Abstract Background RNA interference (RNAi) is a powerful platform utilized to target transcription of specific genes and downregulate the protein product. To achieve effective silencing, RNAi is usually applied to cells or tissue with a transfection reagent to enhance entry into cells. A commonly used control is the same transfection reagent plus a “noncoding RNAi”. However, this does not control for the genomic response to the transfection reagent alone or in combination with the noncoding RNAi. These control effects while not directly targeting the gene in question may influence expression of other genes that in turn alter expression of the target. The current study was prompted by our work focused on prevention of vascular bypass graft failure and our experience with gene silencing in human aortic smooth muscle cells (HAoSMCs) where we suspected that off target effects through this mechanism might be substantial. We have used Next Generation Sequencing (NGS) technology and bioinformatics analysis to examine the genomic response of HAoSMCs to the transfection reagent alone (polyethyleneimine (PEI)) or in combination with commercially obtained control small interfering RNA (siRNAs) (Dharmacon and Invitrogen). Results Compared to untreated cells, global gene expression of HAoSMcs after transfection either with PEI or in combination with control siRNAs displayed significant alterations in gene transcriptome after 24 h. HAoSMCs transfected by PEI alone revealed alterations of 213 genes mainly involved in inflammatory and immune responses. HAoSMCs transfected by PEI complexed with siRNA from either Dharmacon or Invitrogen showed substantial gene variation of 113 and 85 genes respectively. Transfection of cells with only PEI or with PEI and control siRNAs resulted in identification of 20 set of overlapping altered genes. Further, systems biology analysis revealed key master regulators in cells transfected with control siRNAs including the cytokine, Interleukin (IL)-1, transcription factor GATA Binding Protein (GATA)-4 and the methylation enzyme, Enhancer of zeste homolog 2 (EZH-2) a cytokine with an apical role in initiating the inflammatory response. Conclusions Significant off-target effects in HAoSMCs transfected with PEI alone or in combination with control siRNAs may lead to misleading conclusions concerning the effectiveness of a targeted siRNA strategy. The lack of structural information about transfection reagents and “non coding” siRNA is a hindrance in the development of siRNA based therapeutics. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2267-9) contains supplementary material, which is available to authorized users. Keywords: RNAi, Transfection reagent, PEI, Control siRNA Background RNAi is an emerging technology using a natural mechanism to inhibit gene expression via the degradation of target mRNAs through siRNA. We have been exploring RNAi for the purpose of modifying the vascular response to injury especially during graft implantation, employing endothelial cells and vascular smooth muscle cells, under various conditions and technology for siRNA delivery [[39]1–[40]4]. Although these studies have displayed promise of RNAi therapy as an alternative means to treat vascular diseases, there is so far no study discussing how transfection reagent alone and in combination with non-targeting (control) siRNAs affect gene expression level. Our group is interested in developing siRNA based therapies to treat vascular bypass grafts, both vein and prosthetic, to prevent vascular graft failure. Injury to the graft and the host artery during graft implantation is the most important trigger for the downstream graft failure. Vascular smooth muscle cells play a major role in the failure process and thus are often targeted for therapeutic intervention. Thus in the present study the cells chosen were HAoSMCs [[41]1–[42]4]. When determining the effectiveness of siRNA silencing of a target gene, the standard control has been a comparison with control siRNA. In our laboratory we have used a series of controls. For example, if using a transfection reagent, our controls would be: 1. Saline alone. 