RESUMO
PURPOSE: Tumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated. METHODS: RNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-ß signaling. RESULTS: We analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-ß signaling pathway due to its inhibitory role on SMAD7. CONCLUSION: ARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.
Assuntos
Neoplasias da Mama , RNA Longo não Codificante , Proteína Smad7 , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Proteínas Ativadoras de GTPase/genética , Humanos , RNA Longo não Codificante/genética , Proteína Smad7/genética , Ubiquitina-Proteína LigasesRESUMO
The adaptation of tumour cells to hypoxic microenvironment is one of the most significant characteristics of many malignant tumour diseases including hepatocarcinoma. Recently, long non-coding RNAs (lncRNAs) have been reported to play important roles in the various levels of gene regulation thus functioning in growth and survival of tumour cells. Here, new hypoxia-related lncRNAs in hepatocarcinoma cells were screened and validated by lncRNA chip-array as well as real-time RT-PCR. Among them, a hypoxia-activated lncRNA that we identified and termed Hypoxia-Activated BNIP3 Overlapping Non-coding RNA (HABON), was not only regulated by hypoxic-induced factor-1α (HIF-1α) but its expression increased significantly under hypoxia in tumour cells. We deciphered the biological characteristics of HABON including its cell localization, genomic location, as well as its full-length sequence, and proved HABON could promote growth, proliferation and clone-formation of hepatocarcinoma cells under hypoxia. Then, we revealed that HABON was transcriptionally activated by HIF-1α in hypoxic cells, furthermore, it could interact with HIF-1α and promote its protein degradation, thus affecting transcription of HIF-1α's target genes to exert its effects on cells. Besides, the elevated expression of HABON under hypoxia could promote the transcriptional activation of BNIP3 through HIF-1α, and increasing the expression level of BNIP3. This research provides a novel clue for the adaptive survival and growth mechanism of tumour under hypoxia, and gives a way to reveal the nature of tumour cells' resistance characteristics to harsh microenvironment.
Assuntos
Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/fisiopatologia , Neoplasias Hepáticas/patologia , RNA Longo não Codificante/genética , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Comunicação Celular , Proliferação de Células , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
The miR-133b, a commonly recognized muscle-specific miRNA, was reported to be deregulated in many kinds of cancers. However, its potential roles in tumorigenesis remain greatly elusive. Herein, we demonstrate that miR-133b is significantly suppressed in human breast cancer specimens, which is reversely correlated to histological grade of the cancer. Ectopic expression of miR-133b suppresses clonogenic ability and metastasis-relevant traits in vitro, as well as carcinogenesis and pulmonary metastasis in vivo. Further studies have identified Sox9, c-MET, and WAVE2 as direct targets of miR-133b, in which Sox9 contributes to all miR-133b-endowed effects including cell proliferation, colony formation, as well as cell migration and invasion in vitro. Moreover, re-expression of Sox9 reverses miR-133b-mediated metastasis suppression in vivo. Taken together, these findings highlight an important role for miR-133b in the regulation of tumorigenesis and metastatic potential of breast cancer and suggest a potential application of miR-133b in cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , MicroRNAs/metabolismo , Fatores de Transcrição SOX9/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Técnicas In Vitro , MicroRNAs/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Fatores de Transcrição SOX9/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
The core-shell boronic-acid functionalized nanoparticles SnO(2)@Poly(HEMA-co-St-co-VPBA) are designed for selectively enriching glycopeptides, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. Such 60 nm sized core-shell nanoparticles are prepared by means of copolymerization between 2-hydroxyethyl methacrylate (HEMA) grafted on SnO(2) nanoparticles, styrene, and 4-vinylphenylboronic acid (VPBA). All of the synthesis procedures are completed within 3 h. Cyclic boronate esters form between boronic-acid groups on the polymer chains and cis-diol groups on glycopeptides, and thus almost all intact glycopeptides from low-abundant horseradish peroxidase (HRP) and bovine asialofetuin (ASF) are enriched with high selectivity and efficiency. After enrichment, both intact N- and O-glycopeptides are characterized by multistage MS. Furthermore, we successfully apply this method to the human serum sample for characterizing the target glycoproteins haptoglobin and alpha-1-acid-glycoprotein. The present selective enriching method followed by multistage-MS analysis is proven to be a good choice for routine glycopeptide characterization.
