RESUMO
BACKGROUND: Colorectal cancer (CRC) has a high mortality rate, and therapeutic approaches to treat these cancers are varied and depend on the metabolic state of the tumour. Profiles of CRC tumours have identified several biomarkers, including microRNAs. microRNA-210 (miR-210) levels are directly correlated with CRC survival. miR-210 expression is higher in metastatic colon cancer cells versus non-metastatic and normal colon epithelium. Therefore, efficient methods to inhibit miR-210 expression in CRC may provide new advances in treatments. METHODS: Expression of miRs was determined in several metastatic and non-metastatic cell lines. miR-210 expression was inhibited using PMIS-miR-210 in transduced cells, which were transplanted into xenograft mice. In separate experiments, CRC tumours were allowed to grow in xenograft mice and treated with therapeutic injections of PMIS-miR-210. Molecular and biochemical experiments identified several new pathways targeted by miR-210 inhibition. RESULTS: miR-210 inhibition can significantly reduce tumour growth of implanted colon cancer cells in xenograft mouse models. The direct administration of PMIS-miR-210 to existing tumours can inhibit tumour growth in both NSG and Foxn1nu/j mouse models and is more efficacious than capecitabine treatments. Tumour cells further transfer the PMIS-miR-210 inhibitor to neighbouring cells by extracellular vesicles to inhibit miR-210 throughout the tumour. miR-210 inhibition activates the cleaved caspase 3 apoptotic pathway to reduce tumour formation. We demonstrate that the long non-coding transcript XIST is regulated by miR-210 correlating with decreased XIST expression in CRC tumours. XIST acts as a competing endogenous RNA for miR-210, which reduces XIST levels and miR-210 inhibition increases XIST transcripts in the nucleus and cytoplasm. The increased expression of NME1 is associated with H3K4me3 and H3K27ac modifications in the NME1 proximal promoter by XIST. CONCLUSION: Direct application of the PMIS-miR-210 inhibitor to growing tumours may be an effective colorectal cancer therapeutic.
Assuntos
Neoplasias do Colo , Vesículas Extracelulares , MicroRNAs , Nucleosídeo NM23 Difosfato Quinases , RNA Longo não Codificante , Animais , Apoptose/genética , Capecitabina , Caspase 3 , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
In this study, we investigated the role of the transcription factor Six2 in palate development. Six2 was selected using the SysFACE tool to predict genes from the 2p21 locus, a region associated with clefting in humans by GWAS, that are likely to be involved in palatogenesis. We functionally validated the predicted role of Six2 in palatogenesis by showing that 22% of Six2 null embryos develop cleft palate. Six2 contributes to palatogenesis by promoting mesenchymal cell proliferation and regulating bone formation. The clefting phenotype in Six2-/- embryos is similar to Pax9 null embryos, so we examined the functional relationship of these two genes. Mechanistically, SIX2 binds to a PAX9 5' upstream regulatory element and activates PAX9 expression. In addition, we identified a human SIX2 coding variant (p.Gly264Glu) in a proband with cleft palate. We show this missense mutation affects the stability of the SIX2 protein and leads to decreased PAX9 expression. The low penetrance of clefting in the Six2 null mouse combined with the mutation in one patient with cleft palate underscores the potential combinatorial interactions of other genes in clefting. Our study demonstrates that Six2 interacts with the developmental gene regulatory network in the developing palate.