2. Transfection reagent alone. 3. Transfection reagent plus control siRNA. We have noticed differences between these controls with regard to expression of the target gene, raising the question of the control effects on total genomic expression. For example, if the transfection reagent alters target gene expression and that is different from the transfection reagent plus the control siRNA, which is the better control? Can we count on the control siRNA to have no effect on target gene expression? Should saline or balanced salt solution be the appropriate control as it is least likely to affect target gene transcription? In this study, experiments were designed to determine the global effects on gene transcription of saline, a transfection reagent, and the transfection reagent plus control siRNA using RNA sequencing. Since our goal is to treat vascular bypass grafts with siRNA, we chose a transfection reagent PEI, as this is frequently used for in vivo applicability. Control siRNAs were obtained from Invitrogen and Dharmacon. Results Unsupervised clustering suggests that untreated (NT) and HAoSMCs treated with P, PI and PD are transcriptionally different HAoSMCs were either treated with PEI alone (P), Invitrogen control siRNA complexed with PEI (PI) or Dharmacon control siRNA complexed with PEI (PD) for 24 h. HAoSMCs that were left untreated (NT) served as the experimental control. After treatment, cells were subjected to RNA isolation as described above. After preprocessing and normalization of RNA sequencing data we performed unsupervised analysis using PCA to determine relationship between different treatment groups as well as samples within each group. The unsupervised analysis demonstrated that samples are separated on the basis of transfection that is NT versus P, PI and PD along primary component (PC) 1 (Fig. [43]1). The samples from PEI alone group depicted maximum transcriptional differences as compared to control NT, PI and PD groups along primary component (PC) 2. Biological replicates from most of the groups clustered together indicating similar transcriptome profile. Fig. 1. Fig. 1 [44]Open in a new tab Principle Component Analysis (PCA) of HAoSMCs that were treated as follows: No Treatment (NT), PEI alone (P), PEI combined with control siRNA from Invitrogen (PI) and PEI combined with control siRNA from Dharmacon siRNA (PD). PCA analysis of three replicates in each treatment group suggests that NT and HAoSMCs treated with P, PI and PD form different clusters indicating that these treatment groups are transcriptionally different from each other Inflammation and apoptosis related genes are upregulated/activated due to PEI transfection PEI (P), which comes in two forms; linear and branched polymer, has been extensively used as a non-viral cationic carrier to deliver drugs or genes into the cells via proton sponge effects [[45]5]. The supervised comparison of NT samples with P only samples identified 213 significantly differentially expressed genes with false discovery rate <5 % and at least 2-fold change. Out of 213 differentially expressed genes, 115 and 98 were significantly downregulated and upregulated respectively (Fig. [46]2a and Additional file [47]1: Table S2). Treatment with P upregulated multiple genes linked to cell-mediated immune response and inflammatory response including Prostaglandin-Endoperoxide Synthase (PTGS) 2, Nicotinamide phosphoribosyltransferase (NAMPT), most prominently several chemokines and chemokine receptors genes such as Chemokine (C-X-C motif) Ligand (CXCL)-2, CXCL-11, CXCL-8, IL-1A, IL-11, Chemokine (C-C motif) Ligand (CCL)-5 and Colony Stimulating Factor (CSF)-2 (Fig. [48]2a). Fig. 2. Fig. 2 [49]Open in a new tab Transcriptional and biological characterization of alteration in HAoSMC induced by PEI. (a) Heatmap of genes that are significantly differentially expressed due to treatment with PEI alone (P) compared to No Treatment (NT). In the heatmap, rows depict differentially expressed genes and columns depict three replicates each of NT and P treated HAoSMC. The relative expression level of genes is shown using a pseudocolor scale from −3 to +3. Colors