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. 2015 Jun 1;8(6):6377-86.
eCollection 2015.

Knockdown of Nestin inhibits proliferation and migration of colorectal cancer cells

Jing Li  1 , Rui Wang  1 , Lei Yang  2 , Qi Wu  1 , Qinwan Wang  3 , Zhengchao Nie  1 , Yongchun Yu  4 , Ji Ma  3 , Qiuhui Pan  3
Affiliations

Knockdown of Nestin inhibits proliferation and migration of colorectal cancer cells

Jing Li et al. Int J Clin Exp Pathol. .

Abstract

Nestin, a member of type VI intermediate filament protein family, is widely expressed in mammalian nervous tissue and stem/precursor cells of non-neuronal normal tissues. Nestin has also been investigated to determine possible tumor-promoting functions. However, whether Nestin is involved in colorectal cancer (CRC) cells remains unclear. In this report, Nestin expression was upregulated in stromal cells of human CRC tissues. Endogenous Nestin expression in CRC cell lines SW480 and HCT116 was knocked down by a lentivirus. MTT and colony formation assays revealed that Nestin deletion significantly inhibits the proliferation of CRC cell lines; flow cytometer analysis showed that Nestin deletion causes cell cycle arrest at S phase. Transwell chamber and wound healing scratch assays also revealed that Nestin deletion suppresses cell migration. Our findings indicated that Nestin plays an essential role in CRC progression; thus, Nestin can be applied as a therapeutic target of CRC.

Keywords: Nestin; colorectal cancer; metastasis.

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Figures

Figure 1
Figure 1
Nestin is upregulated in human CRC clinical specimens. (A) Nestin expression was determined through qRT-PCR in tissues of patients with CRC compared with adjacent normal tissues. Data are shown as 2-∆∆Ct values relative to 18S rRNA expression in box plots. P=0.017 compared with adjacent normal tissues. (B) Representative immunohistochemistry images showing Nestin expression in (A) adjacent normal tissues and (B) CRC tissues. Magnification =200 ×.
Figure 2
Figure 2
Relative Nestin expression in SW480 and HCT116 cells infected by shRNA-targeting Nestin. Representative images of (A) SW480 and (B) HCT116 cells infected with lentiviral-EGFP. mRNA expression of Nestin in (C) SW480 and (D) HCT116 cells determined through qRT-PCR. Data are shown as 2-∆∆Ct values standardized to 18SrRNA expression and normalized to 1.0 in shCON-transduced cells. *P<0.05, **P<0.01 compared with shCON. (E) Protein levels of Nestin in SW480 and HCT116 cells revealed through Western blot analysis. Data represent at least three independent experiments with similar results.
Figure 3
Figure 3
Knockdown of Nestin suppressed the proliferation and colony-formation ability of CRC cells. MTT analysis performed in (A) SW480 and (B) HCT116 cells after a time course of transfection. Data represent mean ± SD from three independent experiments. (C-F) Colony-forming efficiency of transfected cells. Representative images of the plates used for enumeration of colonies are shown in (C and D) Values of colony-forming efficiency (E and F) represent the means ± SD of three independent experiments. **P<0.01, ***P<0.001 compared with shCON.
Figure 4
Figure 4
Knockdown of Nestin inhibited CRC cell migration. (A, B) Treated cells were re-plated in serum-free medium layered onto the top chambers and invaded the bottom chamber containing serum-supplemented medium for 18 h at 37°C. (C, D) Quantification of cell invasion of (A) and (B) by detecting absorbance at 570 nm. Data represent at least three independent experiments with similar results.*P<0.05 compared with shCON. (E) Phase-contrast microscopy images of the same wounded area at 0, 24, and 48 h after scratching. (F) Cell migration quantification of (E) was assessed on the basis of the scratch area at 0 h normalized to 24 h or 48 h. Data represent at least three independent experiments with similar results.*P<0.05, **P<0.01 compared with shCON.
Figure 5
Figure 5
Knockdown of Nestin arrested cell cycle progression. (A) Transduced cells harvested at 0, 24, and 48 h after pure cells were cultured in a complete medium; cell cycle distribution in SW480 cells was analyzed through FACS. (B) Percentage of transduced cells at S phase. *P<0.05 compared with shCON.
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