing 126 bp core promoter region that retained ~ 58% activity of the full length promoter. In silico analysis revealed the presence of four putative NFκB binding motifs in the promoter. Sequential deletion of these motifs from the full-length promoter reporter construct coupled with luciferase assays revealed an 82% decrease in promoter activity after deletion of the first (-1358/-1347) motif and 99% reduction after the deletion of second motif (-1052/-1041). In-vivo binding of NFκB (RelA) to these two motifs in SCC-4 cells was confirmed by ChIP assays. Site directed mutagenesis of even one of these two motifs completely abolished promoter activity, while mutagenesis of the remaining two motifs had marginal effect on the same. Consistent with these findings, treatment of SCC-4 cells with PDTC, a known inhibitor of NFκB dramatically reduced the levels hnRNPD mRNA and protein. Finally, the expression of hnRNPD and NFκB in clinical specimen from 37 oral cancer patients was assessed and subjected to Spearmen's Correlation analysis which revealed a strong positive correlation between the two. Thus, results of the present study for the first time convincingly demonstrate NFκB (RelA) mediated transcriptional upregulation of hnRNPD expression in oral cancer.
Assuntos
Carcinoma de Células Escamosas , Ribonucleoproteína Nuclear Heterogênea D0 , Neoplasias Bucais , Fator de Transcrição RelA , Sequência de Bases , Carcinoma de Células Escamosas/genética , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Luciferases , Neoplasias Bucais/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Ativação TranscricionalRESUMO
Active breast cancer-associated fibroblasts (CAFs), the most influential cells in breast tumor microenvironment, express/secrete high levels of the proinvasive/metastatic interleukin-6 (IL-6). Therefore, we have tested here the effect of the IL-6 receptor (IL-6R) inhibitor tocilizumab (TCZ; Actemra) on different active breast CAFs. We have shown that TCZ potently and persistently suppresses the expression of various CAF biomarkers, namely α-SMA, SDF-1 as well as the STAT3 pathway and its downstream target AUF1. TCZ also inhibited the proliferation, migration and invasion abilities of active breast CAF cells. Additionally, TCZ repressed the ability of CAF cells in promoting epithelial-to-mesenchymal transition, and enhancing the migratory/invasive and proliferative capacities of breast cancer cells in vitro. Importantly, these findings were confirmed in orthotopic humanized breast tumors in mice. Furthermore, TCZ suppressed the expression of the pro-angiogenic factor VEGF-A and its transactivator HIF-1α in CAF cells, and consequently inhibited the angiogenic-promoting effect of active CAFs both in vitro and in orthotopic tumor xenografts. These results indicate that inhibition of the IL-6/STAT3/AUF1 pathway by TCZ can normalize active breast CAFs and suppress their paracrine pro-carcinogenic effects, which paves the way toward development of specific CAF-targeting therapy, badly needed for more efficient breast cancer treatments.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Interleucina-6/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Long non-coding RNAs (lncRNAs) have emerged as critical regulators in human disease including atherosclerosis. However, the mechanisms involved in the post-transcriptional regulation of the expression of disease-associated lncRNAs are not fully understood. Gene expression studies revealed that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) lncRNA expression was increased by >2-fold in peripheral blood mononuclear cells (PBMCs) derived from patients with coronary artery disease (CAD) or in carotid artery atherosclerotic plaques. We observed a linear association between NEAT1 lncRNA expression and prevalence of CAD which was independent of age, sex, cardiovascular traditional risk factors and renal function. NEAT1 expression was induced by TNF-α, while silencing of NEAT1 profoundly attenuated the TNF-α-induced vascular endothelial cell pro-inflammatory response as defined by the expression of CXCL8, CCL2, VCAM1 and ICAM1. Overexpression of the RNA editing enzyme adenosine deaminase acting on RNA-1 (ADAR1), but not of its editing-deficient mutant, upregulated NEAT1 levels. Conversely, silencing of ADAR1 suppressed the basal levels and the TNF-α-induced increase of NEAT1. NEAT1 lncRNA expression was strongly associated with ADAR1 in CAD and peripheral arterial vascular disease. RNA editing mapping studies revealed the presence of several inosines in close proximity to AU-rich elements within the AluSx3+/AluJo- double-stranded RNA complex. Silencing of the stabilizing RNA-binding protein AUF1 reduced NEAT1 levels while silencing of ADAR1 profoundly affected the binding capacity of AUF1 to NEAT1. Together, our findings propose a mechanism by which ADAR1-catalyzed A-to-I RNA editing controls NEAT1 lncRNA stability in ASCVD.
