Abstract
Background and Aims: Small interfering RNAs (shRNAs) are able to suppress gene expression through the endogenous cellular process of RNA interference pathway. Given the ability to knock-down essentially any gene, shRNAs have been used in many studies to screen for therapeutic targets in various pathological conditions. However, most of these studies have been carried out in vitro and the results cannot always be translated into an in vivo environment. In this project, we aimed to develop an efficient in vivo cancer target validation method using a lentiviral inducible-knockdown shRNA. To verify our approach, we used an essential cell-cycle protein polo-like kinase 1 (PLK1) as a proof of concept target.
Methods: TRIPZ inducible shRNAmir construct (Open Biosystems) carrying shRNA against PLK1 was transfected into the colon carcinoma cell line SW620 using lentiviral transduction. PLK1 gene knock-down following induction with doxycycline was quantified by real-time PCR. Inducible shRNAmir expression (detected by turboRFP) was monitored by fluorescence microscopy and quantified using Flow-cytometry or a fluorescence microplate reader.
Nude mouse xenografts were established by subcutaneous injection of 5×106 PLK1 shRNA transfected SW620 cells into the left flank of female nude mice. Doxycycline treatment (80mg/kilogram daily via oral gavage) was initiated when the mean tumour volume reached 500mm3. Mice were sacrificed 3 weeks following treatment and tumours were collected. PLK1 gene expression was assessed by RT-PCR and protein levels investigated using immunohistochemistry. Assessment of angiogenesis (micro-vessel density) was carried out using CD34 staining.
Results:
Following doxycycline induction in vitro, PLK1 inducible-knockdown SW620 cells showed dose and time-dependent PLK1 down-regulation, which was consistent with shRNAmir expression as marked by turboRFP. Flow-cytometry analysis showed about 95% of cells expressed the shRNAmir, 72 hours after induction.
In the in vivo model, the doxycycline-treated group showed significantly lower PLK1 gene expression compared with untreated control (60% reduction, p<0.003). The doxycycline-treated group also showed significantly lower PLK1 protein expression compared with the untreated control (p<0.001).
There was a decline in tumour growth rate in the treated group compared with the control group, which confirmed that knock-down of PLK1 slowed down tumour growth.
We also observed that, within the tumours from the doxycycline-treated group, there appeared to be reduced PLK1 expression in areas adjacent to the blood vessels compared with other areas of the tumour.
Conclusion: The results support the anti-tumour effects of PLK1 down-regulation and confirm an efficient methodology for cancer target screening using a lentiviral inducible-knockdown shRNA system.
Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 752.
- ©2010 American Association for Cancer Research
Volume 70, Issue 8 Supplement
