RESUMO
Gallbladder carcinoma (GBC), the most common malignant tumour of the bile duct, is highly aggressive and has a poor prognosis. MicroRNA-30a-5p (miR-30a-5p) is an important tumour suppressor that participates in many aspects of carcinogenesis and cancer development. However, the role of miR-30a-5p in GBC development remains to be determined, as do the mechanisms underlying its effects in GBC. Using samples collected from 42 subjects with gallbladder carcinoma (GBC), we showed decreased miR-30a-5p expression in the primary lesions vs. non-tumour adjacent tissues (NATs). Decreased miR-30a-5p was associated with shorter disease-free survival (DFS) and overall survival (OS). Inhibiting miR-30a-5p expression in 2 representative GBC cell lines (GBC-SD and NOZ) increased cell proliferation, migration, invasiveness, as well as ß-catenin nuclear translocation, vice versa. In nude mice, NOZ cells transfected with miR-30a-5p mimics grew slower (vs. miR-NC) upon subcutaneous inoculation, and had lower rate of hepatic metastasis upon spleen inoculation. Dual luciferase assay confirmed that E2F transcription factor 7 (E2F7) was a direct target of miR-30a-5p and antagonized the effects induced by miR-30a-5p downregulation in GBC cells. MiR-30a-5p attenuates the EMT and metastasis in GBC cells by targeting E2F7, suggesting miR-30a-5p is a tumour suppressor that may serve as a novel potential prognostic biomarker or molecular therapeutic target for GBC.
Assuntos
Fator de Transcrição E2F7/genética , Neoplasias da Vesícula Biliar/genética , MicroRNAs/metabolismo , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Fator de Transcrição E2F7/metabolismo , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Neoplasias da Vesícula Biliar/fisiopatologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Pessoa de Meia-Idade , Metástase NeoplásicaRESUMO
Gallbladder cancer (GBC) is the most common malignant tumour of the biliary track system. Angiogenesis plays a pivotal role in the development and progression of malignant tumours. miR-143-3p acts as a tumour suppressor in various cancers. Their role in GBC is however less well defined. Here we show that the expression levels of miR-143-3p were decreased in human GBC tissues compared with the non-tumour adjacent tissue (NAT) counterparts and were closely associated with overall survival. We discovered that miR-143-3p was a novel inhibitor of tumour growth and angiogenesis in vivo and in vitro. Our antibody array, ELISA and PLGF rescue analyses indicated that PLGF played an essential role in the antiangiogenic effect of miR-143-3p. Furthermore, we used miRNA target-prediction software and dual-luciferase assays to confirm that integrin α6 (ITGA6) acted as a direct target of miR-143-3p. Our ELISA and western blot analyses confirmed that the expression of PLGF was decreased via the ITGA6/PI3K/AKT pathway. In conclusion, miR-143-3p suppresses tumour angiogenesis and growth of GBC through the ITGA6/PI3K/AKT/PLGF pathways and may be a novel molecular therapeutic target for GBC.
Assuntos
Neoplasias da Vesícula Biliar/genética , Integrina alfa6/metabolismo , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Placentário/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Regulação para Baixo , Neoplasias da Vesícula Biliar/irrigação sanguínea , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/patologia , Xenoenxertos , Humanos , Integrina alfa6/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Fator de Crescimento Placentário/genética , TransfecçãoRESUMO
The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Sítios de Ligação , Éxons , Células HeLa , Humanos , Fatores de Processamento de RNA , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
OBJECTIVE: To study the effect of thioridazine on the proliferation and apoptosis of human colorectal cancer SW480 cells. METHODS: SW480 cells were treated with different concentrations of thioridazine, and MTT assay was used to evaluate the cell inhibition rate. Hoechst 33342 staining was performed to demonstrate the cell morphology changes. Flow cytometry was used to determine the cell apoptosis and cell cycle changes. RT-qPCR was used to detect PDCD4, c-MYC, BCL2, CCND1, CASPASE3, PARP1, CDK4 and EIF4A mRNA expressions, and Western blotting was employed to assay AKT, p-AKT, and PDCD4 protein expression levels. RESULTS: MTT results showed that thioridazine inhibits the proliferation of SW480 cells. SW480 cells treated with thioridazine presented with such typical features of apoptosis of karyopyknosis, chromatin condensation and nuclear fragmentation. Flow cytometry showed that thioridazine was a cell cycle-specific drug and caused cell cycle arrest at G(1)/G(0) phase and an increased cell apoptosis rate. Thioridazine treatment of the cells resulted in up-regulated PDCD4 mRNA expression and down-regulated mRNA expressions of CCND1, CDK4, c-MYC, BCL2, CASPASE3, PARP1 and EIF4A, increased PDCD4 protein expression and reduced p-AKT protein expression. CONCLUSION: Thioridazine inhibits the proliferation and induces apoptosis of SW480 cells by up-regulating PDCD4 and inhibiting PI3K/Akt pathway.
Assuntos
Apoptose , Neoplasias Colorretais/patologia , Tioridazina/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Regulação para Baixo , Humanos , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5'- and 3'-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.