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Rajamani, Deepa

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Rajamani

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Deepa

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Rajamani, Deepa

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20163

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Author's Publications: search.filters.isAuthorOfPublication.0403712f-e901-4e03-bbd2-7f59de923f03

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    The effects of transfection reagent polyethyleneimine (PEI) and non-targeting control siRNAs on global gene expression in human aortic smooth muscle cells
    (BioMed Central, 2016) Raof, Nurazhani A.; Rajamani, Deepa; Chu, Hsun-Chieh; Gurav, Aniket; Johnson, Joel M.; Logerfo, Frank; Pradhan-Nabzdyk, Leena; Bhasin, Manoj
    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.
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    Temporal retinal transcriptome and systems biology analysis identifies key pathways and hub genes in Staphylococcus aureus endophthalmitis
    (Nature Publishing Group, 2016) Rajamani, Deepa; Singh, Pawan Kumar; Rottmann, Bruce G.; Singh, Natasha; Bhasin, Manoj; Kumar, Ashok
    Bacterial endophthalmitis remains a devastating inflammatory condition associated with permanent vision loss. Hence, assessing the host response in this disease may provide new targets for intervention. Using a mouse model of Staphylococcus aureus (SA) endophthalmitis and performing retinal transcriptome analysis, we discovered progressive changes in the expression of 1,234 genes. Gene ontology (GO) and pathway analyses revealed the major pathways impacted in endophthalmitis includes: metabolism, inflammatory/immune, antimicrobial, cell trafficking, and lipid biosynthesis. Among the immune/inflammation pathways, JAK/Stat and IL-17A signaling were the most significantly affected. Interactive network-based analyses identified 13 focus hub genes (IL-6, IL-1β, CXCL2, STAT3, NUPR1, Jun, CSF1, CYR61, CEBPB, IGF-1, EGFR1, SPP1, and TGM2) within these important pathways. The expression of hub genes confirmed by qRT-PCR, ELISA (IL-6, IL-1β, and CXCL2), and Western blot or immunostaining (CEBP, STAT3, NUPR1, and IGF1) showed strong correlation with transcriptome data. Since TLR2 plays an important role in SA endophthalmitis, counter regulation analysis of TLR2 ligand pretreated retina or the use of retinas from TLR2 knockout mice showed the down-regulation of inflammatory regulatory genes. Collectively, our study provides, for the first time, a comprehensive analysis of the transcriptomic response and identifies key pathways regulating retinal innate responses in staphylococcal endophthalmitis.
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    Identification of key regulators of pancreatic cancer progression through multidimensional systems-level analysis
    (BioMed Central, 2016) Rajamani, Deepa; Bhasin, Manoj
    Background: Pancreatic cancer is an aggressive cancer with dismal prognosis, urgently necessitating better biomarkers to improve therapeutic options and early diagnosis. Traditional approaches of biomarker detection that consider only one aspect of the biological continuum like gene expression alone are limited in their scope and lack robustness in identifying the key regulators of the disease. We have adopted a multidimensional approach involving the cross-talk between the omics spaces to identify key regulators of disease progression. Methods: Multidimensional domain-specific disease signatures were obtained using rank-based meta-analysis of individual omics profiles (mRNA, miRNA, DNA methylation) related to pancreatic ductal adenocarcinoma (PDAC). These domain-specific PDAC signatures were integrated to identify genes that were affected across multiple dimensions of omics space in PDAC (genes under multiple regulatory controls, GMCs). To further pin down the regulators of PDAC pathophysiology, a systems-level network was generated from knowledge-based interaction information applied to the above identified GMCs. Key regulators were identified from the GMC network based on network statistics and their functional importance was validated using gene set enrichment analysis and survival analysis. Results: Rank-based meta-analysis identified 5391 genes, 109 miRNAs and 2081 methylation-sites significantly differentially expressed in PDAC (false discovery rate ≤ 0.05). Bimodal integration of meta-analysis signatures revealed 1150 and 715 genes regulated by miRNAs and methylation, respectively. Further analysis identified 189 altered genes that are commonly regulated by miRNA and methylation, hence considered GMCs. Systems-level analysis of the scale-free GMCs network identified eight potential key regulator hubs, namely E2F3, HMGA2, RASA1, IRS1, NUAK1, ACTN1, SKI and DLL1, associated with important pathways driving cancer progression. Survival analysis on individual key regulators revealed that higher expression of IRS1 and DLL1 and lower expression of HMGA2, ACTN1 and SKI were associated with better survival probabilities. Conclusions: It is evident from the results that our hierarchical systems-level multidimensional analysis approach has been successful in isolating the converging regulatory modules and associated key regulatory molecules that are potential biomarkers for pancreatic cancer progression. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0282-3) contains supplementary material, which is available to authorized users.