RESUMEN
BACKGROUND: Colorectal cancer is the most common malignancy and the third leading cause of cancer-related death worldwide. This study aimed to identify potential diagnostic biomarkers for colorectal cancer by genome-wide plasma cell-free DNA (cfDNA) methylation analysis. METHODS: Peripheral blood from colorectal cancer patients and healthy controls was collected for cfDNA extraction. Genome-wide cfDNA methylation profiling, especially differential methylation profiling between colorectal cancer patients and healthy controls, was performed by methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq). Logistic regression models were established, and the accuracy of this diagnostic model for colorectal cancer was verified using tissue-sourced data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) due to the lack of cfDNA methylation data in public datasets. RESULTS: Compared with the control group, 939 differentially methylated regions (DMRs) located in promoter regions were found in colorectal cancer patients; 16 of these DMRs were hypermethylated, and the remaining 923 were hypomethylated. In addition, these hypermethylated genes, mainly PRDM14, RALYL, ELMOD1, and TMEM132E, were validated and confirmed in colorectal cancer by using publicly available DNA methylation data. CONCLUSIONS: MeDIP-seq can be used as an optimal approach for analyzing cfDNA methylomes, and 12 probes of four differentially methylated genes identified by MeDIP-seq (PRDM14, RALYL, ELMOD1, and TMEM132E) could serve as potential biomarkers for clinical application in patients with colorectal cancer.
Asunto(s)
Neoplasias Colorrectales , Metilación de ADN , Biomarcadores , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ADNRESUMEN
5-Fluorouracil (5-FU)-based chemotherapy is the first-line option for patients with advanced colorectal cancer (CRC). However, the development of chemoresistance is the primary cause of treatment failure. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been demonstrated to exert strong anti-proliferative effects. However, to the best of our knowledge, whether HF inhibits the progression of 5-FU-resistant human CRC HCT-15/FU cells, and the underlying mechanisms, remain unknown. In the present study, the effects of HF on HCT-15/FU cells were assessed in vitro. The results revealed that HF inhibited HCT-15/FU cell viability as demonstrated by the MTT and colony formation assays. Following treatment of HCT-15/FU cells with HF, the migratory and invasive capacities of the cells were significantly decreased. MicroRNA (miRNA/miR)-sequencing data, subsequent miRNA trend analysis and reverse transcription-quantitative PCR all demonstrated that miR-132-3p expression was increased following treatment with HF in a dose-dependent manner. Western blot analysis indicated that following treatment with HF, the expression levels of proteins associated with proliferation, invasion and metastasis in cells were markedly downregulated. These results suggested that HF inhibited the proliferation, invasion and migration of HCT-15/FU cells by upregulating the expression levels of miR-132-3p. Therefore, miR-132-3p may serve as a molecular marker, which may be used to predict CRC resistance to 5-FU, and HF may serve as a novel clinical treatment for 5-FU-resistant CRC.
RESUMEN
AIM: The accumulation of disease-causing proteins is a common hallmark of many neurodegenerative disorders. Measuring the degradation of such proteins using high-throughput-compatible assays is highly desired for the identification of genetic and chemical modulators of degradation. For example, Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder caused by the cytotoxicity of mutant huntingtin protein (mHTT). The high-throughput measurement of mHTT degradation is important in HD drug discovery and research. Existing methods for such purposes have limitations due to their dependence on protein tags or pan protein synthesis inhibitors. Here, we report a high-throughput-compatible pulse-chase method (CH-chase) for the measurement of endogenous tag-free huntingtin protein (HTT) degradation based on Click chemistry and Homogeneous Time Resolved Fluorescence (HTRF) technologies. METHODS: The pulsed-labeled proteins were conjugated with biotin using the click reaction strain-promoted alkyne-azide cycloaddition (SPAAC), and the chase signals were calculated by measuring the reduction percentage of the HTT HTRF signals after pull-down with streptavidin beads. RESULTS: We validated that the signals were within the linear detection range and were HTT-specific. We successfully measured the degradation of endogenous HTT in a high-throughput-compatible format using 96-well plates. The predicted changes of HTT degradation by known modifiers were observed, which confirmed that the assay is suitable for the identification of HTT degradation modifiers. CONCLUSION: We have established the first high-throughput-compatible assay capable of measuring endogenous, tag-free HTT degradation, providing a valuable tool for HD research and drug discovery. The method could be applied to other proteins and can facilitate research on other neurodegenerative disorders and proteinopathies.
Asunto(s)
Proteína Huntingtina/metabolismo , Animales , Línea Celular , Química Clic , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , ProteolisisRESUMEN
This study investigated the effect of homoharringtonine (HHT) on bone marrow CD34 + CD117 + cells in patients with chronic myelogenous leukemia (CML). Fifty-seven patients with CML who could not receive either imatinib or interferon-α were treated with HHT (n = 31) or hydroxycarbamide (HU) (n = 26) to induce and maintain remission, and bone marrow CD34 + CD117 + cells were assayed with flow cytometry. The proportion of CD34 + CD117 + cells was higher in untreated patients (24.7 ± 6.4%) than in donors (4.4 ± 1.1%) and decreased remarkably in patients who had hematological remission (11.2 ± 3.1%) and cytogenetic response (8.9 ± 3.1%) as compared with those without (27.8 ± 7.3% and 18.5 ± 5.3%, respectively) at 12 months after therapy. The hematological remission rate for patients with proportion of CD34 + CD117 + cells ≥ 20% prior to treatment was lower (41.7%) than in patients with CD34 + CD117 + cells < 20% (78.9%; p < 0.05). However, there was no such difference of CD34 + CD117 + cell percentage in the HU group. It can be concluded that high expression of CD117 on CD34 + cells is a marker of poor prognosis and is also a target for the anti-CML effect of HHT.