RESUMO
The incidence of allergic rhinitis (AR) is increasing worldwide. Human nasal epithelial cells (HNECs) are the key cells in the occurrence of AR. Antisense non-coding RNA in the INK4 locus (ANRIL) was discovered to be involved in the progression of AR. However, the mechanism by which ANRIL mediates the progression of AR remains to be determined. The present study aimed to further explore the mechanism by which ANRIL regulates AR. Thereby, HNECs were treated with IL-13 to mimic AR in vitro. The mRNA expression levels of ANRIL, microRNA (miR)-15a-5p, JAK2, mucin 5AC (MUC5AC), granulocyte-macrophage colony-stimulating factor (GM-CSF) and eotaxin-1, and protein expression levels of JAK2, STAT3 and phosphorylated-STAT3 in HNECs were analyzed using reverse transcription-quantitative PCR and western blotting, respectively. ELISAs were used to detect the secretory levels of inflammatory cytokines and mucin in cell supernatants. In addition, a dual luciferase reporter assay was used to confirm the downstream target of ANRIL and the target gene of miR-15a-5p. The results revealed that the secretory levels of eotaxin-1, GM-CSF and MUC5AC were significantly upregulated by IL-13 in the supernatant of HNECs. The expression levels of ANRIL and JAK2 were also upregulated in IL-13-induced HNECs, while the expression levels of miR-15a-5p were downregulated. In addition, ANRIL was identified to bind to miR-15a-5p. The IL-13-induced upregulation of eotaxin-1, GM-CSF and MUC5AC mRNA expression and secretory levels was significantly inhibited by the genetic knockdown of ANRIL, while the miR-15a-5p inhibitor effectively reversed this effect. JAK2 was also discovered to be directly targeted by miR-15a-5p. The overexpression of JAK2 significantly suppressed the therapeutic effect of miR-15a-5p mimics on IL-13-induced inflammation in vitro. In conclusion, the findings of the present study suggested that the genetic knockdown of ANRIL may suppress the production of inflammatory cytokines and mucin in IL-13-treated HNECs via regulation of the miR-15a-5p/JAK2 axis. Thus, ANRIL may serve as a novel target for AR treatment.
Assuntos
Citocinas/genética , Células Epiteliais/metabolismo , Janus Quinase 2/metabolismo , MicroRNAs/metabolismo , Mucina-5AC/genética , RNA Longo não Codificante/metabolismo , Rinite Alérgica/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Inflamação , RNA Longo não Codificante/genética , Rinite Alérgica/genética , Transdução de SinaisRESUMO
Nasopharyngeal carcinoma (NPC) is an epithelial cancer emerging from the lining of nasopharyngeal mucosa, with extremely frequent occurrence in east and southeast Asia. For the purpose of exploring roles of the dysregulated long non-coding RNA (lncRNA) in NPC, we identified a novel lncRNA LINC00669 with an apparent negative correlation to the overall survival from human NPC mRNA expression profiling databases. We further performed RNA pulldown coupled with mass spectrum to find out its target protein, and applied a series of in vitro and in vivo loss-and-gain-of function assays to investigate its oncogenic roles in NPC tumor development and progression. Our results demonstrated that LINC00669 competitively binds to the key JAK/STAT signaling pathway suppressor SOCS1, and insulates it from imposing ubiquitination modification on the pathway component of STAT1, which leads to its abnormal stabilization and activation. The activated STAT1 is then transferred into the nucleus and initiates the transcription of genes related to proliferation and invasion. In summary, our study reveals that the cytoplasmic resident lncRNA LINC00669 confers malignant properties on NPC cancer cells by facilitating a persistent activation of the JAK/STAT signaling pathway. Findings in the current study shed lights on prospects for treating NPC using strategies targeting the novel regulator of the JAK/STAT signaling.
Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Janus Quinase 1/metabolismo , Neoplasias Nasofaríngeas/patologia , RNA Longo não Codificante/genética , Fator de Transcrição STAT1/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Humanos , Janus Quinase 1/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Invasividade Neoplásica , Prognóstico , Fator de Transcrição STAT1/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To observe the expression of human beta-defensin-1 (hBD-1) and human beta-defensin-2 (hBD-2) in recurrent nasal polyps, and to investigate the role of beta-defensin in the recurrent nasal polyps. METHODS: Tissues of nasal polyps was obtained from 10 patients with nasal polyps undergoing endoscopic sinus surgery, recurrent nasal polyps from 10 patients 6 months after surgery, nasal mucosa from 10 recovered patients with nasal polyps postoperatively and,10 control subjects. hBD-1 mRNA and hBD-2 mRNA levels of tissue specimens in all groups were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: There was no significant difference in hBD-1 mRNA level between the 4 groups (P>0.05). Expression of hBD-2 mRNA was detected in patients with nasal polyps and recurrent nasal polyps, but rare in the recovered patients and the control subjects. CONCLUSION: hBD-1 is a constitutive expression and hBD-2 is an induced expression. beta-Defensin may play an important role in forming the nasal polyps.
Assuntos
Mucosa Nasal/metabolismo , Pólipos Nasais/metabolismo , beta-Defensinas/metabolismo , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem , beta-Defensinas/genéticaRESUMO
OBJECTIVE: To establish 2-dimensional polyacrylamide gel electrophoresis (2-DE) map from human nasal polyps and normal nasal mucosa, and to identify differential expression proteins of 2-DE map. METHODS: Samples of nasal polyps and nasal mucosa (each sample group containing 7 cases) were obtained. The total proteins were extracted and separated by immobilized pH gradient (IPG)-based 2-DE. The silver-stained 2-DE was scanned with digital Imagescanner and analyzed with ImageMaster 2-DE Elite 4.01 software. To obtain peptide mass fingerprint (PMF) of differential protein spots, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used. The PMF was searched in Swiss-Prot and TreMBL database by Pept-Ident software, to identify differential expression proteins. RESULTS: The well-resolved, reproducible 2-DE maps of nasal polyps and nasal mucosa were established. For the polyps tissues, the average proteins spot of three 2-DE maps was 825+/-78; and 682+/-96 spot was matched with the average matching rate of 82.7%. The average deviations of matched spot position were (1.13+/-0.16) mm in IEF direction and (1.45+/-0.21) mm in SDS-PAGE direction, respectively. For the nasal mucosa tissues, the average proteins spot of three 2-DE maps was 936+/-62; and 821+/-78 spots were matched with the average matching rate of 87.7%. After comparing the 2-DE maps of nasal polyps and nasal mucosa tissues, the protein spots were 1,458 and 1,617 respectively; and 1,026 protein spots were matched. Forty differential expression protein spots were incised from silver staining gel randomly and digested in the gel by TPCK-Trypsin. Thirty-four PMFs were obtained by MALDI-TOF-MS and 24 differential proteins were identified. CONCLUSION: The well-resolved, reproducible 2-DE maps of human nasal polyps and nasal mucosa have been successfully established. Certain differential proteins related to the pathogenesis of human nasal polyps are identified.
