title: The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells.
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.
authors.affiliation: The Frank W. LoGerfo Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Stoneman 8 M-10E, Boston, 02215, MA, USA. nurazhani@gmail.com.
authors.forename: Nurazhani A
authors.initials: NA
authors.lastname: Raof
authors.affiliation: Division of Interdisciplinary Medicine and Biotechnology, Genomics and Proteomics Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA, 02215, USA. drajaman@bidmc.harvard.edu.
authors.forename: Deepa
authors.initials: D
authors.lastname: Rajamani
authors.affiliation: The Frank W. LoGerfo Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Stoneman 8 M-10E, Boston, 02215, MA, USA. Hsunchien@gmail.com.|Department of Medicine, National Yang-Ming University, School of Medicine, Taipei City, Taiwan. Hsunchien@gmail.com.
authors.forename: Hsun-Chieh
authors.initials: HC
authors.lastname: Chu
authors.affiliation: The Frank W. LoGerfo Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Stoneman 8 M-10E, Boston, 02215, MA, USA. aniket.gurav@gmail.com.
authors.forename: Aniket
authors.initials: A
authors.lastname: Gurav
authors.affiliation: The Frank W. LoGerfo Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Stoneman 8 M-10E, Boston, 02215, MA, USA. jjohns18@bidmc.harvard.edu.
authors.forename: Joel M
authors.initials: JM
authors.lastname: Johnson
authors.affiliation: The Frank W. LoGerfo Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Stoneman 8 M-10E, Boston, 02215, MA, USA. flogerfo@bidmc.harvard.edu.
authors.forename: Frank W
authors.initials: FW
authors.lastname: LoGerfo
authors.affiliation: The Frank W. LoGerfo Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Stoneman 8 M-10E, Boston, 02215, MA, USA. lpradhan@bidmc.harvard.edu.
authors.forename: Leena
authors.initials: L
authors.lastname: Pradhan-Nabzdyk
authors.affiliation: Division of Interdisciplinary Medicine and Biotechnology, Genomics and Proteomics Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA, 02215, USA. mbhasin@bidmc.harvard.edu.
authors.forename: Manoj
authors.initials: M
authors.lastname: Bhasin
mesh_terms: D001011:Aorta / Q000187:drug effects* / Q000378:metabolism; D019295:Computational Biology; D000071221:Enhancer of Zeste Homolog 2 Protein; D050991:GATA4 Transcription Factor / Q000096:biosynthesis; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D020868:Gene Silencing; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D063151:Polycomb Repressive Complex 2 / Q000096:biosynthesis; D011094:Polyethyleneimine / Q000008:administration & dosage; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D050991:GATA4 Transcription Factor; D007375:Interleukin-1; D034741:RNA, Small Interfering; D011094:Polyethyleneimine; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D063151:Polycomb Repressive Complex 2