scholarly journals Down-regulation of Fra a 1.02 in strawberry fruits causes transcriptomic and metabolic changes compatible with an altered defense response

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Begoña Orozco-Navarrete ◽  
Jina Song ◽  
Ana Casañal ◽  
Rosangela Sozzani ◽  
Victor Flors ◽  
...  
Keyword(s):  
Redox State ◽  
Biological Process ◽  
Differentially Expressed ◽  
Strawberry Fruit ◽  
Rna Seq ◽  
Strawberry Plant ◽  
Oxidation Reduction ◽  
Pathogenesis Related ◽  
Transgenic Lines ◽  
Glutathione S Transferases

AbstractThe strawberry Fra a 1 proteins belong to the class 10 Pathogenesis-Related (PR-10) superfamily. In strawberry, a large number of members have been identified, but only a limited number is expressed in the fruits. In this organ, Fra a 1.01 and Fra a 1.02 are the most abundant Fra proteins in the green and red fruits, respectively, however, their function remains unknown. To know the function of Fra a 1.02 we have generated transgenic lines that silence this gene, and performed metabolomics, RNA-Seq, and hormonal assays. Previous studies associated Fra a 1.02 to strawberry fruit color, but the analysis of anthocyanins in the ripe fruits showed no diminution in their content in the silenced lines. Gene ontology (GO) analysis of the genes differentially expressed indicated that oxidation/reduction was the most represented biological process. Redox state was not apparently altered since no changes were found in ascorbic acid and glutathione (GSH) reduced/oxidized ratio, but GSH content was reduced in the silenced fruits. In addition, a number of glutathione-S-transferases (GST) were down-regulated as result of Fra a 1.02-silencing. Another highly represented GO category was transport which included a number of ABC and MATE transporters. Among the regulatory genes differentially expressed WRKY33.1 and WRKY33.2 were down-regulated, which had previously been assigned a role in strawberry plant defense. A reduced expression of the VQ23 gene and a diminished content of the hormones JA, SA, and IAA were also found. These data might indicate that Fra a 1.02 participates in the defense against pathogens in the ripe strawberry fruits.

scholarly journals Comprehensive Analysis of lncRNAs and circRNAs Reveals the Metabolic Specialization in Oxidative and Glycolytic Skeletal Muscles

2019 ◽  
Vol 20 (12) ◽  
pp. 2855 ◽  
Author(s):  
Linyuan Shen ◽  
Mailin Gan ◽  
Qianzi Tang ◽  
Guoqing Tang ◽  
Yanzhi Jiang ◽  
...  
Keyword(s):  
Meat Quality ◽  
Skeletal Muscles ◽  
Target Genes ◽  
Fiber Type ◽  
Muscle Metabolism ◽  
Reduction Process ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Oxidation Reduction ◽  
Regulatory Analysis

The biochemical and functional differences between oxidative and glycolytic muscles could affect human muscle health and animal meat quality. However, present understanding of the epigenetic regulation with respect to lncRNAs and circRNAs is rudimentary. Here, porcine oxidative and glycolytic skeletal muscles, which were at the growth curve inflection point, were sampled to survey variant global expression of lncRNAs and circRNAs using RNA-seq. A total of 4046 lncRNAs were identified, including 911 differentially expressed lncRNAs (p < 0.05). The cis-regulatory analysis identified target genes that were enriched for specific GO terms and pathways (p < 0.05), including the oxidation-reduction process, glycolytic process, and fatty acid metabolic. All these were closely related to different phenotypes between oxidative and glycolytic muscles. Additionally, 810 circRNAs were identified, of which 137 were differentially expressed (p < 0.05). Interestingly, some circRNA-miRNA-mRNA networks were found, which were closely linked to muscle fiber-type switching and mitochondria biogenesis in muscles. Furthermore, 44.69%, 39.19%, and 54.01% of differentially expressed mRNAs, lncRNAs, and circRNAs respectively were significantly enriched in pig quantitative trait loci (QTL) regions for growth and meat quality traits. This study reveals a mass of candidate lncRNAs and circRNAs involved in muscle physiological functions, which may improve understanding of muscle metabolism and development from an epigenetic perspective.