Assuntos
Adenosina/metabolismo , Elementos Alu/genética , Aterosclerose/sangue , Doença da Artéria Coronariana/sangue , Inosina/metabolismo , Placa Aterosclerótica/sangue , Edição de RNA/genética , Estabilidade de RNA/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aterosclerose/genética , Sítios de Ligação , Células Cultivadas , Estudos de Coortes , Doença da Artéria Coronariana/genética , Feminino , Inativação Gênica , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/genética , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , TransfecçãoRESUMO
SChLAP1 is recently reported as a key oncogenic long non-coding RNA in human cancer. However, whether SChLAP1 functions in non-small cell lung cancer (NSCLC) and its specific potential regulatory mechanism remain unexplored. In this study, we found that depletion of SChLAP1 significantly inhibited NSCLC cell proliferation, migration and invasion in vitro, and retarded tumour growth and lung metastasis in vivo. SChLAP1 facilitated NSCLC cell immune evasion against CD8+ T cells through PD-1/PD-L1 immune checkpoint. In detail, SChLAP1 was able to directly interact with AUF1, antagonizing the binding between AUF1 and PDL1 mRNA 3'-UTR, resulting in increasing PDL1 mRNA stability and expression, thereby repressing CD8+ T cell function. Consistently, anti-PD-1/PD-L1 treatment evidently blocked the enhanced cell proliferation and invasion caused by SChLAP1 overexpression. Importantly, SChLAP1 was significantly upregulated in NSCLC cell lines, serum and tissues, which was identified as an excellent indicator for the diagnosis and prognosis of NSCLC. In conclusion, our data for the first time uncover that SChLAP1 functions an oncogene in NSCLC by promoting cancer cell immune evasion via regulating the AUF1/PDL1 axis, targeting of SChLAP1 may be a potential approach to improve the efficacy of immunotherapy in NSCLC patients.
Assuntos
Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas , Ribonucleoproteína Nuclear Heterogênea D0/genética , Neoplasias Pulmonares , RNA Longo não Codificante , Antígeno B7-H1/genética , Linfócitos T CD8-Positivos , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Evasão da Resposta Imune , Neoplasias Pulmonares/patologia , RNA Longo não Codificante/genéticaRESUMO
Long noncoding RNAs (lncRNAs) play an essential role in tumor progression. Few researches focused on the clinical and biological relevance of lncRNAs in peripheral T cell lymphoma (PTCL). In this research, a novel lncRNA (ENST00000503502) was identified overexpressed in the main subtypes of PTCL, and designated as T cell lymphoma-associated lncRNA1 (TCLlnc1). Serum TCLlnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLlnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLlnc1 using small interfering RNAs. As the mechanism of action, TCLlnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLlnc1/HNRNPD/YBX1 complex upregulated transcription of TGFB2 and TGFBR1 genes, activated the tumor growth factor-ß signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.
