Lentivirus-mediated silencing of Tiam1 gene influences multiple functions of a human colorectal cancer cell line

Abstract

T lymphoma invasion and metastasis 1 (Tiam1) is a metastasis-related gene of T lymphoma that is also involved in the metastasis of a variety of other cancers. In this study, we tested the hypothesis that Tiam1 is a determinant of proliferation and metastasis in colorectal cancer, and we examined the effect of the inhibition of Tiam1 expression on proliferation and metastasis. We succeeded in establishing the Tiam1 knockdown colorectal cancer cell line using human immunodeficiency virus lentivirus-mediated RNA interference (RNAi) and found that the silencing of Tiam1 resulted in the effective inhibition of in vitro cell growth and of the invasive ability of colorectal cancer cells. Using an orthotopic xenograft model in nude mice, we confirmed that Tiam1 silencing could reduce tumor growth by subcutaneous injection and could suppress lung and liver metastases of colorectal cancer cells. Our results suggest that Tiam1 truly plays a causal role in the metastasis of colorectal cancer and that RNAi-mediated silencing of Tiam1 may provide an opportunity to develop a new treatment strategy for colorectal cancer.

Figure 1

Titering lentiviral stock. Cells were either transduced with 10-fold serial dilutions of lentiviral supernatant (10^-2-10^-6 dilutions) or untransduced (Mock cells). Forty-eight hours posttransduction, the cells were placed under blasticidin selection. After 14 days of selection, the cells were stained with crystal violet, and colonies were counted. The virus titer was 7 x 10⁵ TU/ml.

Figure 2

Screening of the most effective targeting site for the Tiam1 gene by real-time PCR and Western blot analysis. (A) Quantification of Tiam1 mRNA expression in cells of different interference sites relative to controls (M, pLenti6/U6 Mock virus-transduced cells), as detected by real-time PCR. (B) Tiam1 protein expression in cells of different interference sites, as detected by Western blot analysis. Lane L shows that a greater degree of Lamin A/C knockdown was observed after transduction with a lentivirus containing the human Lamin A/C shRNA-expressing cassette. Lane B shows that lentiviral vectors were effective and that site B was the most effective construct.

Figure 3

Confirmation of Tiam1 expression in different clones by real-time PCR and Western blot analysis. (A) Quantification of Tiam1 mRNA expression in clones transduced by the pLenti6/Tiam1 (site B) lentivirus relative to controls (clone transduced by pLenti6/U6 lentivirus). (B) Western blot analysis shows marked reduction of Tiam1 protein expression in clones 2 and 8. Lane 1, SW480/EGFP cells; lane 2, pLenti6/Tiam1 lentivirus-transduced clone 2; lane 3, pLenti6/Tiam1 lentivirus-transduced clone 8; lane 4, pLenti6/Tiam1 lentivirus-transduced clone 4; lane 5, pLenti6/U6 lentivirus-transduced clone.

Figure 4

Tiam1 gene silencing suppresses cell proliferation in vitro. (A) The in vitro proliferative abilities of WT cells, Mock cells, Tiam1KD/clone 2, Tiam1KD/clone 6, and Tiam1KD/clone 8 cells were evaluated by MTT assay. Each value represents the mean ± SD of the absorbance value (OD). Results showed that all Tiam1 knockdown cells grew significantly more slowly than WT and Mock cells, and Tiam1 protein expression correlated with cell proliferation. (B) The plate colony formation efficiency of WT cells, Mock cells, Tiam1KD/clone 2, Tiam1KD/clone 6, and Tiam1KD/clone 8 cells. Data represent the mean ± SD of triplicate dishes. Compared with WT and Mock cells, Tiam1KD/clone 2 had significantly reduced ability for colony formation (P < .01), and Tiam1KD/clone 8 also showed decreased clonogenicity (P < .05). Tiam1KD/clone 6, compared with WT and Mock cells, displayed no difference in clonogenicity (P > .05). Results showed that plate colony formation efficiency also correlated with Tiam1 protein expression.

Figure 5

Effect of Tiam1 knockdown on the cell cycle detected by flow cytometry analysis. Tiam1 silencing cells showed G₀/G₁ phase arrest and G₂/M and S phase reduction. The difference was statistically significant.

Figure 6

Effect of Tiam1 knockdown on the invasive potential of colorectal cancer cells. In vitro invasion assay was carried out to compare and quantify the invasiveness of WT cells, Mock cells, Tiam1KD/clone 2, Tiam1KD/clone 6, and Tiam1KD/clone 8. Results are representative of three independent experiments, and bars represent the mean ± SD. Tiam1 silencing cells showed decreased invasion that correlated with Tiam1 protein expression.

Figure 7

Tiam1 gene silencing suppresses cell proliferation in vivo. (A) Consecutive external whole-body fluorescence images of Mock cells and Tiam1KD/clone 2 tumors were obtained from days 2 to 30 after subcutaneous injection into nude mice. (B) Tumor areas were calculated by IPP5.0 software and were indicated as the mean ± SD of six mice. Compared with Mock cells, Tiam1KD/clone 2 had a significantly reduced in vivo proliferative ability (F = 2.256, P < .05).

Figure 8

Tiam1 silencing reduces metastatic lesions in vivo. We assessed the effect of Tiam1 silencing on metastasis using an orthotopic xenograft model in nude mice. After 2 months, the colon, lungs, and liver of mice were resected and analyzed for metastasis. Left panel: Direct image of colon, lungs, and liver. Middle panel: External fluorescent image of colon, lungs, and liver. Right panel: Histologic photomicrographs of colon, lung, and liver tissue sections stained with H&E (original magnification, x400).