scholarly journals Spliced genes in muscle from Nelore Cattle and their association with carcass and meat quality

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Danielly B. S. Silva ◽  
Larissa F. S. Fonseca ◽  
Daniel G. Pinheiro ◽  
Ana F. B. Magalhães ◽  
Maria M. M. Muniz ◽  
...  
Keyword(s):  
Biological Process ◽  
Gene Families ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Splice Sites ◽  
Protein Ubiquitination ◽  
Alternatively Spliced ◽  
Nelore Cattle ◽  
Go Terms ◽  
Intramuscular Fat Content

Abstract Transcript data obtained by RNA-Seq were used to identify differentially expressed alternatively spliced genes in ribeye muscle tissue between Nelore cattle that differed in their ribeye area (REA) or intramuscular fat content (IF). A total of 166 alternatively spliced transcripts from 125 genes were significantly differentially expressed in ribeye muscle between the highest and lowest REA groups (p ≤ 0.05). For animals selected on their IF content, 269 alternatively spliced transcripts from 219 genes were differentially expressed in ribeye muscle between the highest and lowest IF animals. Cassette exons and alternative 3′ splice sites were the most frequently found alternatively spliced transcripts for REA and IF content. For both traits, some differentially expressed alternatively spliced transcripts belonged to myosin and myotilin gene families. The hub transcripts were identified for REA (LRRFIP1, RCAN1 and RHOBTB1) and IF (TRIP12, HSPE1 and MAP2K6) have an important role to play in muscle cell degradation, development and motility. In general, transcripts were found for both traits with biological process GO terms that were involved in pathways related to protein ubiquitination, muscle differentiation, lipids and hormonal systems. Our results reinforce the biological importance of these known processes but also reveal new insights into the complexity of the whole cell muscle mRNA of Nelore cattle.

scholarly journals Differential Expression Analysis of ZIKV Infected Human RNA Sequence Reveals Potential Biomarkers

2018 ◽  
Author(s):  
Almas Jabeen ◽  
Nadeem Ahmad ◽  
Khalid Raza
Keyword(s):  
Differential Expression ◽  
Differentially Expressed Genes ◽  
Zika Virus ◽  
Biological Process ◽  
Differential Expression Analysis ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Analysis Pipeline ◽  
Viral Outbreak ◽  
Potential Biomarkers

Zika virus (ZIKV) is considered to be an emerging viral outbreak due to its link to diseases like microcephaly, Guillain-Barre Syndrome in human. In this paper, we identify differentially expressed genes (DEGs) using RNA-seq data. In this study, we adopted the RNA-seq analysis pipeline to quantify RNA-seq data into read counts. Our analysis uncovers the significant DEGs which may be involved in the altered biological process somehow. Here, we report the list of significant DEGs, out of which three genes are found to be highly differentially expressed. In addition, our analysis also predicts other moderate DEGs, low DEGs whose differential expression was induced due to ZIKV infections.

scholarly journals OP0021 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN EARLY RHEUMATOID ARTHRITIS PATIENTS RESPONDING TO TOCILIZUMAB

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 12.2-12
Author(s):  
I. Muller ◽  
M. Verhoeven ◽  
H. Gosselt ◽  
M. Lin ◽  
T. De Jong ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
T Cells ◽  
Gene Ontology ◽  
Regulatory T Cells ◽  
Early Rheumatoid Arthritis ◽  
Whole Blood ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Double Blind

