Human cytomegalovirus promoting endothelial cell proliferation by targeting regulator of G-protein signaling 5 hypermethylation and downregulation

Abstract

Interactions between human cytomegalovirus (HCMV) infection and environmental factors can increase susceptibility to essential hypertension (EH). Although endothelial dysfunction is the initial factor of EH, the epigenetic mechanisms through which HCMV infection induces endothelial cell dysfunction are poorly understood. Here, we evaluated whether HCMV regulated endothelial cell function and assessed the underlying mechanisms. Microarray analysis in human umbilical vein endothelial cells (HUVECs) treated with HCMV AD169 strain in the presence of hyperglycemia and hyperlipidemia revealed differential expression of genes involved in hypertension. Further analyses validated that the regulator of G-protein signaling 5 (RGS5) gene was downregulated in infected HUVECs and showed that HCMV infection promoted HUVEC proliferation, whereas hyperglycemia and hyperlipidemia inhibited HUVEC proliferation. Additionally, treatment with decitabine (DAC) and RGS5 reversed the effects of HCMV infection on HUVEC proliferation, but not triggered by hyperglycemia and hyperlipidemia. In summary, upregulation of RGS5 may be a promising treatment for preventing HCMV-induced hypertension.

Figure 1

mRNA microarray expression data from four groups (HCMV-infected group (HCMV), high glucose (HG) and oxidized low-density lipoprotein (ox-LDL) group (HG + ox-LDL), HCMV-infected plus HG and ox-LDL group (HCMV + HG + ox-LDL), control). (A) Heat map of significantly-changed, the expression of mRNA is hierarchically clustered on the y axis, and four groups are hierarchically clustered on the x axis. The relative mRNA expression is depicted according to the color scale shown on the Left. Red indicates upregulation; green, downregulation. (B) The scatter plot of signal value between the groups of control and HCMV. (C) Control compared with HG + ox-LDL. (D) HCMV + HG + ox-LDL compared with HCMV. (E) HCMV + HG + ox-LDL compared with HG + ox-LDL. Red indicates upregulation; green, downregulation.

Figure 2

Hypertension related target gene screening. (A) The significant GO terms that conformed to a P < 0.05 were screened. (B) Fisher’s exact test was used to select the significant pathway, identified by a P < 0.05. GO, Gene ontology; BP, Biological process; CC, Cellular component; MF, Molecular function. (C) Venn diagrams of DGEs mRNA in the hypertension related gene set, Online Mendelian Inheritance in Man (OMIM), Genetic Association Database (GAD), the Human Gene Database (MalaCards), Disease Gene Search with Evidence Sentences (Digsee), genome-wide association studies (GWAS). (D) Venn diagrams of significantly upregulated or downregulated mRNAs in the hypertension related gene control compared with HCMV. (E) Venn diagrams of significantly upregulated or downregulated mRNAs in the hypertension related gene Control compared with HG + ox-LDL. (F) Venn diagrams of significantly upregulated or downregulated mRNAs in the hypertensionrelated gene HCMV + HG + ox-LDL compared with HCMV. (G) Venn diagrams of significantly upregulated or downregulated mRNAs in the hypertensionrelated gene HCMV + HG + ox-LDL compared with HG + ox-LDL.

Figure 3

mRNAs were validated in an expanded cohort using RT-qPCR with mRNA-specific primers, after exclusion of outliers. The p-values were calculated by two-tailed unpaired Student’s t-tests. *P < 0.05 vs control; ^#P < 0.05 vs HCMV or HG + ox-LDL.

Figure 4

Interaction of HCMV and environmental risk factors on endothelial cell proliferation by DNA methylation. (A) Changes in cell proliferation function in each treatment group by CCK-8 analysis. (B) Detection of methyltransferase activity in different treatment groups. (C) The methylation levels of CpG sites in RGS5 promoter regions from the four groups. (D) Effect of DAC on the expression of RGS5mRNA.*P < 0.05 vs control; ^#P < 0.05 HCMV+HG + ox-LDL vs HCMV or HG + ox-LDL. (E) Effect of DAC on cell proliferation. (F,G) Effect of DAC on the expression of PCNA protein. Data are expressed as means ± standard deviations. *P < 0.05 vs control; ^#P < 0.05 HCMV + HG + ox-LDL vs HCMV or HG + ox-LDL. ^ΔP < 0.05 HCMV vs HCMV + DAC, HG + ox-LDL vs HG + ox−LDL + DAC. Student’s t-test.

Figure 5

HCMV infected promotes endothelial cell proliferation through low expression of RGS5. (A) Green fluorescence was observed in both the RGS5-transfected and GFP-positive control groups, while that was not observed in the untransfected group. (B) RGS5 expression levels in HUVECs were detected by real-time PCR. RGS5 expression signifcantly increased after RGS5o/e adenovirus infected. (C) RGS5o/e reversible cell proliferation induced by HCMV infected by CCK-8 analysis. (D,E) RGS5o/e reduces the increase in PCNA induced by HCMV infected. RGS5o/e(RGS5-overexpression vector). *P < 0.05 vs control; ^#P < 0.05 HCMV + HG + ox-LDL vs HCMV or HG + ox-LDL. ^ΔP < 0.05 HCMV vs HCMV + RGS5 was considered statistically signifcant. Student’s t-test.