The Impact of Serum Amyloid P-Component on Gene Expression in RAW264.7 Mouse Macrophages

Abstract

Serum amyloid P-component (SAP) contributes to host defense and prevents fibrosis. Macrophages are the most abundant inflammatory cell type in atherosclerotic plaques. In the present study, using (3)H-cholesterol-labeled counting radioactivity assay, we demonstrated that the apoAI-mediated cholesterol efflux in RAW264.7 macrophages was increased by SAP treatment in a time- and dose-dependent manner. We analyzed global gene expression changes upon SAP treatment using RNA sequencing. As a result, a total of 175 differentially expressed genes were identified, of which 134 genes were downregulated and 41 genes were upregulated in SAP treated cells compared to control cells. Quantitative RT-PCR analysis confirmed decreased expression of 5 genes and an increase in expression of 1 gene upon SAP treatment. Gene ontology analysis showed that genes involved in response to stimulus were significantly enriched in differentially expressed genes. Beyond protein-coding genes, we also identified 8 differentially expressed long noncoding RNAs. Our study may provide new insights into mechanisms underlying the functional role of SAP in macrophages.

Figure 1

Systematic identification of genes that are altered in SAP treated cells compared to control cells. (a) SAP enhanced apoA1-mediated cholesterol efflux in a time-dependent manner in macrophages. (b) SAP enhanced apoA1-mediated cholesterol efflux in a dose-dependent manner in macrophages for 24 h. (c) Scatter plot depicting the expression profiles of all genes. Log₂ transformed FPKM values from RNA sequencing were used in the scatter plot. We added 1 to FPKM value before log₂ transformation to facilitate calculation. Nonchanged genes were shown in blue color while differently expressed genes (fold change > 2 and FDR < 0.05) were denoted in red or green. (d) Distribution of fold change of genes significantly different in SAP treated cells compared to control cells. (e) Validation of RNA sequencing data by quantitative RT-PCR. The GAPDH gene was used as the reference gene for normalization. The statistical significance was tested using unpaired 2-sample t-test. Values were plotted as means ± standard error of the mean (SEM) of triplicate measurements. n = 3. ^∗ p < 0.05.

Figure 2

Functional clustering analysis of differentially expressed genes. (a) Differentially expressed genes were analyzed using BiNGO software. Significantly enriched GOslim categories were highlighted with different colors representing different levels of significance. The size of each circle is correlated to the number of genes. (b) Heat map of the 24 differentially expressed genes that fall into the response to stimulus category. In the heat map, the first and second columns correspond to the absolute gene expression levels (FPKM values) of SAP and control group, respectively. Values are color-coded, with yellow representing low levels and orange representing high levels. The third column of the heat map reports the relative expression levels. The values are the log₂ ratio of SAP versus control. Red indicates increase and green indicates decrease.

Figure 3

Network analysis of SAP-regulated genes. (a) The gene network generated by STRING. (b) Bar plot showing connection degrees for all genes.

Figure 4

Changed expression of noncoding RNA genes. (a) Classification of noncoding RNA genes. Pie chart is displayed on the differently expressed 19 noncoding RNA genes. (b) Heat map of the 8 selected long noncoding RNA genes. The first and second columns correspond to the absolute gene expression levels (FPKM values) in SAP group and control group, respectively. The third column of the heat map reports the relative expression levels. Values are color-coded as indicated by the color bars. (c) Quantitative RT-PCR analysis of 8 selected lncRNAs. The GAPDH gene was used as the reference gene for normalization. Statistical significance was tested using unpaired 2-sample t-test. Values were plotted as means ± standard error of the mean (SEM) of triplicate measurements. n = 3. ^∗ p < 0.05. (d) RNA sequencing coverage plot of Mir17hg lncRNA. Coverage plot is displayed on the reference genome (UCSC mm9). The upper panel represents expression in SAP group and the lower panel represents expression in control group. For both panels, numbers on y-axis refer to RNA sequencing read-depth at a given nucleotide position.