Background:Tocilizumab (TCZ) is a monoclonal antibody that binds to the interleukin 6 receptor (IL-6R), inhibiting IL-6R signal transduction to downstream inflammatory mediators. TCZ has shown to be effective as monotherapy in early rheumatoid arthritis (RA) patients (1). However, approximately one third of patients inadequately respond to therapy and the biological mechanisms underlying lack of efficacy for TCZ remain elusive (1). Here we report gene expression differences, in both whole blood and peripheral blood mononuclear cells (PBMC) RNA samples between early RA patients, categorized by clinical TCZ response (reaching DAS28 < 3.2 at 6 months). These findings could lead to identification of predictive biomarkers for TCZ response and improve RA treatment strategies.Objectives:To identify potential baseline gene expression markers for TCZ response in early RA patients using an RNA-sequencing approach.Methods:Two cohorts of RA patients were included and blood was collected at baseline, before initiating TCZ treatment (8 mg/kg every 4 weeks, intravenously). DAS28-ESR scores were calculated at baseline and clinical response to TCZ was defined as DAS28 < 3.2 at 6 months of treatment. In the first cohort (n=21 patients, previously treated with DMARDs), RNA-sequencing (RNA-seq) was performed on baseline whole blood PAXgene RNA (Illumina TruSeq mRNA Stranded) and differential gene expression (DGE) profiles were measured between responders (n=14) and non-responders (n=7). For external replication, in a second cohort (n=95 therapy-naïve patients receiving TCZ monotherapy), RNA-seq was conducted on baseline PBMC RNA (SMARTer Stranded Total RNA-Seq Kit, Takara Bio) from the 2-year, multicenter, double-blind, placebo-controlled, randomized U-Act-Early trial (ClinicalTrials.gov identifier: NCT01034137) and DGE was analyzed between 84 responders and 11 non-responders.Results:Whole blood DGE analysis showed two significantly higher expressed genes in TCZ non-responders (False Discovery Rate, FDR < 0.05): urotensin 2 (UTS2) and caveolin-1 (CAV1). Subsequent analysis of U-Act-Early PBMC DGE showed nine differentially expressed genes (FDR < 0.05) of which expression in clinical TCZ non-responders was significantly higher for eight genes (MTCOP12, ZNF774, UTS2, SLC4A1, FECH, IFIT1B, AHSP, and SPTB) and significantly lower for one gene (TND2P28M). Both analyses were corrected for baseline DAS28-ESR, age and gender. Expression of UTS2, with a proposed function in regulatory T-cells (2), was significantly higher in TCZ non-responders in both cohorts. Furthermore, gene ontology enrichment analysis revealed no distinct gene ontology or IL-6 related pathway(s) that were significantly different between TCZ-responders and non-responders.Conclusion:Several genes are differentially expressed at baseline between responders and non-responders to TCZ therapy at 6 months. Most notably, UTS2 expression is significantly higher in TCZ non-responders in both whole blood as well as PBMC cohorts. UTS2 could be a promising target for further analyses as a potential predictive biomarker for TCZ response in RA patients in combination with clinical parameters (3).References:[1]Bijlsma JWJ, Welsing PMJ, Woodworth TG, et al. Early rheumatoid arthritis treated with tocilizumab, methotrexate, or their combination (U-Act-Early): a multicentre, randomised, double-blind, double-dummy, strategy trial. Lancet. 2016;388(10042):343-55.[2]Bhairavabhotla R, Kim YC, Glass DD, et al. Transcriptome profiling of human FoxP3+ regulatory T cells. Human Immunology. 2016;77(2):201-13.[3]Gosselt HR, Verhoeven MMA, Bulatovic-Calasan M, et al. Complex machine-learning algorithms and multivariable logistic regression on par in the prediction of insufficient clinical response to methotrexate in rheumatoid arthritis. Journal of Personalized Medicine. 2021;11(1).Disclosure of Interests:None declared

scholarly journals OP0286 PROTEOMICS AND RNA SEQUENCING APPROACHES HIGHLIGHT THE ROLE OF ENDOTHELIAL CELL DYSREGULATION IN IL-1 AND IFN MEDIATED AUTOINFLAMMATORY DISEASES, NOMID AND CANDLE

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 178-179
Author(s):  
S. Alehashemi ◽  
M. Garg ◽  
B. Sellers ◽  
A. De Jesus ◽  
A. Biancotto ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Whole Blood ◽  
Cell Activation ◽  
Immune Cell ◽  
Differentially Expressed ◽  
Neutrophilic Dermatosis ◽  
Healthy Controls ◽  
Differentially Expressed Proteins ◽  
Rna Seq ◽  
Before And After

