RESUMEN
Long non-coding RNAs (lncRNAs) are associated with the occurrence, development and prognoses of non-small cell lung cancer (NSCLC). In the present study, we investigated the functional mechanisms of the lncRNA XIST in two human NSCLC cell lines, A549 and NCI-H1299. In all the 5 NSCLC cell lines (NL9980, NCI-H1299, NCI-H460, SPC-A-1 and A549) tested, the expression levels of XIST were significantly elevated, as compared with those in normal human bronchial epithelial cell line BEAS-2B. In A549 and NCI-H1299 cells, knockdown of XIST by siRNA significantly inhibited the cell proliferation, migration and invasion, and promoted cell apoptosis. Furthermore, XIST knockdown elevated the expression of E-cadherin, and suppressed the expression of Bcl-2. Moreover, knockdown of XIST significantly suppressed the tumor growth in NSCLC A549 xenograft mouse model. Bioinformatic analysis and luciferase reporter assays revealed that XIST was negatively regulated by miR-449a. We further identified reciprocal repression between XIST and miR-449a, which eventually influenced the expression of Bcl-2: XIST functioned as a miRNA sponge of miR-449a, which was a negative regulator of Bcl-2. These data show that expression of the lncRNA XIST is associated with an increased growth rate and metastatic potential in NSCLC A549 and NCI-H1299 cells partially through miR-449a, and suggest that XIST may be a potential prognostic factor and therapeutic target for patients with NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Largo no Codificante/genética , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Ratones , Invasividad Neoplásica , Trasplante de NeoplasiasRESUMEN
AIM: To investigate the endogenous signaling pathways associated with high proliferation potential of breast cancer cells. METHODS: Breast cancer cell lines LM-MCF-7 and MCF-7 with high and low proliferation capability were used. The promoter activity of fatty acid synthase (FASN) was examined using luciferase reporter gene assay. The expression level of FASN mRNA was measured using RT-PCR and real time PCR, respectively. The level of leukotriene B4 (LTB4) was determined with ELISA. The expression levels of 5-lipoxygenase (5-LOX) was analyzed using RT-PCR and Western blot, respectively. 5-Bromo-20-deoxyuridine (BrdU) incorporation assay was used to study the proliferation of LM-MCF-7 and MCF-7 cells. RESULTS: The promoter activity of FASN was significantly higher in LM-MCF-7 cells than MCF-7 cells. Treatment of LM-MCF-7 cells with ERK1/2 inhibitor PD98059 (30-50 µmol/L) or LOX inhibitor NDGA (25 µmol/L) abolished the activation of FASN. Moreover, treatment of LM-MCF-7 cells with the specific 5-LOX inhibitor MK-886 (20-40 µmol/L) or 5-LOX siRNA (50-100 nmol/L) decreased the promoter activity of FASN. The level of LTB4, the final metabolite produced by 5-LOX, was significantly higher in LM-MCF-7 cells than MCF-7 cells. Administration of exogenous LTB4 (1-10 nmol/L) was able to stimulate the promoter activity of FASN in MCF-7 cells. Treatment of LM-MCF-7 cells with the FASN inhibitor cerulenin (10 µmol/L) reduced all the levels of p-ERK1/2, 5-LOX, and LTB4. Treatment of LM-MCF-7 cells with cerulenin, PD98059, or MK-886 abolished the proliferation. Administration of exogenous LTB4 (10 nmol/L) significantly increased BrdU incorporation in MCF-7 cells. CONCLUSION: THESE results suggest a novel positive feedback loop involving FASN/p-ERK1/2/5-LOX/LTB4/FASN contributes to the sustaining growth of breast cancer LM-MCF-7 cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Retroalimentación Fisiológica , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Transducción de Señal , Araquidonato 5-Lipooxigenasa/genética , Araquidonato 5-Lipooxigenasa/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Cerulenina/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leucotrieno B4/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND & OBJECTIVE: Metastasis associated proteins play important roles in metastasis. This study was to investigate proteins which may be involved in breast cancer cell metastasis and further explore the potential mechanisms. METHODS: LM-MCF-7 and MCF-7, two breast cancer cell lines with different metastatic potentials, derived from the same parent cell line, were used in our study. Proteomics and Western blot were applied to identify differentially expressed proteins. Wound healing assay was performed to observe the effect of survivin gene on breast cancer cell migration by transfecting pcDNA3-Sur plasmid into MCF-7 cells. RESULTS: Eight differently expressed proteins, which were correlated with cell structures, cellular metabolism, apoptosis, protein enfold or interaction, were identified. Protein expression of nm23 and p27 was relatively higher in MCF-7 cells; while the expression of survivin, Bcl-2 and myosin light chain kinase was relatively higher in LM-MCF-7 cells. Increased migration ability was observed in MCF-7 cells which were transfected with pcDNA3-Sur. CONCLUSION: Metastasis associated proteins exist in breast cancer cell lines with different metastatic abilities. Survivin is closely related to the metastasis in breast cancer cells.
