Polymorphisms in KSHV-encoded microRNA sequences affect levels of mature viral microRNA in Kaposi Sarcoma lesions

BACKGROUND: We previously reported Kaposi sarcoma-associated herpesvirus (KSHV) microRNA sequence variants in clinical samples correlated with increased risk of multicentric Castleman's disease (MCD). We then demonstrated that microRNAs with variant sequence have different maturation and mature microRNA expression in vitro. Here, we illustrate the association between microRNA sequence and changes in mature microRNA levels within Kaposi sarcoma (KS) lesions. METHODS: KSHV microRNA sequences were determined from 20 KS lesions and 4 control skin biopsies from individuals evaluated for KS. Levels of mature KSHV microRNAs were measured with 21 custom small RNA qRT-PCR assays using RNA RNU6B as endogenous control. RESULTS: The levels of 13 KSHV-encoded microRNAs were elevated in KS lesions compared to control biopsies. MicroRNA 9-5p was strongly down regulated in South African vs. US biopsies. Low levels of K12-9-5p were associated with single nucleotide polymorphisms (SNPs) in miR-K12-9-5p, 4-5p, 5-3p, 7-3p and pri-miR-K12-3. One SNP in pri-miR-K12-3 resulted in down regulation of miR-K12-6-3p, 8-3p, 10-3p, 12-5p and the upregulation of 5-5p, illustrating sequence variants outside pre-microRNAs were also associated with changes in mature microRNA levels. CONCLUSIONS: The levels of mature KSHV-encoded microRNAs in KS lesions correlate with sequence variation reflecting changes in secondary and tertiary RNA structure.