Off-target effects of plasmid-transcribed shRNAs on NFκB signaling pathway and cell survival of human melanoma cells

Abstract

Signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) are transcription factors involved in cell survival, inflammation and metastasis. Constitutively activated STAT3 is found in many cancers, including melanoma. To study the crosstalk between STAT3 and NFκB signaling and its role in regulation of cancer cell survival, we used RNA interference (RNAi) to down-regulate STAT3 expression in human melanoma cells. RNAi strategies including double-stranded RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA are widely used to knock down disease-causing genes in a targeted fashion. We found that shRNAs up-regulate non-specific NFκB activity, while siRNA directed against STAT3 specifically increase NFκB activity. The basal survival of melanoma cells is unaffected by STAT3 knockdown-likely due to activation of pro-survival NFκB signaling. Whereas, owing to off-target effects, plasmid-transcribed shRNA affects melanoma survival. Our data show that shRNA-mediated gene silencing induces non-specific or off-target effects that may influence cell functions.

Fig. 1

Plasmid transcribed shRNAs nonspecifically induce NFκB activity in melanoma cells. a–b Representative pictures of WM239 melanoma cells 48 h after co-transfection with p-Silencer encoding STAT3 shRNA and EGFP plasmids. Using phase contrast (a) and fluorescence microscopy (b), transfection efficiency was evaluated as ~65 %. c The level of STAT3 silencing at the RNA expression level was evaluated with qPCR and related to STAT3 expression in the control shRNA transfected cells. d The levels of phosphorylated and total STAT3 and IκB proteins in cells expressing control shRNA or STAT3 specific shRNAs were examined by Western blotting. Immunoblots were re-probed with an antibody recognizing β-actin to ensure equal loading. Similar results were obtained in four independent experiments. Densitometry of immunoblots using Image J was performed. The analysis of the Western blots was done using β-actin as the loading control. e–f Increase of the NFκB transcriptional activity in WM239 melanoma cells transfected with plasmids encoding shRNA against STAT3 or control shRNAs. The cells were co-transfected with plasmids encoding STAT3 shRNA and NFκB reporter plasmids using Lipofectamine 2000. Luciferase activity was determined after 48 h using a dual light luciferase assay and normalized to the protein content determined in the sample. The results indicated that the activation of NFκB transcriptional activity is not specific for shRNA against STAT3. Two commercial control shRNAs cause similar up-regulation of NFκB-driven transcription (f). The results of three separate experiments are presented as mean ± S.D.

Fig. 2

STAT3 knockdown with siRNA leads to NFκB activation in human melanoma cells. a–b Representative pictures of WM239 melanoma cells 48 h after transfection with siRNA labeled with Rhodamine (siGlo) using AMAXA electroporation. Using fluorescence microscopy (a) and flow cytometry analysis (b) the efficacy of siRNA transfection was determined as 84 %. c Effective STAT3 knockdown in WM239 cells. The STAT3 mRNA level was determined using qPCR and was related to its level in control siRNA transfected cells. d The levels of phosphorylated and total STAT3 and IκB proteins in cells transfected with control or STAT3 specific siRNA were examined by Western blotting. Immunoblots were re-probed with an antibody recognizing β-actin to ensure equal loading. Similar results were obtained in three independent experiments. The right panel shows quantification of the Western blots from three experiments using Image J with β-actin as the loading control. e The increase of the NFκB transcriptional activity in melanoma cells transfected with STAT3 specific siRNA. Cells growing onto 24-wells plates were co-transfected with the NFκB-luc plasmid and control or STAT3 siRNA using AMAXA electroporation. The luciferase activity was measured 48 h after transfection. The bars indicate mean values of luciferase activity in the mock transfected cells and cells transfected with the control or STAT3 siRNA. Data are presented as mean ± S.D. from three experiments, each in duplicate. f Evaluation of NFκB DNA binding by ELISA. Cells (1 × 10⁷ cells/per group) were mock transfected or were co-transfected with a control or STAT3 siRNA using AMAXA electroporation. Cell nuclei extracts were collected 48 h after transfection and 2 μg of nuclear extract was subjected to an NFκB DNA binding assay (Active Motif—TransAM^® NFκB Family Kit, Carlsbad, USA). The NFκB -ELISA assay results demonstrated an increase in the NFκB binding to DNA after silencing the expression of STAT3. Data are presented as mean ± S.D. from three experiments. g–h. STAT3 knockdown with siRNA induces specifically NFκB-Luciferase activity in WM209 and T1 melanoma cell lines. The NFκB-Luciferase activity measured in WM209 and T1 cells untreated (mock), treated with electroporation only or in cells transfected with two empty plasmids such as p-Super and PCMV6-XL5, or plasmids coding for shRNAs or siRNAs. An increase of the NFκB transcriptional activity was observed in melanoma cells transfected with plasmids encoding shRNA against STAT3 and control shRNAs and siRNA against STAT3 and control. Electroporation itself or transfection with empty plasmids do not induce NFκB activation. Data are presented as mean ± S.D. from three experiments

Fig. 3

STAT3 knockdown with specific shRNA, but not siRNA, reduces cell survival and induces DNA fragmentation in melanoma cells. a–b Cells (1 × 10⁷ cells/per group) were mock transfected or were transfected with a control or STAT3 siRNA or plasmids coding for control or STAT3 specific shRNAs. Cell viability was determined using the MTT metabolism assay 48 h after the transfection of the indicated shRNA or siRNA. Measurements were related to respective controls (cells transfected with ctr siRNA or shRNA were taken as 100 %) and data represent mean ± S.D. from three experiments, each in triplicate. c STAT3 knockdown with two specific shRNAs results in induction of apoptotic cell death. The levels of phosphorylated and total STAT3 and cleaved PARP proteins in cells transfected with plasmids coding for control or STAT3 specific shRNA were examined by Western blotting. Immunoblots were re-probed with an antibody recognizing β-actin to ensure equal loading. Similar results were obtained in three independent experiments. d Quantification of the Western blots by using β-actin as the loading control using Image J. e DNA fragmentation occurs in cells transfected with STAT3 specific shRNA. TUNEL staining was performed on cells 48 h after transfection and then observed using a fluorescence microscope. TUNEL staining is shown in green. In the top right is the positive control with cells incubated with Dnase1 and the negative control below are cells labeled with Label solution (without terminal transferase). f The percentage of TUNEL-positive cells was defined as the number of TUNEL-stained cells divided by the total number of cells seen with bright field. At least 600 cells in three randomly selected fields of view from each well were counted for three independent experiments