Upregulation of nucleostemin in colorectal cancer and its effects on cell malignancy

Abstract

Objective: Nucleostemin (NS) is a new protein localized in the nucleolus of most stem cells and tumor cells, which regulates their self-renewal and cell cycle progression. The aim of this study was to investigate the expression of NS in colorectal cancer (CRC) and the effects of NS knockdown in the Sw620 cell line to provide basis for clinical target therapy.

Methods: NS expression in 372 patients with CRC and 367 normal participants was assessed using immunohistochemistry. The expression level of NS gene was evaluated by polymerase chain reaction. Then, the relationship among NS expression, clinicopathological features, and prognosis was analyzed. Silencing of NS expression was achieved by using NS-specific small-interfering RNAs. The viability and growth rate of Sw620 cells were determined by proliferation and invasion assays. Cell cycle distribution of the cells was analyzed by flow cytometry.

Results: High NS expression was positively related with node metastasis, distant metastasis, and TNM stage. In Kaplan-Meier survival analysis, patients with low NS expression always had significantly longer survival time than those with high expression. Moreover, our results showed that knockdown of NS expression inhibited proliferation and viability of Sw620 cells in a time-dependent manner. Cell cycle studies revealed that NS depletion resulted in G1 cell cycle arrest at short times of transfection (24 hours), followed with apoptosis at longer times (48 hours and 72 hours), suggesting that post-G1 arrest apoptosis occurred in Sw620 cells.

Conclusion: Overall, these results point to the essential role of NS in Sw620 cells; thus, this gene might be considered a promising target for treatment of CRC.

Keywords: Sw620; apoptosis; colorectal cancer; nucleostemin; small interfering RNA; target therapy.

Figure 1

Elevated level of NS in CRC. Notes: (A) On qPCR analysis, NS expression levels were found to be elevated in the CRC specimen, compared with paired normal colon tissue (n=30, P<0.01). (B) In CRC cell lines, NS expression was also increased (*P<0.05). (C) Northern blotting assay revealed that the NS blot was significantly denser in CRC samples and cell lines, than in normal tissue and colon cells. (D) Immunohistochemical nuclear staining of NS in CRC. CRC samples were classified as having (a and b) low or (c and d) high NS expression. Original magnifications: ×100 (A and C); ×400 (B and D). Abbreviations: CRC, colorectal cancer; NS, nucleostemin; qPCR, quantitative polymerase chain reaction.

Figure 2

Kaplan–Meier curves showing NS expression and overall survival in patients with CRC. Notes: (A) Patients with high NS expression levels had poorer overall survival compared with patients with low NS expression. Data are representative of 372 CRC tissues (P<0.001). (B) Patients with high NS expression in metastatic lymph nodes had poorer overall survival than patients with low metastatic lymph node NS expression. Data are representative of 88 metastatic lymph node tissues (P=0.003). Abbreviations: NS, nucleostemin; CRC, colorectal cancer.

Figure 3

Knockdown of NS expression by siRNA in Sw620 cell line. Notes: (A) NS-siRNA delivery into Sw620 cells. After 24 hours of transfection with fluorescein-labeled NS-siRNA, Sw620 cells were harvested and analyzed by fluorescence microscopy. (B) Protein levels of NS gene expression in Sw620 cells were detected by Western blot. (C) Analysis of NS mRNA level in Sw620 cells. The densitometry analysis of NS mRNA relative to β2-microglobulin mRNA data was studied by UVItec software. Each value represents the mean ± SEM of three independent experiments, and *P<0.05 and **P<0.01 were considered statistically significant. Abbreviations: NS, nucleostemin; SEM, standard error of the mean; siRNA, small interfering RNA.

Figure 4

NS-siRNA significantly reduces proliferation and invasion in colorectal cancer cells. Notes: (A) NS expression in LoVo, Caco2, and Sw620 cells detected using Western blot analysis. (B) siRNA-induced NS silencing confirmed using (a) quantitative polymerase chain reaction and (b) Western blot analyses. (C) NS-siRNA inhibited the proliferation of (a) Caco2 and (b) Sw620 cells. (D) NS-siRNA was observed to significantly reduce proliferation. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05 for the difference between the two groups. Abbreviations: NS, nucleostemin; OD, optical density; siRNA, small interfering RNA.

Figure 5

Knockdown of NS leads to profound morphological changes and biological characteristics of Sw620 cells. Notes: (A) Apoptotic effects of NS-siRNA in Sw620 cells. Forty-eight hours after transfection of Sw620 cells with 200 nM IR- and NS-siRNAs, the cells were collected. Control (IR-siRNA-transfected) and NS-siRNA-transfected Sw620 cells were double stained with acridine orange/ethidium bromide and studied by fluorescence microscopy (magnification, ×40). Viable cells are equally green, early apoptotic cells are green and contain bright green dots in their nuclei (short arrows), and late apoptotic cells are orange (long arrows). (B) Morphological changes of Sw620 cells after transfection with NS-siRNA. The cells were transfected with 200 nM NS-siRNA for 24–72 hours, and then morphological changes were studied using light microscopy (magnification: ×40). After 24 hours, cell aggregation (white arrow) was observed in NS-siRNA-transfected Sw620 cells, whereas after longer times (48–72 hours), cell shrinking (long black arrows) and apoptotic bodies (short black arrows) were clearly observed. (C) Effects of NS-siRNA on cell cycle distribution of Sw620 cells. Following NS-siRNA transfection, the Sw620 cells were collected at different time intervals (24–72 hours) and their DNA contents were analyzed by flow cytometry, as mentioned in the “Materials and methods” section. The results are from a typical experiment. Abbreviations: FL2, Fluorescence 2; NS, nucleostemin; siRNA, small interfering RNA; IR, insulin receptor.