Assuntos
Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Linfoma de Células T Periférico/metabolismo , RNA Longo não Codificante/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Feminino , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Células Jurkat , Linfoma de Células T Periférico/sangue , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , RNA Longo não Codificante/sangue , RNA Longo não Codificante/genética , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
Studies have found that RNA-binding proteins (RBPs) are dysfunctional and play a significant regulatory role in the development of glioma. Based on The Cancer Genome Atlas database and the previous studies, we selected heterogeneous nuclear ribonucleoprotein (HNRNPD) as the research candidate and sought its downstream targeted genes. In the present study, HNRNPD, linc00707, and specific protein 2 (SP2) were highly expressed, while zinc fingers and homeboxes 2 (ZHX2) and miR-651-3p were remarkedly downregulated in glioma tissues and cells. HNRNPD, linc00707, and SP2 knockdown or ZHX2 and miR-651-3p overexpression suppressed glioma cells proliferation, migration, and invasion and vasculogenic mimicry (VM) formation. Knockdown of HNRNPD increased the stability of ZHX2 mRNA. ZHX2 bound to the promoter region of linc00707 and negatively regulate its expression. Linc00707 could bind with miR-651-3p, while miR-651-3p bound to the 3' untranslated region (3'UTR) of SP2 mRNA to negatively regulate its expression. The transcription factor SP2 directly bound to the promoter regions of the VM formation-related proteins MMP2, MMP9, and VE-cadherin, playing a role in promoting transcription in order to regulate the VM formation ability of glioma cells.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Mimetismo Molecular , Neovascularização Patológica , RNA Longo não Codificante/metabolismo , Fator de Transcrição Sp2/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/patologia , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea D0/genética , Proteínas de Homeodomínio/genética , Humanos , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica , RNA Longo não Codificante/genética , Transdução de Sinais , Fator de Transcrição Sp2/genética , Fatores de Transcrição/genéticaRESUMO
Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.
Assuntos
Ribonucleoproteína Nuclear Heterogênea D0/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNARESUMO
BACKGROUND: AUF1 is one of the AU-rich binding proteins, which promotes rapid ARE-mRNA degradation. Recently, it has been reported that AUF1 is involved in regulating the antioxidant system because of its capacity to bind specifically to RNA containing oxidized bases and degrade oxidized RNA. Many antioxidant proteins have been reported to be overexpressed in colorectal cancer (CRC), however, the role of AUF1 in the progression of CRC has not been explored. METHODS: The expression level of AUF1 protein in human CRC cell lines and CRC tissues was detected by western blotting and immunohistochemistry (IHC. The effects of AUF1 knockdown on CRC cell proliferation, migration, invasion and changes in the signaling pathways were evaluated using a cell counting kit-8 (CCK-8), Transwell assays and western blotting. Subcutaneous xenograft tumor model was employed to further substantiate the role of AUF1 in CRC. RESULTS: AUF1 protein was upregulated in CRC tissues and CRC cells, and high expression of AUF1 was significantly associated with advanced AJCC stage (P = .001), lymph node metastasis (P = .007), distant metastasis (P = .038) and differentiation (P = .009) of CRC specimens. CRC patients with the high expression of AUF1 had an extremely poor prognosis. The knockdown of AUF1 suppressed CRC cell line proliferation, migration and invasion, inhibited CRC cells tumorigenesis and growth in nude mice, and reduced phosphorylated-ERK1/2 and phosphorylated AKT in CRC cells. CONCLUSION: Our findings demonstrate that AUF1 is probably involved in the progression of CRC via the activation of the ERK1/2 and AKT pathways. AU-rich RNA-binding factor 1 could be used as a novel prognostic biomarker and a potential therapeutic target for CRC.
Assuntos
Neoplasias Colorretais/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Idoso , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Progressão da Doença , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Fosforilação , Transdução de Sinais , Carga Tumoral , Regulação para CimaRESUMO
Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.
