RESUMO
Colorectal cancer is a malignant tumor with higher morbidity and mortality. The purpose of this study is to investigate whether inhibition of Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1) affects tumor cell proliferation, survival and migration in colon tumors with high Cyclin E1 (CCNE1) expression. PcDNA3.1-CCNE1 vector and si-PKMYT1 were transfected in SW480 cells by Lipofectamine 2000. Q-PCR and western blot assay were processed to detect the expression. Transwell assay and Edu assay were undertaken to verify the migration and proliferation. CCNE1 promotes the proliferation and migration of SW480. Silencing of PKMYT1 inhibited the proliferation of tumor cells. Silencing the expression of PKMYT1 under the premise of overexpression of CCNE1, the level of Cyclin Dependent Kinase 1 (CDK1)-PT14 was reduced, indicating that the cell cycle was blocked. The expression of γH2AX increased significantly, indicating that the DDR pathway of tumor cells was activated and DNA damage accumulated. The results of immunofluorescence microscopy showed significantly increased expression of DNA damage-associated marker (γH2AX: H2AX Variant Histone). In CCNE1 amplificated colorectal tumor cells, knockdown of PKMYT1 reduced cells in S phase, inhibited cell proliferation and promoted cell apoptosis, confirming that PKMYT1 was a potential therapeutic target for colorectal tumor. This study may verify a potential therapeutic target and provide a new idea for the treatment of colorectal cancer in the future.
Assuntos
Neoplasias Colorretais , Humanos , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.