RGC32 induces epithelial-mesenchymal transition by activating the Smad/Sip1 signaling pathway in CRC

Abstract

Response gene to complement 32 (RGC32) is a transcription factor that regulates the expression of multiple genes involved in cell growth, viability and tissue-specific differentiation. However, the role of RGC32 in tumorigenesis and tumor progression in colorectal cancer (CRC) has not been fully elucidated. Here, we showed that the expression of RGC32 was significantly up-regulated in human CRC tissues versus adjacent normal tissues. RGC32 expression was significantly correlated with invasive and aggressive characteristics of tumor cells, as well as poor survival of CRC patients. We also demonstrated that RGC32 overexpression promoted proliferation, migration and tumorigenic growth of human CRC cells in vitro and in vivo. Functionally, RGC32 facilitated epithelial-mesenchymal transition (EMT) in CRC via the Smad/Sip1 signaling pathway, as shown by decreasing E-cadherin expression and increasing vimentin expression. In conclusion, our findings suggested that overexpression of RGC32 facilitates EMT of CRC cells by activating Smad/Sip1 signaling.

Figure 1. RGC32 was overexpressed and was associated with poor survival in CRC patients.

(A) The expression of RGC32 mRNA in normal and tumor tissues of patients with CRC was detected with real-time PCR. (B) Kaplan–Meier analysis of overall survival in patients with CRC. (C) Representative images of RGC32 expression in normal intestinal epithelium and CRC specimens examined by IHC. RGC32 was not detected (a, b) in normal intestinal epithelial cells, whereas it was detected in CRC cells (c and d display weak signals, e and f display moderate signals, and g and h display strong signals). *P < 0.05, ***P < 0.001 compared with the score of RGC32 in normal tissue.

Figure 2. RGC32 promoted migration and invasion of CRC cell lines in vitro.

(A,B) Overexpression of RGC32 markedly increased SW480 cell migration and invasion as measured with wound-healing (A) and with Transwell invasion assays (B). (C,D) Knockdown of RGC32 markedly decreased SW620 cell migration and invasion as measured by wound-healing (C) and Transwell invasion assays (D). **P < 0.01 vs vector or scramble; ***P < 0.001 vs vector or scramble.

Figure 3. RGC32 induced EMT phenotype marker alterations in colorectal cancer.

(A) The morphology of SW480 cells transfected with vector or RGC32 plasmid (RGC32) and SW620 cells transfected with scramble or RGC32 shRNA (shRGC32). (B) The expression of mesenchymal and epithelial markers in SW480 cells transfected with vector or RGC32 plasmid (RGC32) and SW620 cells transfected with scramble or RGC32 shRNA (shRGC32) was detected by western blotting. (C) Confocal microscopy of E-cadherin and vimentin staining in SW620 cells transfected with scramble or RGC32 shRNA(shRGC32). (D) Immunohistochemical staining of the EMT-related proteins E-cadherin and Vimentin in primary tumor tissues derived from scramble or RGC32-containing SW620 cells as indicated. Magnification: 400x; *P < 0.05 vs scramble.

Figure 4. RGC32 regulated the Smad/Sip1 signaling pathway.

(A) Coimmunoprecipitation assay to test the correlation between RGC32, Smad2/3 and Sip1. (B) The expression of Smad signaling and Sip1 in SW480 cells transfected with vector or RGC32 plasmid (RGC32) and SW620 cells transfected with scramble or RGC32 shRNA(shRGC32) was detected by western blotting. (C) The expression of p-Smad2 and p-Smad3 in cytolymph and the nucleus detected by immunofluorescence. *P < 0.05, ***P < 0.001 vs scramble.

Figure 5. The Smad/Sip1 signaling pathway mediated RGC32-induced EMT in CRC.

(A,B) Knockdown of endogenous Sip1 in SW620 cells shown by RT-PCR and western blotting. (C) The expression of mesenchymal and epithelial markers in SW620 cells transfected with scramble (Ctrl) or Sip1 siRNA (siSip1) was detected by western blotting. (D,E) In vitro scratch wound-healing assays and migration assays were performed to investigate the role of Sip1 in CRC cell migration. *P < 0.05, **P < 0.01, ***P < 0.001 vs ctrl.