engleski

RNA-Sequencing Analysis Identifies Differentially Expressed Annotated and Novel 5' Capped RNAs During Acute Hepatitis C Infection in Cultured Hepatocytes

Purpose: To use next-generation RNA Sequencing (RNA-Seq) of 5' capped RNAs to identify annotated and novel Pol II RNAs that are differentially expressed after acute infection of hepatocytes with the hepatitis C virus (HCV). Methods: 5' capped (Pol II) RNAs were isolated from triplicate samples of acutely HCV- (JFH1) and mock-infected Huh 7.5 cells at 6, 48, and 72 hours ; the Pol II RNAs were subsequently analyzed by RNA-Seq. Several transcripts that were differentially expressed in acute HCV infection were confirmed by qPCR in additional HCV and control hepatocytes. Results: Analysis of 5' capped RNAs identified 100, 684, and 1844 differentially expressed annotated genes in acutely infected Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change >50% and adjusted p-values <0.05). Examples include two upregulated transcription factors, MYC (↑2X) and ANKRD1 (↑5X) ; we confirmed the differential expression of these genes by qPCR analysis. There were 1, 4, and 15 differentially expressed KEGG pathways at the same time points ; these included RIG-I-like receptor signaling, MAPK signaling, apoptosis, and NOD-like receptor signaling. Analyzing 5' capped RNAs also identified many novel Pol II RNAs that were differentially expressed after HCV infection (p-values <0.05). For example, 2 novel transcripts, located on chromosomes 8 and 17, were upregulated at both 48 and 72 hours after acute HCV infection (↑3X and ↑2X, respectively) ; this was also confirmed by qPCR analysis. Conclusions: Analyzing 5' capped RNAs by RNA-Seq after acute HCV infection revealed genes and biological pathways not identified by previous gene array studies. These genes and pathways represent critical components of the host immune, antiviral, and inflammatory responses. These studies also suggest that MYC and ANKRD1 may regulate transcription of novel Pol II RNAs during acute HCV infection. We are currently testing the ability of some of these RNAs to alter HCV replication in cell culture.

RNA-Sequencing; Gene Expression; Acute Hepatitis C; 5'cap

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