Background:Systemic Autoinflammatory diseases present with sterile inflammation. NOMID (Neonatal-Onset Multisystem Inflammatory Disease) is caused by gain-of-function mutations inNLRP3and excess IL-1 production, presents with fever, neutrophilic dermatosis, aseptic meningitis, hearing loss and eye inflammation; CANDLE (Chronic Atypical Neutrophilic Dermatosis, Lipodystrophy and Elevated Temperature) is caused by loss-of-function mutations in proteasome genes that lead to type-1 interferon signaling, characterized by fever, panniculitis, lipodystrophy, cytopenia, systemic and pulmonary hypertension and basal ganglia calcification. IL-1 blockers are approved for NOMID and JAK-inhibitors show efficacy in CANDLE treatment.Objectives:We used proteomic analysis to compare differentially expressed proteins in active NOMID and CANDLE compared to healthy controls before and after treatment, and whole blood bulk RNA seq to identify the immune cell signatures.Methods:Serum samples from active NOMID (n=12) and CANDLE (n=7) before and after treatment (table 1) and age matched healthy controls (HC) (n=7) were profiled using the SomaLogic platform (n=1125 proteins). Differentially expressed proteins in NOMID and CANDLE were ranked after non-parametric tests for unpaired (NOMIDp<0.05, CANDLE,p<0.1) and paired (p<0.05) analysis and assessed by enriched Gene Ontology pathways and network visualization. Whole blood RNA seq was performed (NOMID=7, CANDLE=7, Controls =5) and RPKM values were used to assess immune cells signatures.Table 1.Patient’s characteristicsNOMIDN=12, Male =6CANDLEN=7, Male =6AgeMedian (range)12 (2, 28)16 (3, 20)Ethnicity%White (Hispanic)80 (20)100 (30)GeneticsNLRP3mutation(2 Somatic, 10 Germline)mutations in proteasome component genes(1 digenic, 6 Homozygous/compound Heterozygous)Before treatmentAfter treatmentBefore treatmentAfter treatmentCRPMedian (range) mg/L52 (16-110)5 (0-23)5 (0-101)1 (0-4)IFN scoremedian (range)0NA328 (211-1135)3 (0-548)Results:Compared to control, 205 proteins (127 upregulated, 78 downregulated) were significantly different at baseline in NOMID, compared to 163 proteins (101 upregulated, and 62 downregulated) in CANDLE. 134 dysregulated proteins (85 upregulated, 49 downregulated) overlapped in NOMID and CANDLE (Figure 1). Pathway analysis identified neutrophil and monocyte chemotaxis signature in both NOMID and CANDLE. NOMID patients had neutrophilia and active neutrophils. CANDLE patients exhibited active neutrophils in whole blood RNA. Endothelial cell activation was the most prominent non-hematopoietic signature and suggest distinct endothelial cell dysregulation in NOMID and CANDLE. In NOMID, the signature included neutrophil transmigration (SELE) endothelial cell motility in response to angiogenesis (HGF, VEGF), while in CANDLE the endothelial signatures included extracellular matrix protein deposition (COL8A) suggesting increased vascular stiffness. CANDLE patients had higher expression of Renin, 4 out of 7 had hypertension, NOMID patients did not have hypertension. Treatment with anakinra and baricitinib normalized 143 and 142 of dysregulated proteins in NOMID and CANDLE respectively.Conclusion:Differentially expressed proteins in NOMID and CANDLE are consistent with innate immune cell activation. Distinct endothelial cell signatures in NOMID and CANDLE may provide mechanistic insight into differences in vascular phenotypes. Treatment with anakinra and Baricitinib in NOMID and CANDLE leaves 30% and 13% of the dysregulated proteins unchanged.Acknowledgments:This work was supported by Intramural Research atNational Institute of Allergy Immunology and Infectious Diseases of National Institutes of Health, Bethesda, Maryland, the Center of Human Immunology and was approved by the IRB.Disclosure of Interests:None declared

scholarly journals High heterogeneity undermines generalization of differential expression results in RNA-Seq analysis

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Weitong Cui ◽  
Huaru Xue ◽  
Lei Wei ◽  
Jinghua Jin ◽  
Xuewen Tian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Expression ◽  
Small Sample ◽  
Differentially Expressed ◽  
Cancer Type ◽  
Rna Seq ◽  
Sample Sizes ◽  
Large Sample ◽  
Expression Levels ◽  
Gene Expression Levels

