MicroRNA Profile of MA-104 Cell Line Associated With the Pathogenesis of Bovine Rotavirus Strain Circulated in Chinese Calves

Abstract

Bovine rotavirus (BRV) causes massive economic losses in the livestock industry worldwide. Elucidating the pathogenesis of BRV would help in the development of more effective measures to control BRV infection. The MA-104 cell line is sensitive to BRV and is thereby a convenient tool for determining BRV-host interactions. Thus far, the role of the microRNAs (miRNAs) of MA-104 cells during BRV infection is still ambiguous. We performed Illumina RNA sequencing analysis of the miRNA libraries of BRV-infected and mock-infected MA-104 cells at different time points: at 0 h post-infection (hpi) (just after 90 min of adsorption) and at 6, 12, 24, 36, and 48 hpi. The total clean reads obtained from BRV-infected and uninfected cells were 74,701,041 and 74,184,124, respectively. Based on these, 579 were categorized as known miRNAs and 144 as novel miRNAs. One hundred and sixty differentially expressed (DE) miRNAs in BRV-infected cells in comparison with uninfected MA-104 cells were successfully investigated, 95 of which were upregulated and 65 were downregulated. The target messenger RNAs (mRNAs) of the DE miRNAs were examined by bioinformatics analysis. Functional annotation of the target genes with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) suggested that these genes mainly contributed to biological pathways, endocytosis, apoptotic process, trans-Golgi membrane, and lysosome. Pathways such as the mammalian target of rapamycin (mTOR) (mml-miR-486-3p and mml-miR-197-3p), nuclear factor kappa B (NF-κB) (mml-miR-204-3p and novel_366), Rap1 (mml-miR-127-3p), cAMP (mml-miR-106b-3p), mitogen-activated protein kinase (MAPK) (mml-miR-342-5p), T-cell receptor signaling (mml-miR-369-5p), RIG-I-like receptor signaling (mml-miR-504-5p), AMP-activated protein kinase (AMPK) (mml-miR-365-1-5p), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling (mml-miR-299-3p) were enriched. Moreover, real-time quantitative PCR (qPCR) verified the expression profiles of 23 selected DE miRNAs, which were consistent with the results of deep sequencing, and the 28 corresponding target mRNAs were mainly of regulatory pathways of the cellular machinery and immune importance, according to the bioinformatics analysis. Our study is the first to report a novel approach that uncovers the impact of BRV infection on the miRNA expressions of MA-104 cells, and it offers clues for identifying potential candidates for antiviral or vaccine strategies.

Keywords: bovine rotavirus G8P[7] isolate; deep sequencing; miRNA; miRNA-mRNA interaction; signaling pathway.

FIGURE 1

Parametric analysis process of animal small RNA.

FIGURE 2

Morphological observation of the cytopathic effect (CPE) of bovine rotavirus (BRV) in MA-104 cells. (A) Uninfected MA-104 cells with an intact cell sheet. (B) CPE of infected MA-104 cells at 48 h post-infection (hpi) with BRV. Partial cells in the cell sheet became lysed and were detached from the growth surface. (C) CPE of infected MA-104 cells at 72 hpi. The cells showed complete lysis and detachment from the growth surface. Bar indicates × 4 magnification.

FIGURE 3

PCR detection of the bovine rotavirus (BRV) isolate VP6 gene during the course of the virus inside the cell after infection (0–72 hpi) and detection of the 11 genome segments of BRV by SDS–PAGE. (A) Amplicon of the VP6 gene segment (294 bp). M: DNA ladder DL5000bp; lanes 1–7, samples from cell culture harvest. The bands were visualized in 1% TS-GelRed stain in agarose gel. (B) RNA profile of group A BRV isolate. Typical RNA migration pattern showed that segments 7, 8, and 9 very close to each other, producing one segment, while segments 3 and 4 appeared as one segment. (C) Negative control of the SDS–PAGE image.

FIGURE 4

MicroRNA (miRNA) expression profiles of bovine rotavirus (BRV)-infected cells in comparison with the control (24 hpi). (A) Length distributions of total small RNA (sRNA) segments: the length of reads proportional to the reads of that length (23 nt was the main observed length). (B) Pie chart showing the total read distributions of sRNAs and their categories (e.g., known miRNAs, novel miRNAs, rRNA, tRNA, snRNA, snoRNA, repeats, exons, introns, etc.). (C) Volcano chart representing the expression change of the miRNAs between the virus-treated and normal non-treated cells. The x-axis shows the log2(fold change) and the y-axis shows the −log10 (q-Value). The horizontal dotted lines in the figure correspond to the q-Value (default) or the adjusted p-Value (FDR), with 0.05 as the significant difference threshold. Significant difference thresholds: log2(fold change) > 1 and FDR-adjusted p = 0.05. Upregulated miRNAs are represented by red dots, downregulated miRNAs by green dots, and miRNAs with no significant changes by blue dots.

FIGURE 5

(A–F) Gene Ontology (GO) enrichment histogram of candidate target genes between the infected and control groups from 0 to 48 hpi. The proportions of the three categories of GO [biological process (BP), cellular component (CC), and molecular function (MF)] and the number of candidate target genes annotated are shown.

FIGURE 6

(A–F) Rich distribution points of candidate target genes with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment between the infected and control groups from 0 to 48 hpi. The vertical axis represents the pathway name, the horizontal axis represents the Rich factor, the size of the dots indicates the number of candidate target genes in this pathway, and the color of the dots corresponds to the different Q-value ranges.

FIGURE 7

(A–F) Validation of the differentially expressed (DE), microRNAs (miRNAs) by real-time quantitative PCR (qPCR) from 0 to 48 hpi. The differences in expression were determined using the 2^{– ΔΔCT} method and with unpaired, two-tailed Student’s t-test for grouped data. All experiments were performed in triplicate (n = 3). U6 was used as an internal reference. Data represent the mean ± SD (error bar). Significant difference thresholds: log2(fold change) > 2 and FDR-adjusted p < 0.05.

FIGURE 8

(A–F) Validation of target messenger RNAs (mRNAs) by real-time quantitative PCR (qPCR) from 0 to 48 hpi. The differences in expression were determined using the 2^{– ΔΔCT} method and with unpaired, two-tailed Student’s t-test for grouped data. All experiments were performed in triplicate (n = 3). β-actin was used as an internal reference. Data represent the mean ± SD (error bar). Significant difference thresholds: log2(fold change) > 2 and of FDR-adjusted p < 0.05.

FIGURE 9

(A–F) Expression levels of the common differentially expressed (DE) microRNAs (miRNAs) at different time points of bovine rotavirus (BRV) infection. The differences in expression were determined using the 2^{– ΔΔCT} method and with unpaired, two-tailed Student’s t-test for grouped data. All experiments were performed in triplicate (n = 3). U6 was used as an internal reference. Data represent the mean ± SD (error bar). Significant difference thresholds: log2(fold change) > 2 and FDR-adjusted p < 0.05.

FIGURE 10

Predicted crosstalk between the differentially expressed (DE) microRNAs (miRNAs), target messenger RNAs (mRNAs), and the signaling pathways during bovine rotavirus (BRV) infection.