RESUMO
While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, we show that the microRNA-(miR)203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. Reciprocally, loss of miR-203 function in placode, but not neural crest, cells perturbs trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses a miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.
RESUMO
Helicobacter pylori colonizes the stomach and induces an inflammatory response that can develop into gastric pathologies including cancer. The infection can alter the gastric vasculature by the deregulation of angiogenic factors and microRNAs. In this study, we investigate the expression level of pro-angiogenic genes (ANGPT2, ANGPT1, receptor TEK), and microRNAs (miR-135a, miR-200a, miR-203a) predicted to regulate those genes, using H. pylori co-cultures with gastric cancer cell lines. In vitro infections of different gastric cancer cell lines with H. pylori strains were performed, and the expression of ANGPT1, ANGPT2, and TEK genes, and miR-135a, miR-200a, and miR-203a, was quantified after 24 h of infection (h.p.i.). We performed a time course experiment of H. pylori 26695 infections in AGS cells at 6 different time points (3, 6, 12, 28, 24, and 36 h.p.i.). The angiogenic response induced by supernatants of non-infected and infected cells at 24 h.p.i. was evaluated in vivo, using the chicken chorioallantoic membrane (CAM) assay. In response to infection, ANGPT2 mRNA was upregulated at 24 h.p.i, and miR-203a was downregulated in AGS cells co-cultured with different H. pylori strains. The time course of H. pylori 26695 infection in AGS cells showed a gradual decrease of miR-203a expression concomitant with an increase of ANGPT2 mRNA and protein expression. Expression of ANGPT1 and TEK mRNA or protein could not be detected in any of the infected or non-infected cells. CAM assays showed that the supernatants of AGS-infected cells with 26695 strain induced a significantly higher angiogenic and inflammatory response. Our results suggest that H. pylori could contribute to the process of carcinogenesis by downregulating miR-203a, which further promotes angiogenesis in gastric mucosa by increasing ANGPT2 expression. Further investigation is needed to elucidate the underlying molecular mechanisms.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , MicroRNAs , Neoplasias Gástricas , Humanos , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/complicações , Helicobacter pylori/genética , MicroRNAs/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMO
PURPOSE: Altered miRNAs play a crucial role in the emergence of the breast cancer stem cell (BCSC) phenotype. The interplay between miRNAs and methylation enzymes has been documented. One of the most aggressive breast cancer cell lines, MDA-MB-231, has expressed much more DNMT3B than DNMT3A. This study aims to evaluate the ability of miR-203 restoration and miR-150 inhibition to regulate DNMT3B and DNMT3A to modify the methylation level of BCSC-associated genes. METHODS: MDA-MB-231 cells were transfected with miR-203 mimic or miR-150 inhibitor or DNMT3B siRNA, and downstream analysis was performed by flow cytometry, real-time PCR and Western blotting. RESULTS: DNMT3A and DNMT3B are regulated both by miR-203a-3p and miR-150-5p. Transfection with miR-203 mimic and miR-150 inhibitor significantly reduced the CD44+CD24- subpopulation and down-regulated the expression of CD44 mRNA by increasing promoter methylation levels. SiRNA knockdown of DNMT3B increased the CD44+CD24- subpopulation and the expression of CD44 and ALDH1A3 by decreasing methylation density. The inhibition of miR-150 down-regulated OCT3/4 and SOX2 expression without affecting methylation levels, while miR-203 restoration and miR-150 inhibition down-regulated NANOG expression by elevating the methylation level. A positive-feedback loop was found between miR-203 and its target DNMT3B, as restoring miR-203 suppressed DNMT3B, while knocking down DNMT3B up-regulated miR-203. The restoration of miR-203 and knockdown of DNMT3B decreased methylation levels and increased the expression of miR-141 and miR-200c. CONCLUSIONS: The study concluded that miR-203 and miR-150 play a role in the regulation of genes involved in BCSC methylation, including other miRNAs, by targeting DNMT3B and DNMT3A.
Assuntos
Neoplasias da Mama/genética , DNA (Citosina-5-)-Metiltransferases/fisiologia , DNA Metiltransferase 3A/fisiologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Feminino , Humanos , Metilação , Células-Tronco Neoplásicas , Células Tumorais Cultivadas , DNA Metiltransferase 3BRESUMO
miR-203 is a tumor-suppressor microRNA with known functions in cancer metastasis. Here, we explore its normal developmental role in the context of neural crest development. During the epithelial-to-mesenchymal transition of neural crest cells to emigrate from the neural tube, miR-203 displays a reciprocal expression pattern with key regulators of neural crest delamination, Phf12 and Snail2, and interacts with their 3'UTRs. We show that ectopic maintenance of miR-203 inhibits neural crest migration in chick, whereas its functional inhibition using a 'sponge' vector or morpholinos promotes premature neural crest delamination. Bisulfite sequencing further shows that epigenetic repression of miR-203 is mediated by the de novo DNA methyltransferase DNMT3B, the recruitment of which to regulatory regions on the miR-203 locus is directed by SNAIL2 in a negative-feedback loop. These findings reveal an important role for miR-203 in an epigenetic-microRNA regulatory network that influences the timing of neural crest delamination.
