RESUMO
PURPOSE: Cancer development remains the most challenging obstacle in colorectal cancer (CRC) treatment. The current study aims to identify and demonstrate novel oncogenes for CRC. METHODS: The CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database were subjected to bioinformatics analysis to identify the novel potential diagnostic and prognostic biomarkers for CRC. Immunohistochemical assay, western blot, and quantitative PCR (qPCR) were used to analyze hydroxyacylglutathione hydrolase-like (HAGHL) gene expression in CRC tissues and cultured CRC cells. D-Lactate colorimetric assay was applied to determine concentration of D-lactate in supernatants from CRC tissues and cell culture medium. Cell counting kit-8 (CCK-8) assay, flow cytometry, tumor xenografts experiment, and TUNEL staining analysis were performed to evaluate the function of HAGHL in CRC. RESULTS: We comprehensively analyzed the CRC data of the Cancer Genome Atlas database and the Gene Expression Omnibus database, and identified several novel potential diagnostic and prognostic biomarkers for CRC, including HAGHL, DNTTIP1, DHX34, and AP1S3. The expression of HAGHL, the strongest oncogenic activity gene, is positively related to D-lactate levels in CRC tissues and negatively associated with patient prognosis. HAGHL downregulation suppressed the production of D-lactate and induced apoptosis, resulting in inhibition of cell proliferation in vitro. In vivo experiment showed that knockdown of HAGHL induced cell apoptosis and inhibited tumor growth. CONCLUSION: These findings suggest that HAGHL acts as a novel metabolic oncogene and demonstrate the underlying mechanism by which HAGHL regulates CRC progression, highlighting its utility as a diagnostic and prognostic factor and as a potential therapeutic target for the treatment of CRC.
Assuntos
Neoplasias Colorretais , MicroRNAs , Humanos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Oncogenes , Lactatos , Biomarcadores , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Movimento Celular , RNA Helicases/genética , RNA Helicases/metabolismoRESUMO
OBJECTIVE: To evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. METHODS: Based on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. RESULTS: DNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. CONCLUSIONS: The results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.
Assuntos
Humanos , Antituberculosos/farmacologia , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , DNA Bacteriano/análise , Testes de Sensibilidade Microbiana , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase Multiplex/economia , Mycobacterium tuberculosis/efeitos dos fármacos , Mutação Puntual , Sensibilidade e Especificidade , Análise de Sequência de DNA , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
OBJECTIVE: To evaluate multiplex allele specific polymerase chain reaction as a rapid molecular tool for detecting multidrug-resistant tuberculosis. METHODS: Based on drug susceptibility testing, 103 isolates were multidrug-resistant tuberculosis and 45 isolates were sensitive to isonicotinylhydrazine and rifampin. Primers were designed to target five mutations hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and multiplex allele specific polymerase chain reaction was performed. Whole-genome sequencing confirmed drug resistance mutations identified by multiplex allele specific polymerase chain reaction. RESULTS: DNA sequencing revealed that 68.9% of multidrug-resistant strains have point mutations at codon 315 of the katG gene, 19.8% within the mabA-inhA promoter, and 98.0% at three hotspots within rpoB. Multiplex allele specific polymerase chain reaction detected each of these five mutations, yielding 82.3% sensitivity and 100% specificity for isoniazid resistance, and 97.9% sensitivity and 100% specificity for rifampin resistance as compared to drug susceptibility testing. CONCLUSIONS: The results show that multiplex allele specific polymerase chain reaction is an inexpensive and practical method for rapid detection of multidrug-resistant tuberculosis in developing countries.