Targeting Six1 by lentivirus-mediated RNA interference inhibits colorectal cancer cell growth and invasion

Abstract

The Six1 homeodomain protein is a developmental transcription factor that has been implicated in tumor onset and progression. Recently, it's reported that overexpression of Six1 is sufficient to induce epithelial-to-mesenchymal transition (EMT) and metastasis of colorectal cancer. Moreover, its expression is significantly associated with poorer overall survival probability in advanced-stage colorectal cancer. To address whether Six1 could serve as a therapeutic target for human colorectal cancer, we used a lentivirus-mediated short hairpin RNA (shRNA) gene knockdown method to suppress the expression of Six1 in colorectal cancer cells. We showed that lentivirusmediated shRNA targeted to Six1 gene efficiently reduced its expression in colorectal cancer cells at both mRNA and protein levels. In vitro functional assays revealed that knockdown of Six1 significantly suppressed cell proliferation, and inhibited cell migration and invasion of colorectal cancer cells. Furthermore, tumor xenograft model demonstrated that downregulation of Six1 dramatically inhibited colorectal cancer growth in vivo. In conclusion, these findings suggest that lentivirus-mediated Six1 inhibition may represent a novel therapeutic approach for treatment of colorectal cancer.

Keywords: Six1; cell growth; colorectal cancer; invasion.

Figure 1

Suppression of Six1 expression in colorectal cancer cells by lentivirus-mediated RNA interference. (A) Western blot analysis showed the expression of Six1 in colorectal cancer cell lines. β-actin was used as an internal control. (B and C) Both SW620 and LOVO cells were transduced with lentiviral vectors encoding either a scrambled control sequence (scrambled shRNA) or two different shRNAs targeting Six1 (Six1-shRNA-1/2). 72 hours after transduction, the relative Six1 mRNA and protein expression was determined by quantitative real-time RT-PCR (B) and western blot analysis (C), respectively. β-actin was used as an internal control. Data represent the mean ± SEM of three independent experiments. *, P<0.05, compared with the scrambled shRNA group.

Figure 2

Knockdown of Six1 inhibited colorectal cancer cells growth in vitro. (A and B) Cell proliferation was determined by cell count assay once daily for 5 days in both SW620 (A) and LOVO cells (B). (C) DNA synthesis was measured by BrdU incorporation assay in the two cell lines after transduction. (D) Detection of cell proliferation by colony formation assay in both SW620 and LOVO cells. Representative photographs showed cell colony in 6-well plate. Cell colonies were scored visually and counted using a light microscopy. Data represent the mean ± SEM of three independent experiments. *, P<0.05, compared with the scrambled shRNA group.

Figure 3

Six1 silencing decreased cell motility and invasion of colorectal cancer cells. A: Wound healing assay was used to evaluate the migration of both SW620 and LOVO cells after silencing Six1. Monolayers of cells were mechanically wounded with a pipette tip and monitored with a microscope every 12 hours. The migration was determined by the rate of cells filling the scratched area. The normalized wound area was calculated by the software TScratch (right panel). B: Cell invasion was determined by matrigel-coated transwell assay. Cells crossed the Matrigel-coated filter were fixed, stained and counted. Data represent the mean ± SEM of three independent experiments. *, P<0.05, compared with the scrambled shRNA group.

Figure 4

Knockdown of Six1 inhibited tumor growth in colorectal cancer xenografts. SW620 cells transduced with either scrambled shRNA or Six1-shRNA-1 were injected subcutaneously into two groups of nude mice (n=5). Tumor volume was determined on day 7, 14, 21 and 28. Growth curve of tumor xenografts was assessed by serial microcaliper measurements (A). Average Weights of tumor xenografts 28 days after inoculation were recorded (B). Data represent the mean ± SEM. of three independent experiments. *, P<0.05, compared with the scrambled shRNA group.