Targeting MACC1 by RNA interference inhibits proliferation and invasion of bladder urothelial carcinoma in T24 cells

Abstract

The purpose of this article is to research on whether MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma (BUC). In this study, the expression of MACC1 gene was knocked down by RNA interference (RNAi) in the T24 cell (human BUC cell). The transcription level of MACC1 was detected by RT-PCR. Activities of MACC1, caspase-3, caspase-8, Bax and Met (mesenchymal-epithelial transition factor) protein were measured by Western blot. The cell proliferation and apoptosis were detected by MTT and flow cytometry. The cell's invasion ability was performed on Matrigel transwell assay. We also detect MMP2 (metalloproteinase-2) proteins by ELISA. The results showed that the level of MACC1 mRNA and protein was significantly reduced after RNAi. MTT assay showed that the proliferation of T24 cell was decreased due to RNA interference. Apoptosis studies also showed that MACC1 gene interference in T24 loses its anti-apoptotic effects. The expression of apoptosis proteins (Caspase-3, Caspase-8 and Bax) increased significantly due to the MACC1 RNAi. The level of Met protein was down-regulated obviously due to RNAi. Transwell assay showed that invasion abilities of T24 cells were reduced obviously due to MACC1 RNAi. Further studies showed that the secretion of MMP-2 was reduced by RNAi. It can conclude that the ability of proliferation and invasion in T24 cells can be inhibited by RNAi-targeting MACC1. As a result, MACC1 can serve as a potential target for gene therapy of human bladder urothelial carcinoma.

Keywords: Metastasis-associated in colon cancer 1; RNA interference; bladder cancer; carcinoma; gene therapy; metalloproteinase-2.

Figure 1

RT-PCR was used for detection the mRNA expression of MACC1 in cells (A). Data were expressed as mean ± S.E.M from three separate experiments (B). *(P < 0.05) indicates a significant difference compared with the SV-HUC-1 cells.

Figure 2

Effect of MACC1 RNAi on the expression of MACC1 in T24 cells. RT-PCR was used for detection the effect of MACC1 RNAi on the mRNA expression of MACC1. Data were expressed as mean ± S.E.M from three separate experiments (A). The protein level of MACC1 in T24 cells was detected by western blot. Data were expressed as mean ± S.E.M from three separate experiments (B). Statistical analyses were performed using the t-test and one-way ANOVA. *(P < 0.05) indicates a significant difference compared with the control groups.

Figure 3

Effect of MACC1 RNAi on the apoptosis of T24 cells. The apoptosis of T24 cells were detected by FITC-Annexin V staining (A). Data were expressed as mean ± S.E.M from three separate experiments (B). Cell proliferation was monitored with MTT assay (C).*(P < 0.05) indicates a significant difference compared with the control groups.

Figure 4

Effect of MACC1 RNAi on the proteins of T24 cells. The apoptosis proteins (Caspase-3, Caspase-8 and Bax) and Met were detected by western blot (A). Data were expressed as mean ± S.E.M from three separate experiments (B). *(P < 0.05) indicates a significant difference compared with the control groups.

Figure 5

Effect of MACC1 RNAi on the invasion of T24 cells. T24 cell invasion ability was monitored with transwell assay (A) and the invasion ration was expressed as mean ± S.E.M from three separate experiments (B). The concentration of MMP2 in cell culture medium was analyzed with ELISA assay (C).*(P < 0.05 vs. control groups).