Simultaneous siRNA-mediated knockdown of antiapoptotic BCL2, Bcl-xL, XIAP and survivin in bladder cancer cells

Abstract

Bladder cancer (BCa) represents the ninth most common malignancy worldwide. Despite intensive treatment with surgery and chemotherapy the prognosis for BCa patients particularly at advanced stages is poor. The ability to evade apoptosis is a hallmark of cancer cells. Since the antiapoptotic genes BCL2, Bcl-xL, XIAP and survivin are frequently upregulated in BCa tissues, their combined siRNA-mediated knockdown might be more potent in decreasing BCa growth than the single inhibition of one target. Against each target two siRNAs were selected that specifically reduced the mRNA and protein levels of their appropriate target in EJ28 and J82 BCa cells. Inhibition of survivin provoked the strongest antiproliferative effect of all single target treatments, for example cell counts decreased by 50%. Simultaneous targeting of all four antiapoptotic genes downregulated expression levels of all targets and mediated significant reductions in cell viability and cell counts as well as induction of apoptosis. In EJ28 cells, combined knockdown of BCL2, Bcl-xL, XIAP and survivin caused a 2.5-fold enhancement in apoptosis rate and reduced cellular viability by 40%. Therefore, simultaneous knockdown of antiapoptotic BCL2, Bcl‑xL, XIAP and survivin may represent a promising treatment option for bladder cancer.

Figure 1

Reduction of BCL2, Bcl-xL, XIAP and survivin mRNA levels dependent on the concentration of the siRNA 24 h after transfection with the appropriate target-specific siRNA in EJ28 BCa cells. Cells were transfected with 2, 5, 10, 20 or 40 nM target-specific or control siRNA. Values are normalised to the reference gene TBP and are shown relative to the respective control siRNA treatment (100%). Values represent averages of two independent experiments with their mean deviation.

Figure 2

Relative target mRNA expression levels of EJ28 and J82 bladder cancer cells 48 h after transfection with a total of 40 nM siRNA. Expression values are normalised to the reference gene TBP and are shown relative to the control siRNA (100%). Values represent averages of two independent experiments with their mean deviation.

Figure 3

Time-dependent reduction of BCL2, Bcl-xL, XIAP and survivin mRNA levels after transfection with 40 nM of the appropriate target-specific siRNA in EJ28 BCa cells. Values are normalised to the reference gene TBP and are shown relative to the control siRNA (100%). Values represent averages of two independent experiments with their mean deviation.

Figure 4

Detection of BCL2, Bcl-xL, XIAP and survivin protein content by western blotting 48 h after transfection with a total of 40 nM siRNA in EJ28 bladder cancer cells. Beta-actin was used for loading control.

Figure 5

Viability of EJ28 and J82 bladder cancer cells 96 h after transfection with a total of 40 nM siRNA. Values shown are relative to the control siRNA ‘ns-si’ (100%) and are averages of a fourfold determination. Error bars represent the 95% confidence interval. An unpaired Student’s t-test was used to compare the differences in cell viability between target-directed-siRNA and ns-si treated cells (^*p≤0.05, ^**p≤0.01, ^***p≤0.001).

Figure 6

Cell count of EJ28 and J82 bladder cancer cells 48 h after transfection with a total of 40 nM siRNA. Values shown are relative to the control siRNA ‘ns-si’ (100%) and are averages of two independent experiments. Error bars represent the mean deviation.

Figure 7

Percentage of early and late apoptotic cells 48 h after treatment of (A) EJ28 and (B) J82 cells with a total of 40 nM siRNAs. Values shown are representatives of two independent experiments.