Assuntos
Proteínas Argonautas/genética , RNA Helicases DEAD-box/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Neoplasias/genética , Ribonuclease III/genética , Regiões 3' não Traduzidas/genética , Células A549 , Proteínas Argonautas/metabolismo , Sítios de Ligação/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismoRESUMO
BACKGROUND: Although trastuzumab provides significant clinical benefit for HER2-positive breast cancers, responses are limited by the emergence of resistance. Recent evidence suggests that long noncoding RNAs (lncRNAs) play important roles in tumorigenesis and chemoresistance. However, the regulatory mechanism of lncRNAs in trastuzumab resistance is not well established to date. In this research, we identified the differentially expressed lncRNA and investigated its regulatory role in trastuzumab resistance of breast cancer. METHODS: LncRNA microarray and qRT-PCR were performed to identify the dysregulated lncRNAs. Transmission electron microscopy, differential ultracentrifugation and qRT-PCR were used to verify the existence of exosomal AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1). Bioinformatics prediction, RNA fluorescence in situ hybridization (RNA-FISH) and immunoprecipitation assays were performed to identify the direct interactions between AFAP1-AS1 and other associated targets, such as AU-binding factor 1 (AUF1) and ERBB2. Finally, a series gain- or loss-functional assays were done to prove the precise role of AFAP1-AS1 in trastuzumab resistance. RESULTS: AFAP1-AS1 was screened out due to its higher expression in trastuzumab-resistant cells compared to sensitive cells. Increased expression of AFAP1-AS1was associate with poorer response and shorter survival time of breast cancer patients. AFAP1-AS1 was upregulated by H3K27ac modification at promoter region, and knockdown of AFAP1-AS1 reversed trastuzumab resistance. Moreover, extracellular AFAP1-AS1 secreted from trastuzumab resistant cells was packaged into exosomes and then disseminated trastuzumab resistance of receipt cells. Mechanically, AFAP1-AS1 was associated with AUF1 protein, which further promoted the translation of ERBB2 without influencing the mRNA level. CONCLUSION: Exosomal AFAP1-AS1 could induce trastuzumab resistance through associating with AUF1 and promoting ERBB2 translation. Therefore, AFAP1-AS1 level may be useful for prediction of trastuzumab resistance and breast cancer treatment.
Assuntos
Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos , Exossomos/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , RNA Longo não Codificante/genética , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologia , Animais , Antineoplásicos Imunológicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proliferação de Células , Exossomos/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Biossíntese de Proteínas , Receptor ErbB-2/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Enterovirus A71 (EV-A711) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1-4) from the EV-A71 5'UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.
Assuntos
Bioensaio/métodos , Enterovirus Humano A/genética , Regulação Viral da Expressão Gênica , Pequeno RNA não Traduzido/análise , Regiões 5' não Traduzidas/genética , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , RNA Helicases DEAD-box/metabolismo , Técnicas de Silenciamento de Genes , Ribonucleoproteína Nuclear Heterogênea D0/análise , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal/genética , Biossíntese de Proteínas/genética , Pequeno RNA não Traduzido/metabolismo , Ribonuclease III/metabolismo , Células VeroRESUMO
BACKGROUND: The CDKN2A/B locus contains crucial tumor suppressors and a lncRNA gene ANRIL. However, the mechanisms that coordinately regulate their expression levels are not clear. METHODS: Novel RNAs transcribed from the CDKN2A gene were screened by CDKN2A-specific RNA capture deep-sequencing and confirmed by Northern blotting and clone-sequencing. Long non-coding RNA (lncRNA) binding proteins were characterized by RNA pull-down combined with mass spectrometry and RNA immunoprecipitation. LncRNA functions in human cells were studied using a set of biological assays in vitro and in vivo. RESULTS: We characterized a novel lncRNA, P14AS with its promoter in the antisense strand of the fragment near CDKN2A exon 1b in human cells. The mature P14AS is a three-exon linear cytoplasmic lncRNA (1043-nt), including an AU-rich element (ARE) in exon 1. P14AS decreases AUF1-ANRIL/P16 RNA interaction and then increases ANRIL/P16 expression by competitively binding to AUF1 P37 and P40 isoforms. Interestingly, P14AS significantly promoted the proliferation of cancer cells and tumor formation in NOD-SCID mice in a P16-independent pattern. Moreover, in human colon cancer tissues, the expression levels of P14AS and ANRIL lncRNAs were significantly upregulated compared with the paired normal tissues. CONCLUSION: A novel lncRNA, P14AS, transcribed from the antisense strand of the CDKN2A/P14 gene, promotes colon cancer development by cis upregulating the expression of oncogenic ANRIL.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , RNA Longo não Codificante/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Feminino , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 3'-terminal stem-loop (3'SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3'SL was proposed to act as a replication silencer. The lower part of the 3'SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3'SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3'SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3'SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3'SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3'SL acts as an RNA thermometer that modulates flavivirus replication during host switching.
