Background: Human microRNA (miRNA) can suppress gene expression through binding to the 3' untranslated region (UTR) of the target mRNA. In silico analysis shows that, hsp-miR-200b-3p, -200c-3p and -429 are predicted to bind the 3' UTR of CMV immediate early protein 2 (IE2) with perfect seed complementarity. IE2 plays an important role in early replication and latent-to-lytic switch of CMV. We hypothesized that expression of these miRNAs in the pre-transplant period could predict CMV reactivation after solid organ transplantation (SOT).

Methods: We analyzed peripheral blood mononuclear cells (PBMCs) that were collected pre-transplant from SOT recipients enrolled in the Swiss Transplant Cohort. All patients were CMV seropositive pre-transplant and were managed using a pre-emptive strategy (no prophylaxis). PBMCs were stimulated with live CMV Towne strain at 0.03 MOI, followed by isolation of small RNAs at day 1. We measured quantitative miRNA levels using real-time PCR. Using in-vitro experiments, we transfected miR-200b-3p and -200c-3p into human foreskin fibroblasts (HFF)-1 and infected with CMV AD169 strain at 0.1 MOI. The protein was collected at 72 hours and tested for IE2 by western blot.

Results: The median of miR-200c-3p was 17-fold higher compared to 200b-3p in total 272 recipients (median [IQR], 7354 [155-37853] vs. 432 [49-2582] copies/[mu]L of total small RNA, p<0.001). MiR-429 was measured with low level of 193 (0-116) copies/[mu]L. MiR-200b-3p and -200c-3p were significantly lower in recipients with CMV reactivation after SOT (n=144) compared to recipients without reactivation (200b-3p, 120.4 [17.5-430.1] vs. 258.4 [66.9-811.3], p=0.004; 200c-3p, 1262.9 [69-7297.2] vs. 3977.4 [215-12599.6] copies/[mu]L, p=0.029, respectively). MiR-429 levels were no different between the two groups. The multivariate regression model revealed that 200b-3p of ≥ 100 copies/[mu]L was an independent predictor of post-SOT CMV reactivation (OR, 0.542, 95% CI, 0.328-0.895, p=0.017). Targeting of IE2 was supported by in-vitro analysis, which showed that IE2/β-actin in HFF-1 transfected by 200b-3p was 2.8-fold lower compared to mock HFF-1.

Conclusions: This study provides both clinical and in-vitro evidence that host miRNAs may play an important biological role in preventing reactivation of CMV from latency post-SOT.

CITATION INFORMATION: Han S, Kumar D, Ferreira V, Husain S, Mueller N, Humar A, Mian M, Ashton P. Human MicroRNA Responses Are Predictive of CMV Reactivation Following Solid Organ Transplant. Am J Transplant. 2016;16 (suppl 3).

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