CRTC2 promotes non-small cell lung cancer A549 migration and invasion in vitro

Abstract

Background: CRTC2 is highly expressed in lung cancer and contributes to lung cancer pathogenesis; however, whether CRTC2 promotes lung cancer metastasis remains unknown. In the present study, we investigated the role of CRTC2 in lung cancer metastasis in vitro.

Methods: CRTC2 stable knockdown of lung cancer cell A549 was generated with small hairpin RNA and confirmed by quantitative reverse transcription-PCR and Western blot. Wound healing and invasion transwell assays were performed to explore migration and invasion activity, and Western blot was conducted to detect the expression of related proteins.

Results: Suppression of CRTC2 significantly inhibited A549 cell migration and invasion in vitro. Mechanistic studies showed that knockdown of CRTC2 greatly downregulated MMP2 and MMP9 expression. CRTC2 silencing remarkably suppressed epithelial-mesenchymal transition by modulating the expression of E-cadherin and vimentin. Furthermore, suppression of CRTC2 expression significantly reduced MAPK/c-Jun N-terminal kinase activity.

Conclusion: CRTC2 may promote A549 migration and invasion by modulation of c-Jun N-terminal kinase-mediated epithelial-mesenchymal transition and matrix metalloproteinase expression.

Keywords: CRTC2; MMPs; epithelial-mesenchymal transition; lung cancer; metastasis.

Figure 1

Suppression of CRTC2 expression inhibits A549 migration and invasion. (a,b) Silence of CRTC2 was confirmed by quantitative reverse transcription‐PCR and Western blot. (c) Wound healing assay. Cell cultures in confluence photographed immediately after scratching the wound (0 hours) or 16 hours post scratching. Representative images are shown. (d,e) Transwell invasion assay. A549 wild‐type or CRTC2 knockdown cells (5 × 10⁴ in 200 μL serum‐free medium) were seeded in the top chamber containing a layer of Matrigel; the lower chamber contained medium with 10% fetal bovine serum as a chemoattractant. After incubation for 24 hours, invaded cells on the lower membrane surface were detected. Representative images of migrated A549 wild‐type and CRTC2 cells are shown. Quantification of cell migration of each line is shown (right). Data shown are mean ± standard deviation. *P < 0.05; **P < 0.01. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; mRNA, messenger RNA; NC, negative control; shRNA, small hairpin RNA.

Figure 2

Knockdown of CRTC2 suppresses matrix metalloproteinase (MMP) expression. The indicated proteins were detected by Western blot. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was detected as an input control. NC, negative control; shRNA, small hairpin RNA.

Figure 3

CRTC2 knockdown suppresses epithelial‐mesenchymal transition of A549. The indicated proteins were detected by Western blot. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was detected as an input control. NC, negative control; shRNA, small hairpin RNA.

Figure 4

CRTC2 silencing downregulates c‐Jun N‐kinase (JNK) activity in A549 CRTC2 knockdown cells. The indicated proteins were detected by Western blot. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was detected as an input control. NC, negative control; p, phosphorylated; shRNA, small hairpin RNA.