Panoply™ Human TGFB2 Knockdown Stable Cell Line

Increasing evidence indicates that microRNAs (miRNAs) are closely associated with metastasis in gallbladder cancer (GBC). Recent studies have shown that miR-324-5p functions as a tumor suppressor in various malignancies. In this study, researchers found that miR-324-5p expression was significantly downregulated in GBC tissues and cells compared to normal controls. Downregulation of miR-324-5p correlated with local invasion and lymph node metastasis, and predicted a poor prognosis for GBC patients. Further functional assays demonstrated that restoring miR-324-5p expression significantly inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of GBC cells in vitro, and suppressed metastasis in vivo. Additionally, RNA immunoprecipitation (RIP) and dual-luciferase reporter assays confirmed that transforming growth factor-β2 (TGFB2) is a direct target of miR-324-5p in GBC cells. Mechanistic studies revealed that silencing TGFB2 expression using small interfering RNA (siRNA) partially mimicked the inhibitory effects of miR-324-5p overexpression on the metastatic phenotype of GBC cells. Collectively, these findings demonstrate that miR-324-5p inhibits GBC cell metastasis by targeting TGFB2, suggesting that miR-324-5p holds promise as a potential biomarker for GBC diagnosis and therapeutic strategies.

Dual-luciferase reporter assays confirmed that TGFB2 is a direct target of miR-324-5p in gallbladder cancer (GBC). Consequently, the researchers investigated whether TGFB2 knockdown exerted similar functional effects in GBC. To explore the impact of TGFB2 on GBC cell migration and invasion, they generated TGFB2-knockdown GBC-SD and SGC-996 cell lines. As shown in Figure 1A, TGFB2 protein levels were significantly reduced in the knockdown cells compared to control cells. Using these transfected cells, the researchers performed Transwell migration and invasion assays. The results demonstrated that TGFB2 knockdown significantly inhibited cell migration compared to control cells (Figure 1B). Furthermore, the number of invading TGFB2-knockdown GBC-SD and SGC-996 cells was significantly lower than that of control cells (Figure 1C). Additionally, TGFB2 knockdown promoted E-cadherin expression while suppressing N-cadherin and Vimentin expression in SGC-996 cells (Figure 1D). In vivo liver metastasis assays revealed that TGFB2 knockdown reduced liver metastatic foci formed by SGC-996 cells (Figure 1E, F). These data strongly suggest that silencing TGFB2 recapitulates, to some extent, the inhibitory effect of miR-324-5p overexpression on the metastatic phenotype of GBC cells.

Figure 1. Knockdown of TGFB2 partially phenocopied the inhibitory effects of miR-324-5p overexpression on GBC cell metastatic behaviours. (Zhang X, et al., 2020)