Assuntos
Regiões 3' não Traduzidas , Interações entre Hospedeiro e Microrganismos/genética , Sequências Repetidas Invertidas , RNA Viral/genética , Replicação Viral/genética , Vírus do Nilo Ocidental/genética , Animais , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Culicidae/citologia , Culicidae/genética , Culicidae/virologia , Genoma Viral , Ribonucleoproteína Nuclear Heterogênea D0/genética , Humanos , Proteínas de Insetos/genética , Mutação , Conformação de Ácido Nucleico , Proteínas de Ligação a RNA/genética , Vírus do Nilo Ocidental/fisiologiaRESUMO
BACKGROUND: Recent studies have shown that gastrokine 1 (GKN1), an important tumor suppressor gene, is downregulated in Helicobacter pylori (H. pylori) infected gastric mucosa and gastric cancer. However, the underlying mechanism is poorly understood. Herein, we investigated the potential mechanism of H. pylori-induced GKN1 downregulation. MATERIALS AND METHODS: GKN1 and AU-rich element RNA-binding factor 1 (AUF1) expressions were assessed by quantitative real-time PCR, Western blot, or immunohistochemistry in H. pylori-infected tissues and H. pylori co-cultured cell lines. The regulation of AUF1 on GKN1 was determined by RNA pulldown assay, RNA immunoprecipitation, mRNA turnover, and luciferase activity assays. The involvement of phosphorylated extra-cellular signal-regulated kinase (p-ERK) or CagA in H. pylori-induced AUF1 expression was verified using p-ERK inhibitor or CagA knockout H. pylori. In addition, the cell proliferation and migration capacities of AUF1-knockdown cells were investigated. RESULTS: GKN1 expression progressively decreased from H. pylori-infected gastritis to gastric cancer tissues. H. pylori co-culture also induced significant GKN1 reduction in GES-1 and BGC-823 cells. Besides, the mRNA level of GKN1 and AUF1 in human gastric mucosa showed negative correlation significantly. AUF1 knockdown resulted in upregulation of GKN1 expression and promoted GKN1 mRNA decay by binding the 3' untranslated region of GKN1 mRNA H. pylori-induced AUF1 expression was associated with p-ERK activation and CagA. Furthermore, knockdown of AUF1 significantly inhibited cell viability, migration ability, and arrested fewer cells in S-phase. CONCLUSION: Our data demonstrated that H. pylori infection downregulated GKN1 expression via the CagA/p-ERK/AUF1 pathway. AUF1 promoted gastric cancer at least partly through downregulating GKN1, which presented a novel potential target for the treatment of gastric cancer.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/metabolismo , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Hormônios Peptídicos/metabolismo , Neoplasias Gástricas/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Ribonucleoproteína Nuclear Heterogênea D0/genética , Interações Hospedeiro-Patógeno , Humanos , Hormônios Peptídicos/genética , Fosforilação , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologiaRESUMO
Alternative splicing (AS) is critically associated with tumorigenesis and patient's prognosis. Here, we systematically analyzed survival-associated AS signatures in oral squamous cell carcinoma (OSCC) and evaluated their prognostic predictive values. Survival-related AS events were identified by univariate and multivariate Cox regression analyses using OSCC data from the TCGA head neck squamous cell carcinoma data set. The Percent Spliced In calculated by SpliceSeq from 0 to 1 was used to quantify seven types of AS events. A predictive model based on AS events was constructed by least absolute shrinkage and selection operator Cox regression assay and further validated using a training-testing cohort design. Patient survival was estimated using the Kaplan-Meier method and compared with Log-rank test. The receiver operating characteristics curve area under the curves was used to evaluate the predictive abilities of these predictive models. Furthermore, gene-gene interaction networks and the splicing factors (SFs)-AS regulatory network was generated by Cytoscape. A total of 825 survival-related AS events within 719 genes were identified in OSCC samples. The integrative predictive model was better at predicting outcomes of patients as compared to those models built with the individual AS event. The predictive model based on three AS-related genes also effectively predicted patients' survival. Moreover, seven survival-related SFs were detected in OSCC including RBM4, HNRNPD, and HNRNPC, which have been linked to tumorigenesis. The SF-AS network revealed a significant correlation between survival-related AS genes and these SFs. Our findings revealed a systemic portrait of survival-associated AS events and the splicing network in OSCC, suggesting that AS events might serve as novel prognostic biomarkers and therapeutic targets for OSCC.