Hepatitis C virus core impacts expression of miR122 and miR204 involved in carcinogenic progression via regulation of TGFBRAP1 and HOTTIP expression

Abstract

Background: Despite the breadth of understanding the noncoding RNAs' function in molecular biology, their functional roles in hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated the effect of hepatitis C virus (HCV) core upon the expression of noncoding RNAs.

Methods: The lncRNAs, mRNAs, and circRNAs were employed for identification of HCV core protein gene expression in human Huh7 hepatoma (Huh7) cell line. In data analysis, we applied a threshold that eliminated all genes that were not increased or decreased by at least a 2-fold change in a comparison between transfected and control cells. Hierarchical Clustering and the Kyoto encyclopedia of genes and genome pathway analyses were performed to show the distinguishable lncRNA, mRNAs, and circRNAs expression pattern among samples.

Results: The array data showed that 4,851 lncRNAs, 4,785 mRNAs, and 823 circRNAs were 2-fold up-regulated but 3,569 lncRNAs, 3,192 mRNAs, and 419 circRNAs were 2-fold down-regulated in Huh 7-core cells. The genes in the enriched set were associated with macromolecule and nucleic acid metabolic processes, DNA damage response and regulation of voltage-gated calcium channel. We identified 10 genes from the selected 14 genes that were higher or lower expression in Huh7-core cells than that of Huh7-vector cells by quantitative real-time polymerase chain reaction. Interestingly, overexpression of miR122 and miR204 partly abrogated the expression of TGFBRAP1 and HOTTIP, and increased the HPCAL1 expression in the predicted carcinogenic pathways.

Conclusion: Our data suggests that the pathways of miR204-HPCAL1-lncRNAHOTTIP and miR122-TGFBRAP1 were likely involved in the carcinogenic progress due to the presence of HCV core, and that overexpression of miR122 and miR204 might inhibit the HCC progress by down-regulation of TGFBRAP1 and HOTTIP expression.

Keywords: Core; HOTTIP; gene expression; hepatitis C virus; hepatocellular carcinoma; lncRNA microarray; miR122.

Figure 1

The hot map of lncRNA array analysis. (A) Hot map was generated for lncRNA expression in Huh7-core cells compared with Huh7-vector cells. (B) Box plot is a convenient way to quickly visualize the distribution of a dataset. It is commonly used for comparing the distributions of the intensities from all samples. (C) Scatter plot is a visualization method used for assessing the lncRNA expression variation between the two compared samples. The values of X and Y axes are the normalized signal values of the samples (log2 scaled). The green lines are fold change lines. The lncRNAs above the top green line and below the bottom green line indicated more than 2-fold change of lncRNAs between the two compared samples. Abbreviation: lncRNA, long noncoding RNA.

Figure 2

The hot map of mRNA array analysis. (A) Hot map was developed for mRNA expression in Huh7-core cells compared with Huh7-vector cells. (B) Box plot is a convenient way to quickly visualize the distribution of a dataset. It is commonly used for comparing the distributions of the intensities from all samples. (C) Scatter plot is a visualization method used for assessing the mRNA expression reproducibility between the two compared samples. The values of X and Y axes in the scatter plot are the normalized signal values of the samples (log2 scaled). The green lines are fold change lines. The mRNAs above the top green line and below the bottom green line indicated more than 2-fold change of mRNAs between the two compared samples.

Figure 3

The hot map of circRNA array analysis. (A) Hot map was produced for circRNA expression in Huh7-core cells compared with Huh7-vector cells. (B) Box plot is a convenient way to quickly visualize the distribution of a dataset. It is commonly used for comparing the distributions of the intensities from all samples. (C) Scatter plot is a visualization method used for assessing the circRNA expression variation between the two compared samples. The values of X and Y axes in the scatter plot are the normalized signal values of the samples (log2 scaled). The green lines are fold change lines. The circRNAs above the top green line and below the bottom green line indicated more than 2-fold change of circRNAs between the two compared samples. Abbreviation: circRNA, circular RNA.

Figure 4

Presence of HCV genotype 1b core causes the gene expression profiling in Huh7 cells by gene ontology analysis. (A) Analysis of biological process. (B) Analysis of cellular component. (C) Analysis of molecular function. Abbreviation: HCV, hepatitis C virus.

Figure 5

Identification of selected gene expression in HCV core and HCV vector cells analyzed by RT-qPCR. (A) Differential ncRNA expression. (B) Differential molecular gene expression. Note: *p<0.05. Abbreviations: HCV, hepatitis C virus; ncRNA, noncoding RNA; RT-qPCR, quantitative real-time PCR.

Figure 6

Effects of overexpression of miRNAs on potential downstream target gene expression analyzed by RT-qPCR. (A) Effects of overexpression of miR 122 on downstream target gene expression. (B) Effects of overexpression of miR 192 on downstream target gene expression. (C) Effects of overexpression of miR 204 on downstream target gene expression. Note: *p<0.05. Abbreviations: miRNAs, microRNAs; RT-qPCR, quantitative real-time PCR.

Figure 7

Predicted and modified HCV core protein-related carcinogenic pathways. (A and B) Predicted HCV genotype 1b core protein-related carcinogenic pathways by the data of bioinformation and the molecular target site predict platform. (C) Modified HCV core protein-related carcinogenic pathways supported by overexpression of miR122 and miR204. Label?: that means questionable or uncertainty. Abbreviation: HCV, hepatitis C virus.