indicate standardized values (green represents down regulation and red represents up regulation). (b) Functional categories enrichment analysis of all significantly differentially expressed genes, and (c) Pathways enrichment analysis of all significantly differentially expressed genes To understand the underlying biological mechanism of alterations induced due to PEI treatment, we performed gene-ontology (GO) categories and canonical pathways analysis. The GO analysis of differentially expressed genes identified significantly affected categories (P value <0.01) that include cytokine activity, inflammatory response, chemokine activity and immune response (Fig. [50]2b). Furthermore, pathways analysis on the list of differentially expressed genes identified significant pathways (multiple test corrected P value <0.01) that include, Granulocyte Adhesion and Diapedesis, peroxisome proliferator-activated receptor (PPAR) signaling, IL-10 signaling, and IL-6 signaling (Fig. [51]2c). These pathways that are triggered by immune systems in response to PEI transfection, play a critical role in multiple diseases including cancer, immunological and neurodegenerative diseases. The activation of immune and inflammatory pathways 24 h after in vitro transfection with PEI at the vendor recommended N/P ratio in HAoSMCs suggests toxicity associated with PEI. Transcriptional alterations due to control siRNA compared to PEI alone To understand the non-specific transcriptional alterations induced by control siRNA when combined with PEI, we performed global RNA sequencing on samples transfected with control unlabeled siRNA from Invitrogen or Dharmacon combined with PEI. The transfection of samples with control unlabeled siRNA from Invitrogen or Dharmacon compared to PEI alone significantly altered (FDR <5 % and FC > ± 2) 132 and 85 genes respectively (Fig. [52]3a and Additional file [53]1: Table S3A, S3B). A significant number (64) of genes were commonly altered by unlabeled siRNA from Invitrogen or Dharmacon (Fig. [54]3b and Additional file [55]1: Table S4) suggesting similar mechanisms that may affect transcriptional profile of the cells. To understand the biological mechanism underlying the alterations induced by control siRNA transfection, we performed functional and pathways enrichment analysis on the genes that were commonly altered by both Invitrogen and Dharmacon control siRNA. The functions uniting commonly differentially expressed genes were dominated by functions involved in cell proliferation and growth and immune/inflammatory response (Fig. [56]3c). Further pathways analysis on commonly affected genes depicted significant enrichment in inflammatory response pathways including Granulocyte/Agranulocyte Adhesion and Diapedesis. These pathways are the primary line of host defense against infection by bacterial pathogens and are rapidly recruited to sites of bacterial invasion suggesting that control siRNAs are probably recognized as foreign entities [[57]6]. Fig. 3. Fig. 3 [58]Open in a new tab Transcriptional characterization of alteration induced in HAoSMC by transfection with PEI combined with control siRNA from Invitrogen (PI) or Dharmacon (PD). (a) Heat map of significantly differentially expressed genes due to transfection with PI or PD, (b) Functional categories significantly altered due to transfection with PI, (c) Functional categories that depict pattern of alteration (Raw P value < .01) due to transfection with PD. No functional category was found significant after multiple test correction, and (d) Venn diagram depicting genes common between HAoSMC treated with PI and PD Control siRNA and PEI lead to immune systems and inflammation related transcriptional changes Compared to NT, transfection of cells with either PI or PD resulted in significant transcriptional changes (Fig. [59]4a). The transfection of cells with PI resulted in dysregulation of 66 genes (Additional file [60]1: Table S5A), mostly dominated by genes linked to inflammation related pathways including “Agranulocyte/Granulocyte Adhesion and Diapedesis”, “Farnesoid X Receptor/ Retinoid X Receptor (FXR/RXR) Activation”, “Acute Phase