RESUMO
BACKGROUND: Chemotherapy is the common treatment for cervical cancer, and the occurrence of drug resistance seriously affects the therapeutic effect of cervical cancer. Our previous study found that PRKD2 mutations occurred only in cervical cancer patients with chemotherapy resistance. However, the relationship between PRKD2 and drug resistance of cervical cancer remains unknown. OBJECTIVE: We aim to clarify the relationship between PRKD2 and drug resistance of cervical cancer. METHODS: Samples of patient tumor tissue were collected before chemotherapy and sequenced by WES. Chemotherapy clinical response was determined by measuring tumor volume. The expression of PRKD2, cell viability, and apoptosis were assessed by qRT-PCR, Western blot, CCK8, and flow cytometry in SiHa and ME180 cells after transfected with siPRKD2. The chemotherapy sensitivity signaling- related proteins were analyzed by Western blot. The expression levels of PRKD2 TP53, and CDKN1A in tissues were detected by immunohistochemistry staining. RESULTS: The expression of PRKD2 was higher in chemotherapy-resistant cervical cancer patients. PRKD2 knockdown increased the chemotherapy sensitivity of cervical cancer cells via the TP53/CDKN1A pathway, which led to G1 arrest and cell apoptosis. Furthermore, downregulation of PRKD2 enhances chemotherapeutic sensitivity in cervical cancer patients through the TP53/CDKN1A pathway. CONCLUSION: In summary, PRKD2 may be a promising therapeutic target to improve the efficacy of chemotherapy.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21 , Proteína Quinase D2 , Proteína Supressora de Tumor p53 , Neoplasias do Colo do Útero , Feminino , Humanos , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Proteína Quinase D2/metabolismoRESUMO
BACKGROUND AND AIMS: N6-Methyladenosine (m6A) modification is involved in many pathological processes, including insulin resistance (IR). Quercetin (Que), a bioactive compound with strong antioxidant activity, has potential therapeutic effects on IR-related metabolic diseases. The aim of this study is to investigate the roles of m6A and Que in hyperinsulinemia. METHODS AND RESULTS: Male C57Bl/6 mice received a high-fat diet (HFD) for 8 weeks to establish an IR model. Que treatment reduced the body weight, blood glucose, plasma triglycerides (TG) and serum insulin, ameliorated IR, and decreased oxidative stress in HFD-fed mice. Cellular IR model was established in C2C12 cells by palmitic acid (PA) stimulation, and a noncytotoxic dose of Que was found to promote glucose uptake and inhibit oxidative stress. Moreover, methyltransferase-like 3 (METTL3) and serine-threonine kinase protein kinase D2 (PRKD2) was downregulated in skeletal muscle of HFD-fed mouse and in PA-induced C2C12 cells. The online bioinformatic tool SRAMP revealed that there were multiple m6A modification sites in the PRKD2 mRNA sequence. Downregulation of METTL3 enhanced PRKD2 expression by reducing m6A level and promoting mRNA stability in PRKD2 mRNA transcript. Que decreased m6A, METTL3, and phosphorylated insulin receptor substrate 1 (p-IRS1) levels, increased the protein expression of PRKD2, glucose transporter type 4 (GLUT4) and p-AKT, promoted glucose uptake, and reduced oxidative stress in PA-induced C2C12 cells. Moreover, METTL3 overexpression or PRKD2 silence reversed the inhibitory effects of Que on the levels of MDA and p-IRS1 and the promotive effects on glucose uptake, superoxide dismutase (SOD), GSH and GLUT4 and p-AKT levels. CONCLUSION: Que promoted glucose uptake, repressed oxidative stress and improved IR through METTL3-mediated m6A of PRKD2 mRNA.
Assuntos
Resistência à Insulina , Metiltransferases , Proteína Quinase D2 , Quercetina , Adenosina/análogos & derivados , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Glicemia/metabolismo , Linhagem Celular , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Insulinas/metabolismo , Masculino , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Ácido Palmítico/farmacologia , Proteína Quinase D2/genética , Proteína Quinase D2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Superóxido Dismutase , Triglicerídeos/metabolismoRESUMO
Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild type up to 36â h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish.This article has an associated First Person interview with the first author of the paper.