Abstract Background RNA sequencing (RNA-Seq) has been widely applied in oncology for monitoring transcriptome changes. However, the emerging problem that high variation of gene expression levels caused by tumor heterogeneity may affect the reproducibility of differential expression (DE) results has rarely been studied. Here, we investigated the reproducibility of DE results for any given number of biological replicates between 3 and 24 and explored why a great many differentially expressed genes (DEGs) were not reproducible. Results Our findings demonstrate that poor reproducibility of DE results exists not only for small sample sizes, but also for relatively large sample sizes. Quite a few of the DEGs detected are specific to the samples in use, rather than genuinely differentially expressed under different conditions. Poor reproducibility of DE results is mainly caused by high variation of gene expression levels for the same gene in different samples. Even though biological variation may account for much of the high variation of gene expression levels, the effect of outlier count data also needs to be treated seriously, as outlier data severely interfere with DE analysis. Conclusions High heterogeneity exists not only in tumor tissue samples of each cancer type studied, but also in normal samples. High heterogeneity leads to poor reproducibility of DEGs, undermining generalization of differential expression results. Therefore, it is necessary to use large sample sizes (at least 10 if possible) in RNA-Seq experimental designs to reduce the impact of biological variability and DE results should be interpreted cautiously unless soundly validated.

scholarly journals Long Intergenic Non-Coding RNAs in the Mammary Parenchyma and Fat Pad of Pre-Weaning Heifer Calves: Identification and Functional Analysis

Animals ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1268
Author(s):  
Shengchao Zhang ◽  
Sibtain Ahmad ◽  
Yuxia Zhang ◽  
Guohua Hua ◽  
Jianming Yi
Keyword(s):  
Mammary Gland ◽  
Crude Protein ◽  
Regulatory Mechanism ◽  
Differentially Expressed ◽  
Milk Replacer ◽  
Rna Seq ◽  
Fat Pad ◽  
Mammary Fat Pad ◽  
Non Coding Rnas ◽  
Gland Development

Enhanced plane of nutrition at pre-weaning stage can promote the development of mammary gland especially heifer calves. Although several genes are involved in this process, long intergenic non-coding RNAs (lincRNAs) are regarded as key regulators in the regulated network and are still largely unknown. We identified and characterized 534 putative lincRNAs based on the published RNA-seq data, including heifer calves in two groups: fed enhanced milk replacer (EH, 1.13 kg/day, including 28% crude protein, 25% fat) group and fed restricted milk replacer (R, 0.45 kg/day, including 20% crude protein, 20% fat) group. Sub-samples from the mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested from heifer calves. According to the information of these lincRNAs’ quantitative trait loci (QTLs), the neighboring and co-expression genes were used to predict their function. By comparing EH vs R, 79 lincRNAs (61 upregulated, 18 downregulated) and 86 lincRNAs (54 upregulated, 32 downregulated) were differentially expressed in MFP and PAR, respectively. In MFP, some differentially expressed lincRNAs (DELs) are involved in lipid metabolism pathways, while, in PAR, among of DELs are involved in cell proliferation pathways. Taken together, this study explored the potential regulatory mechanism of lincRNAs in the mammary gland development of calves under different planes of nutrition.

scholarly journals Identifying an lncRNA-Related ceRNA Network to Reveal Novel Targets for a Cutaneous Squamous Cell Carcinoma

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 432
Author(s):  
Yaqin Xu ◽  
Yingying Dong ◽  
Yunhua Deng ◽  
Qianrong Qi ◽  
Mi Wu ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Biological Process ◽  
Normal Skin ◽  
Expression Patterns ◽  
Therapeutic Targets ◽  
Cutaneous Squamous Cell Carcinoma ◽  
Differentially Expressed ◽  
Cerna Network

