RESUMO
Long noncoding RNA growth arrest specific 5 (GAS5) exerts inhibitory effects through the modulation of several target microRNAs (miRs) in cancer. However, its potential roles and underlying relationship during colorectal cancer (CRC) progression are unclear. Therefore, we explored the role of the negative feedback loop formed by the GAS5/miR34a axis and mammalian target of rapamycin/sirtuin 1 (mTOR/SIRT1) pathway on macroautophagy and apoptosis in CRC. Expression of GAS5, miR34a, SIRT1 and mTOR in CRC patients and cell lines was detected by quantitative reverse transcription polymerase chain reaction. Online bioinformatic analysis was used to predict the downstream miRs of GAS5. Luciferase assay and western blotting were performed to demonstrate miR34a as a downstream target gene of GAS5 in CRC cells. The effects of the GAS5/miR34a axis on apoptosis, macroautophagy, and the mTOR/SIRT1 pathway were assessed by flow cytometry, transmission electron microscopy and western blotting, respectively. Our results suggested that GAS5 was downregulated and acted as a molecular sponge of miR34a during CRC progression. miR34a participated in regulating GAS5suppressed CRC cell macroautophagy and induced apoptosis through the mTOR/SIRT1 pathway. GAS5mediated macroautophagy was maintained in an equilibrium state that might have a protective effect on CRC cell apoptosis. The mTOR signaling pathway suppressed GAS5 expression and formed a negative regulation feedback loop with miR34a in CRC cells. Our results suggested that the GAS5/miR34a/SIRT1/mTOR negative regulatory feedback loop mediated CRC cell macroautophagy, and maintained the cells in an autonomous equilibrium state, but not excessive activation state, which functions as a strong antiapoptotic phenotype during human CRC progression.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Macroautofagia/genética , MicroRNAs/genética , RNA Longo não Codificante/metabolismo , Idoso , Animais , Azoximetano/administração & dosagem , Azoximetano/toxicidade , Linhagem Celular Tumoral , Colo/imunologia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Retroalimentação Fisiológica , Feminino , Humanos , Macroautofagia/efeitos dos fármacos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , RNA Longo não Codificante/genética , Ratos , Transdução de Sinais/genética , Sirolimo/farmacologia , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The present study aimed to investigate the effect of Dermatopontin (DPT) gene silencing on the apoptosis and proliferation of osteosarcoma MG63 cells. Three eukaryotic expression vectors of short hairpin (sh)RNA fragments targeting different loci of DPT were designed and transfected into an osteosarcoma cell line MG63. The cells were assigned to a blank, shRNAcontrol, DPTshRNAa, DPTshRNAb or DPTshRNAc group. The shRNA with the highest silencing efficiency was screened using reverse transcriptionquantitative polymerase chain reaction and western blotting. The screened shRNA was transfected into MG63 cells. The proliferation, cell cycle and apoptosis of MG63 cells were measured using a Cell Counting Kit8 assay, flow cytometry and Annexin Vfluorescein isothiocyanate assay. The recombinant plasmids containing DPT shRNA were successfully constructed. DPT gene silencing was able to significantly reduce the proliferation rate of MG63 cells (P<0.05). The proportion of cells in the G0/G1 phase and in the G2/M phase increased significantly (both P<0.05), while the proportion of cells in the S phase decreased (P<0.05). Furthermore, the cell apoptosis rate increased significantly (P<0.05). These results demonstrate that DPT gene silencing is able to reduce the proliferation of MG63 cells, slow down cell cycle progression and promote apoptosis, hence may become a novel target for the treatment of osteosarcoma.