Résumé
Objective:Vacuolar ATPase (V-ATPase) was found within the membranes and internal organelles of a vast array of eukaryotic cells,and was related to various kinds of highly metastatic tumors.LASS2/TMSG1 gene was a novel tumor metastasis suppressor gene cloned from human prostate cancer cell line PC-3M in 1999 by our laboratory.It was found out that protein encoded by LASS2/TMSG1 could interact with the c subunit of V-ATPase (ATP6V0C).In this study,To use RNA interference to suppress the expression of ATP6V0C and try to further investigate the molecular mechanism of ATP6V0C in tumor metastasis and its relationship with LASS2/TMSG1 gene.Methods and Results:The expression level of ATP6V0C mRNA and protein in high metastatic potential prostate cancer cell lines (PC-3M-1E8 and PC-3M) was significantly higher than that in low metastatic potential prostate cancer cell lines (PC-3M-2B4 and PC-3),the expression level in PC-3M-1E8 being the highest.Follow-up tests selected PC-3M-1E8 cells for gene silencing.The expression and secretion of MMP-2 and the expression of MMP-9 in ATP6V0C siRNA transfected PC-3M-1E8 cells displayed no obvious change,but the activity of secreted MMP-9 was abated noticeably compared with the controls (P ＜ 0.05).Extracellular hydrogen ion concentration and V-ATPase activity in interference group were both reduced significantly compared with the controls (P ＜ 0.05).The migration and invasion capacity of ATP6V0C siRNA interfered cells in vitro were diminished significantly compared with the controls (P ＜ 0.05).Furthermore,a dramatic reduction of LASS2/TMSG1 mRNA and protein level after transfection of siRNA in PC-3M-1 E8 cells was discovered (P ＜ 0.05).Confocal immunofluorescence showed a vast co-localization of ATP6V0C protein and LASS2/TMSG1 protein in plasma and membrane.The co-localization signals of control group were much stronger than those of interference group.Conclusion:Specific siRNA silencing of ATP6V0C gene inhibits the invasion of human prostate cancer cells in vitro by mechanism of inhibiting V-ATPase activity and then reducing the extracellular hydrogen ion concentration,inhibiting MMP-9 activation and affecting ECM degradation and reconstruction.Meanwhile,ATP6V0C and LASS2/TMSG1 have interaction and it is likely that ATP6V0C functions as a feedback regulator of LASS2/TMSG1.
Résumé
En este trabajo se muestran los resultados obtenidos, después de la transfección en Leishmania (L.) enriettii del gen de ATPasa del tipo vacuolar extraído de Leishmania (L.) mexicana. Los promastigotes transfectados fueron evaluados In vitro, utilizando líneas de macrófagos e In vivo, utilizando dos modelos experimentales (Ratones Balb/c y Hámsteres dorados). El progreso de la infección fue registrado semanalmente por las mediciones realizadas en el sitio de inoculación. Se colectaron muestras de piel, ganglio poplíteo, hígado, bazo, corazón y sangre para realizar el diagnóstico parasitológico; utilizando histopatología y reacción en cadena de la polimerasa (PCR).Los grupos inoculados con L. enriettii transfectadas presentaron diferencias significativas en el tamaño de la lesión respecto al grupo control sin transfección. La PCR fue positiva en piel y ganglios linfáticos las primeras semanas y posteriormente en bazo, hígado, corazón y sangre, lo cual pone en evidencia la migración de los parásitos a otros órganos. En los grupos control los parásitos fueron detectados solamente en el lugar de inoculación y no en otros tejidos. Los resultados demuestran el papel del gen ATPasa del tipo vacuolar en los procesos de invasión de Leishmania a la célula huésped y el incremento de la virulencia de L. enriettii después de la transfección del mencionado gen en estos parásitos.
In this study we examined the effect of the transfection of the vacuolar type ATPase gene from Leishmania (L.) mexicana to Leishmania (L.) enriettii. Transfected promastigotes were evaluated in vitro using macrophages and in vivo using two experimental models (Balb/c mice and Golden Hamsters). The progression of the infection was recorded weekly by measurements taken at the inoculation site. Samples of skin, the popliteal ganglion, liver, spleen, heart and blood were taken for parasitological diagnosis: histopathology and the polymerase chain reaction (PCR). The groups inoculated with transfected L. enriettii showed significant differences in the size of the lesions with respect to the control group (without transfection). The PCR analysis showed positive for L. enriettii in the skin and lymph nodes during the first weeks post-infection and subsequently in the spleen, liver and heart, thus suggesting that the parasites migrate between organs. In the control group, parasites were detected in the skin at the inoculation site but not in the other organs tested. The results demonstrate the role the vacuolar ATPase gene plays in the invasion of the host cells by Leishmania, and the increase in the virulence of L. enriettii after transfection with this gene.