Expression of human miR-200b-3p and -200c-3p in cytomegalovirus-infected tissues

Abstract

Human cytomegalovirus (HCMV) infection can cause inflammatory tissue-invasive end-organ diseases upon lytic replication. In humans, mature miR-200b-3p and -200c-3p suppress the synthesis of HCMV immediate early 2 (IE2) protein by binding to the 3'-UTR of the mRNA encoded by the unique long (UL) 122-123 region in human foreskin fibroblasts and pre-transplant peripheral blood mononuclear cells stimulated with HCMV. The present study aimed to quantitate the expression of Homo sapiens (hsa)-miR-200b-3p and 200c-3p in HCMV-infected tissues. We collected 240 HCMV-infected and 154 HCMV-non-infected, formalin-fixed, paraffin-embedded tissue samples of the gastrointestinal (GI) tract and bronchi/lungs. MiRNAs, HCMV, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantitated by quantitative reverse transcription-PCR (qRT-PCR) and quantitative PCR (qPCR) on the basis of standard curves generated using miRNA mimics, the HCMV strain from National Institute for Biological Standards and Control (NIBSC) 09/162, and GAPDH control. To avoid the effect of cell counts on the qRT-PCR and qPCR results, the data were normalized to GAPDH levels. HCMV-infected tissues had significantly lower levels of 200b-3p/GAPDH (3.03 ± 1.50 compared with 3.98 ± 1.08 log₁₀ copies/μl, P<0.001) and 200c-3p/GAPDH (4.67 ± 1.84 compared with 6.35 ± 1.47 log₁₀ copies/μl, P<0.001) than normal tissues. The values for 200b-3p/GAPDH (r = -0.51, P<0.001) and 200c-3p/GAPDH (r = -0.54, P<0.001) were significantly inversely correlated with HCMV load. Low tissue levels of 200b-3p and 200c-3p in humans are associated with cytopathic inflammation due to HCMV infection.

Keywords: cytomegalovirus; immediate early protein 2; microRNA; tissues.

Figure 1. Differences and correlation between levels of hsa-miR-200b-3p, -200c-3p, and -429 in 394 analyzed tissues samples

(A) Differences in hsa-miR-200b-3p, -200c-3p, and -429 levels after normalization to GAPDH. Bars represent means and S.D. Dotted line indicates limit of detection (100 copies/μl of input nucleic acid). *P-value between three miRs by ANOVA test, **P<0.001 between two miRs by Bonferroni’s multiple comparisons tests. (B) Correlation between 200b-3p and 200c-3p after normalization to GAPDH. The levels of hsa-miR-200b-3p/GAPDH and -200c-3p/GAPDH are strongly correlated, with r = 0.87 (95% CI, 0.86–0.87) and P<0.001. ‘Nucleic acid’ stands for DNA for GAPDH and RNA for miRNAs. Each dot corresponds to the miRNA/GAPDH level expressed as log₁₀ copies/μl of input nucleic acid in all figures.

Figure 2. Difference of hsa-miR-200b-3p and -200c-3p levels between HCMV-infected (n=240) and non-infected (n=154) FFPE tissues

(A), (B) Bars indicate means and S.D. Dotted lines represent limit of detection (100 copies/μl of input nucleic acid). ‘Nucleic acid’ stands for DNA for GAPDH and RNA for miRNAs. Each dot corresponds to the miRNA/GAPDH level expressed log₁₀ copies/μl of input nucleic acid in all figures.

Figure 3. Difference in hsa-miR-200b-3p and -200c-3p levels according to tissue type

(A) Upper-GI tract samples originating from esophagus and stomach, including 67 HCMV-infected and 56 non-infected FFPE tissues. The percentage of samples with undetectable level of hsa-miR- 200b-3p/GAPDH (21.7 compared with 0.0%) was significantly higher in HCMV-infected than non-infected upper-GI tract tissues (P<0.001). However, the percentage of undetectable 200c-3p/GAPDH (4.3 compared with 0.0%) was similar between the two groups (P=0.121). (B) Lower-GI tract samples from duodenum, cecum, terminal ileum, colon, and rectum, including 168 HCMV-infected and 79 normal FFPE tissues. The percentage of samples with undetectable level of hsa-miR-200b-3p/GAPDH (23.0 compared with 0.0%) was significantly higher in HCMV-infected than non-infected lower-GI-tract tissues (P<0.001). For 200c-3p, values normalized to GAPDH had a significantly high undetectable percentage (12.1 compared with 0.0%, P<0.001). (C) Lung samples, including 5 HCMV-infected and 19 normal FFPE tissues. In lung, the 200b-3p/GAPDH and 200c-3p/GAPDH levels did not show significant difference between two groups (P=0.297 and 0.120, respectively). *P<0.001. P-values for GI tract and lung samples were obtained through independent t test and Mann–Whitney U test, respectively. In (A,B), height of boxes and upper/lower lines indicate mean and S.D. In (C), bars represent median and 25 or 75 percentile values. Each dot corresponds to the miRNA/GAPDH levels expressed as log₁₀ copies/μl of input nucleic acid in all figures.

Figure 4. Correlation between HCMV and hsa-miR-200b-3p or -200c-3p levels in HCMV-infected FFPE tissues

Straight and dotted lines represent each equation according to r coefficient and 95% CI values, respectively. Each dot corresponds to the miRNA/GAPDH or HCMV level expressed as log₁₀ copies/μl of input nucleic acid or DNA in all figures. ‘Nucleic acid’ stands for DNA for GAPDH and RNA for miRNAs.