RESUMEN
OBJECTIVE: To investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells. METHODS: shRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. RESULTS: ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01). CONCLUSIONS: ASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/efectos de los fármacos , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/fisiología , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Glutamina/efectos de los fármacos , Antígenos de Histocompatibilidad Menor/efectos de los fármacos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/fisiología , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Línea Celular Tumoral/fisiología , Neoplasias Colorrectales/fisiopatología , Regulación hacia Abajo/efectos de los fármacos , Técnicas de Silenciamiento del Gen/métodos , Glutamina/genética , Glutamina/fisiología , Humanos , Oncogenes/efectos de los fármacos , Oncogenes/genética , ARN Mensajero/fisiología , ARN Interferente Pequeño/farmacología , TransfecciónRESUMEN
Long noncoding RNAs (lncRNAs) are important regulators in cancer progression. Deregulation of the lncRNA taurine upregulated gene 1 (TUG1) predicts poor prognosis and is implicated in the development of several cancers. In this study, we investigated the role of TUG1 in the pathogenesis of intrahepatic cholangiocarcinoma (ICC). We found that TUG1 is upregulated in ICC samples, which correlates with poor prognosis and adverse clinical pathological characteristics. Knockdown of TUG1 inhibited the proliferation, motility, and invasiveness of cultured ICC cells, and decreased tumor burden in a xenograft mouse model. When we explored the mechanisms underlying these effects, we found that TUG1 acts as an endogenous competing RNA (ceRNA) that 'sponges' miR-145, thereby preventing the degradation of Sirt3 mRNA and increasing expression of Sirt3 and GDH proteins. Accordingly, glutamine consumption, α-KG production, and ATP levels were dramatically decreased by TUG1 knockdown in ICC cells, and this effect was reversed by miR-145 inhibition. These findings indicate that the TUG1/miR-145/Sirt3/GDH regulatory network may provide a novel therapeutic strategy for treatment of ICC.
