microRNA-4717 differentially interacts with its polymorphic target in the PD1 3' untranslated region: A mechanism for regulating PD-1 expression and function in HBV-associated liver diseases

Abstract

Programmed cell death-1 (PD-1) is involved in hepatitis B virus (HBV) infection, the leading cause of hepatocellular carcinoma (HCC) worldwide. Single-nucleotide polymorphism, rs10204525, located in the PD1 3' untranslated regions (UTR), is associated with chronic HBV infection. MicroRNAs (miRNAs) regulate gene expression via specific binding to the target 3'UTR of mRNA. In this study, three miRNAs were predicted to putatively interact with PD1 rs10204525 polymorphic site of allele G. One of them, miRNA-4717, was demonstrated to allele-specifically affect luciferase activity in a dose-dependent manner in cells transfected with vectors containing different rs10204525 alleles. In lymphocytes from chronic HBV patients withrs10204525 genotype GG, miR-4717 mimics significantly decreased PD-1 expression and increased (TNF)-α and interferon (IFN)-γ production. miR-4717 inhibitor significantly increased PD-1 expression and decreased TNF-α and IFN-γ production although not significantly. In lymphocytes from chronic HBV patients with rs10204525 genotype AA, no similar effects were observed. miR-4717 levels in peripheral lymphocytes from patients with HBV-related chronic hepatitis, cirrhosis and HCC were significantly decreased. In conclusion, miR-4717 may allele-specifically regulate PD-1 expression through interaction with the 3' UTR of PD1 mRNA, leading to the alteration of immune regulation and affecting the susceptibility and disease course of chronic HBV infection.

Keywords: 3’-untranslated region; hepatitis B virus infection; microRNA; programmed cell death-1; single nucleotide polymorphism.

Figure 1. Predicted miRNAs putatively bining the 3′UTR of PD1 mRNA with rs10204525 allele G

The PD1 rs10204525 site is represented by an underscore.

Figure 2. Effects of the predicted miRNAs on the luciferase activity of pMIR-A or pMIR-G bearing segments from PD1 3′UTR of rs10204525 allele A and allele G, respectively

(A) Effects of miR-302c. (B) Effects of miR-541. (C) Effects of miR-4717. (D) Dose-response relationship between miR-4717 mimics concentration and luciferase activity of pMIR-G. Statistical analysis was performed by using one-way univariate analysis of variance (ANOVA) and the least significant difference (LSD) test.

Figure 3. Effects of miR-4717 mimics or miR-4717 inhibitor on PD-1 mRNA expression in lymphocytes from chronic HBV patients with different rs10204525 genotypes assessed by real-time quantitative PCR

Comparisons were performed by using non-parametric tests (Kruskal-Wallis H test and Mann-Whitney U test).

Figure 4. Effects of miR-4717 mimics or miR-4717 inhibitor on PD-1 protein expression on lymphocytes from chronic HBV patients with different rs10204525 genotypes assessed by flow cytometry

(A) Genotype AA + miR control. (B) Genotype AA + miR-4717 mimics. (C) Genotype AA + miR-4717 inhibitor. (D) Genotype GG + miR control. (E) Genotype GG + miR-4717 mimics. (F) Genotype GG + miR-4717 inhibitor. (G) Comparison of PD-1 on lymphocytes from genotypes AA and GG by miR-4717 mimics and miR-4717 inhibitor. Statistical analysis was carried out by using one-way univariate analysis of variance (ANOVA) and Post Hoc test.

Figure 5. Effect of miR-4717 mimics or miR-4717 inhibitor on the production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by lymphocytes from chronic HBV patients with rs10204525 genotypes AA and GG

(A) TNF-α. (B) IFN-γ. The values were log transformed to obtain normal distribution and then analyzed by using ANOVA and Post Hoc test.

Figure 6. miR-4717 levels in patients with chronic HBV infection

(A) Comparison of miR-4717 levels in patients with chronic HBV infection and health controls. (B) miR-4717 levels in HBV infected patients with different clinical diseases. Comparisons were performed by using non-parametric tests (Kruskal-Wallis H test and Mann-Whitney U test). HBV, hepatitis B virus; CH, chronic hepatitis; LC, liver cirrhosis; HCC, hepatocellular carcinoma.

Figure 7. miR-4717 levels in HBV patients according to HBeAg status, HBV DNA and ALT levels

(A) miR-4717 levels between HBeAg + and HBeAg – patients. (B) miR-4717 levels according to HBV-DNA loads. (C) miR-4717 levels according to between ALT concentrations. Comparisons were performed by using non-parametric tests (Kruskal-Wallis H test and Mann-Whitney U test).

Figure 8. miR-4717 levels of peripheral lymphocytes in the study subjects with different PD1 rs10204525 genotypes

(A) miR-4717 levels in healthy controls. (B) miR-4717 levels in patients with chronic HBV infection. Comparisons were performed by using non-parametric tests (Kruskal-Wallis H test and Mann-Whitney U test).