A cutaneous squamous cell carcinoma (cSCC) derived from keratinocytes is the second most common cause of non-melanoma skin cancer. The accumulation of the mutational burden of genes and cellular DNA damage caused by the risk factors (e.g., exposure to ultraviolet radiation) contribute to the aberrant proliferation of keratinocytes and the formation of a cSCC. A cSCC encompasses a spectrum of diseases that range from recursor actinic keratosis (AK) and squamous cell carcinoma (SCC) in situ (SCCIS) to invasive cSCCs and further metastatic SCCs. Emerging evidence has revealed that lncRNAs are involved in the biological process of a cSCC. According to the ceRNA regulatory theory, lncRNAs act as natural miRNA sponges and interact with miRNA response elements, thereby regulating the mRNA expression of their down-stream targets. This study was designed to search for the potential lncRNAs that may become potential therapeutic targets or biomarkers of a cSCC. Considering the spirit of the study to be adequately justified, we collected microarray-based datasets of 19 cSCC tissues and 12 normal skin samples from the GEO database (GSE42677 and GSE45164). After screening the differentially expressed genes via a limma package, we identified 24 differentially expressed lncRNAs (DElncRNAs) and 3221 differentially expressed mRNAs (DEmRNAs). The miRcode, miRTarBase, miRDB and TargetScan databases were used to predict miRNAs that could interact with DElncRNAs and DEmRNAs. A total of 137 miRNA-lncRNA and 221 miRNA-mRNA pairs were retained in the ceRNA network, consisting of 31 miRNAs, 11 DElncRNAs and 155 DEmRNAs. For the functional analysis, the top enriched biological process was enhancer sequence-specific DNA binding in Gene Ontology (GO) terms. The FoxO signaling pathway, autophagy and cellular senescence were the top enrichment terms based on a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The combination of a STRING tool and Cytoscape software (plug-in MCODE) identified five core mRNAs and built a core mRNA-associated ceRNA network. The expression for five identified core mRNAs and their related nine lncRNAs was validated using the external dataset GSE7553. Finally, one lncRNA HLA-F-AS1 and three mRNAs named AGO4, E2F1 and CCND1 were validated with the same expression patterns. We speculate that lncRNA HLA-F-AS1 may sponge miR-17-5p or miR-20b-5p to regulate the expression of CCND1 and E2F1 in the cSCC. The present study may provide potential diagnostic and therapeutic targets for cSCC patients.

scholarly journals Dynamic alteration in miRNA and mRNA expression profiles at different stages of chronic arsenic exposure-induced carcinogenesis in a human cell culture model of skin cancer

2021 ◽  
Author(s):  
Mayukh Banerjee ◽  
Ana Ferragut Cardoso ◽  
Laila Al-Eryani ◽  
Jianmin Pan ◽  
Theodore S. Kalbfleisch ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Mrna Expression ◽  
Hacat Cell ◽  
Arsenic Exposure ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Chronic Arsenic Exposure ◽  
Pathway Analyses ◽  
Time Point

AbstractChronic arsenic exposure causes skin cancer, although the underlying molecular mechanisms are not well defined. Altered microRNA and mRNA expression likely play a pivotal role in carcinogenesis. Changes in genome-wide differential expression of miRNA and mRNA at 3 strategic time points upon chronic sodium arsenite (As3+) exposure were investigated in a well-validated HaCaT cell line model of arsenic-induced cutaneous squamous cell carcinoma (cSCC). Quadruplicate independent HaCaT cell cultures were exposed to 0 or 100 nM As3+ for up to 28-weeks (wk). Cell growth was monitored throughout the course of exposure and epithelial-mesenchymal transition (EMT) was examined employing immunoblot. Differentially expressed miRNA and mRNA profiles were generated at 7, 19, and 28-wk by RNA-seq, followed by identification of differentially expressed mRNA targets of differentially expressed miRNAs through expression pairing at each time point. Pathway analyses were performed for total differentially expressed mRNAs and for the miRNA targeted mRNAs at each time point. RNA-seq predictions were validated by immunoblot of selected target proteins. While the As3+-exposed cells grew slower initially, growth was equal to that of unexposed cells by 19-wk (transformation initiation), and exposed cells subsequently grew faster than passage-matched unexposed cells. As3+-exposed cells had undergone EMT at 28-wk. Pathway analyses demonstrate dysregulation of carcinogenesis-related pathways and networks in a complex coordinated manner at each time point. Immunoblot data largely corroborate RNA-seq predictions in the endoplasmic reticulum stress (ER stress) pathway. This study provides a detailed molecular picture of changes occurring during the arsenic-induced transformation of human keratinocytes.

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