Identification and validation of a novel mature microRNA encoded by the Merkel cell polyomavirus in human Merkel cell carcinomas

Abstract

Background: Merkel cell polyomavirus (MCPyV) is present in approximately 80% of human Merkel cell carcinomas (MCCs). A previous in silico prediction suggested MCPyV encodes a microRNA (miRNA) that may regulate cellular and viral genes.

Objectives: To determine the presence and prevalence of a putative MCPyV-encoded miRNA in human MCC tumors.

Study design: Over 30 million small RNAs from 7 cryopreserved MCC tumors and 1 perilesional sample were sequenced. 45 additional MCC tumors were examined for expression of an MCPyV-encoded mature miRNA by reverse transcription real-time PCR.

Results: An MCPyV-encoded mature miRNA, "MCV-miR-M1-5p", was detected by direct sequencing in 2 of 3 MCPyV-positive MCC tumors. Although a precursor miRNA, MCV-miR-M1, had been predicted in silico and studied in vitro by Seo et al., no MCPyV-encoded miRNAs have been directly detected in human tissues. Importantly, the mature sequence of MCV-miR-M1 found in vivo was identical in all 79 reads obtained but differed from the in silico predicted mature miRNA by a 2-nucleotide shift, resulting in a distinct seed region and a different set of predicted target genes. This mature miRNA was detected by real-time PCR in 50% of MCPyV-positive MCCs (n = 38) and in 0% of MCPyV-negative MCCs (n = 13).

Conclusions: MCV-miR-M1-5p is expressed at low levels in 50% of MCPyV-positive MCCs. This virus-encoded miRNA is predicted to target genes that may play a role in promoting immune evasion and regulating viral DNA replication.

Figure 1

Overview of high-throughput sequencing of small human RNAs in Merkel cell carcinoma and data analysis pipeline.

Figure 2. Merkel cell polyomavirus encodes a microRNA expressed in Merkel cell carcinoma tumors

A. MCV-miR-M1 binding site. The 22-nt sequence is complementary to a portion of the large T antigen transcript (nt 1217–1238) that is upstream of the reported deletions and truncations in MCCs. B. MCV-miR-M1 hairpin structure. The pre-miRNA structure is shown, with the mature sequence, MCV-miR-M1-5p, bolded and seed region (nt 2–7) underlined. Mature sequence was determined by sequencing of 22-nt RNAs from MCC tumors. C. MCV-miR-M1 mature miRNA expression in MCC tumors with different levels of detectable MCPyV DNA. No MCV-miR-M1 was detected in tumors without detectable MCPyV DNA. MCV-miR-M1 was detected in 28.6% of weakly MCPyV positive tumors (4/14). MCV-miR-M1 was detected in 57.9% of moderately MCPyV positive tumors (11/19). MCV-miR-M1 was detected in 80% of strongly MCPyV positive tumors (4/5). A significant positive trend was observed (total n=51, including 6 sequenced tumors with 45 additional tumors).