Response Signaling”, “Dendritic Cell Maturation”, “IL-6 Signaling”, “p38 Mitogen-Activated Protein Kinase (MAPK) Signaling”, and “PPAR Signaling” (Fig. [61]4b). Similarly, transfection of cells with PD resulted in dysregulation of 78 genes (Additional file [62]1: Table S5B), which are dominated by genes linked to inflammation related pathways comparable to PI (Fig. [63]4b). Thus, transfection with both Dharmacon and Invitrogen non-targeting control siRNAs resulted in inflammation response similar to host defense response against infection of bacteria’s or viruses described above. The analysis identified a core set of 20 overlapping genes that are significantly altered due to transfection of cells with P, PI or PD (Table [64]1). This core set of overlapping genes is dominated by genes linked to immune/inflammatory and cell proliferation including Kinesin Family Member (KIF)-1A, STAT-4, CCL-8, Superoxide Dismutase (SOD)-2, IL-36B, CSF-2, and IL3-6RN (Fig. [65]4c). These genes have also been linked to vascular dysregulation and the associations are listed in Table [66]2. Fig. 4. Fig. 4 [67]Open in a new tab Transcriptional characterization of alteration induced in HAoSMC by PEI alone (P) or PEI combined with control siRNA from Invitrogen (PI) or Dharmacon (PD). (a) Venn Diagram depicting overlap among the genes that are differentially expressed in HAoSMC due to P alone, PI or PD as compared to no treatment (NT), (b) Functions significantly altered due to transfection with PI and PD, (c) Pathways significantly altered due to transfection with PI and PD, and (d) Heatmap of 20 common significantly differentially expressed genes due to transfection with P, PI or PD Table 1. List of commonly significantly differentially expressed genes due to transfection of PEI alone (P), and PEI with Invitrogen (PI) and Dharmacon (PD) control siRNA respectively Genes P vs. NT PI vs. NT PD vs. NT Log FC P value FDR log FC P value FDR Log FC PV value FDR KRT5 −9.00108542 1.82E-08 9.11E-07 −9.00108542 1.12E-08 1.85E-06 −9.00108542 4.00E-09 5.77E-07 DSC2 −5.547567862 9.33E-06 0.000210938 −7.514505361 3.54E-07 3.79E-05 −5.843576549 2.01E-06 0.00010804 CASP14 −4.866920217 1.36E-06 3.98E-05 −6.718742357 9.08E-09 1.56E-06 −3.84210716 2.16E-05 0.000762738 PKP1 −4.065832231 4.03E-05 0.000734415 −5.837618386 4.72E-07 4.98E-05 −4.812633855 2.41E-06 0.000126389 SFN −3.866190605 1.96E-05 0.000390234 −2.706646929 0.000813618 0.022294014 −5.116141429 1.68E-07 1.47E-05 XIST −2.6415241 0.000447384 0.005355807 −4.189237025 9.64E-07 9.24E-05 −2.564333239 0.000399303 0.007636661 KIF1A −2.37198378 0.001162629 0.011613329 −6.165808144 1.86E-09 3.89E-07 −2.639316704 0.000279027 0.005863775 PABPC1L −1.469888511 5.36E-08 2.38E-06 −1.136168153 1.40E-05 0.000867902 −1.006563861 7.85E-05 0.002217316 MMRN2 −1.148174676 2.51E-05 0.000484772 −1.125962103 2.61E-05 0.00144306 −1.140277865 1.12E-05 0.00044101 STAT4 1.030820203 0.00090642 0.009359621 1.164708993 0.000135637 0.005691294 1.401188438 2.28E-06 0.000120413 ELN 1.327728145 5.88E-23 1.68E-20 1.389401412 3.20E-25 1.34E-21 1.303860319 1.21E-22 1.52E-19 CCL8 1.434021582 2.81E-11 2.43E-09 1.621652693 1.26E-14 8.32E-12 1.716686958 7.40E-17 3.37E-14 SOD2 1.461039459 3.51E-39 2.20E-36 1.007297504 7.52E-20 1.18E-16 1.329823799 5.71E-33 1.79E-29 CNTNAP2 1.550048726 6.82E-17 1.17E-14 1.167033839 4.31E-10 1.13E-07 1.302670483 1.60E-12 4.10E-10 C8orf4 1.657269124 1.89E-46 2.16E-43 1.214785377 1.07E-25 6.74E-22 1.229534359 1.34E-26 3.37E-23 NR3C2 1.839357043 6.15E-12 6.08E-10 1.505931617 3.47E-08 4.93E-06 1.89042933 2.94E-13 7.86E-11 IL36B 2.065054755 1.81E-13 2.09E-11 1.371901177 1.91E-06 0.000158997 1.623123119 7.63E-09 1.01E-06 CSF2 2.157136236 2.41E-36 1.38E-33 1.04449791 5.99E-10 1.46E-07 1.692407468 1.62E-23 2.77E-20 AMH 2.487063814 2.12E-08 1.05E-06 1.631133689 0.00045514 0.014311243 2.135611981 1.50E-06 8.61E-05 IL36RN 4.508694239 2.15E-34 1.12E-31 3.347303687 2.41E-18 2.33E-15 3.737917275 1.76E-23 2.77E-20 [68]Open in a new tab Table 2. Effects of the altered genes after transfection of cells with PEI and control siRNAs on vascular system in the literature Genes Effects on vascular system References