Assuntos
Crista Neural/citologia , Crista Neural/metabolismo , Fatores de Transcrição/metabolismo , Animais , Embrião de Galinha , Epigenômica , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hibridização In Situ , Fatores de Transcrição/genéticaRESUMO
BACKGROUND/AIM: Host molecules disturbed by human papillomavirus (HPV) oncoproteins have been shown to be potential biomarkers of cervical carcinogenesis and represent an alternative or supplementary aid to cytological testing and HPV detection. The miR-203 and one of its targets, ΔNp63, are known to be host molecules that interact with each other to control the proliferation and differentiation of keratinocytes; both have been found to be dysregulated in many cancers. As the role of p63 and miR-203 in cervical carcinogenesis is not yet well-understood, we have, thus, decided to evaluate the changes of expression of both in cervical carcinogenesis. MATERIALS AND METHODS: This study was carried out by obtaining quantitative polymerase chain reaction (qPCR) data from cervical biopsies. RESULTS: miR-203 and ΔNp63 displayed a similar expression pattern across cervical tissues and both targets showed statistically significant differences between low-grade squamous intraepithelial lesion (LSIL) x high-grade squamous intraepithelial lesion (HSIL); HSIL x Cancer. Additionally, we did not observe an inverse correlation between ΔNp63 mRNA and miR-203 levels as expected but, rather, a positive correlation between cervical tissues. CONCLUSION: Although preliminary, the expression levels of ΔNp63 mRNA and miR-203 seem to be promising for cervical cancer screening. In addition, positive correlation between miR-203 and ΔNp63 expression suggests the possible existence of some indirect pathways. However, further studies are needed to clarify the role of ΔNp63 and miR-203 in cervical carcinogenesis and, thus, determine how they can be applied in new strategies for diagnosis.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , Lesões Intraepiteliais Escamosas Cervicais/genética , Neoplasias do Colo do Útero/genética , Biomarcadores Tumorais/genética , Carcinogênese/genética , Detecção Precoce de Câncer , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , Gradação de Tumores , Papillomaviridae/patogenicidade , Lesões Intraepiteliais Escamosas Cervicais/patologia , Lesões Intraepiteliais Escamosas Cervicais/virologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
BACKGROUND AND AIM: Although miR-203 has been proposed as a relevant biomarker for several cancers, the validated prognostic significance of miR-203 in lung cancer remains obscure. Thus, we aimed to identify the relationship between miR-203 expression and clinicopathological significance in non-small cell lung cancer (NSCLC) patients in the current study. METHODS: The expression of miR-203 in 125 cases of NSCLC and their paired adjacent non-cancerous tissues was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Simultaneously, the correlation of miR-203 expression with a variety of clinicopathological factors and patient survival was analyzed. Functionally, in vitro effects of miR-203 on proliferation and viability were explored in lung cancer H460, A549, H1299, PC9 and H292 cells, as assessed by MTS tetrazolium assay and fluorimetric resorufin viability assay, respectively. RESULTS: The relative level of miR-203 was 6.12 ± 6.25 in NSCLC tissues, remarkably downregulated than that of their paired non-tumorous lung tissues (7.88 ± 5.56, P = 0.019). The area under curve (AUC) of low expression of miR-203 to diagnose NSCLC was 0.622 (95 % CI 0.552-0.692, P = 0.001). MiR-203 expression was negatively correlated to lymphatic metastasis (r = -0.334, P < 0.001), tumor size (r = -0.407, P < 0.001) and clinical TNM stages (r = -0.298, P = 0.001). Furthermore, the survival of the low miR-203 expression group was 4.88 ± 4.38 months, markedly shorter than that of the high expression group (23.35 ± 1.12 months, P < 0.001). The level of miR-203 was an independent prognostic indicator of NSCLC using univariate analysis. MiR-203 mimic could suppress the cell growth of five lung cancer cell lines tested to different degrees in vitro. CONCLUSIONS: MiR-203 could become a prognostic predictor in NSCLC and may be a new target for the molecular therapy of NSCLC patients.