Our bioinformatics analysis identified KSHV miRNA binding sites for miR-K12-3 and miR-K12-7 in the 3'UTR of the murine C/EBP-beta gene (Fig.7A), suggesting the possibility of functional repression of this gene by these two miRNAs. Subsequent analyses revealed that miR-K12-3 and miR-K12-7 specifically suppress expression of LIP and only slightly affect expression of the other two C/EBP-beta isoforms (p42 and p35) and that antagomirs targeting miR-K12-3 and miR-K12-7 restored expression of LIP (Fig.7B).
Currently, the validated targets of KSHV miRNAs (Table 1) represent a collection of genes identified by unbiased methods or chosen by previously identified functions.Table 1 KSHVmiRNA validated targets. All targets have been validated using ectopic expression of miRNAs to demonstrate that a luciferase reporter or ectopic reporter are repressed. Additional validations include(1) exogenous reporters have been mutated to disrupt targeting by a ectopic miRNA,(2) ectopic miRNA suppresses endogenous target mRNA and/or protein levels,(3) de novo infection with KSHV represses endogenous target expression,(4) miRNA inhibitors and/or mutant virus display derepression of endogenous target mRNA and/or protein levels, and(5) repression of miRNA target is observed in KSHV clinical samples.
Our dataset also confirmed 12 out of 29 validated KSHV miRNA targets with expression in our dataset(>40%). Confirmed interactions include those with BACH1, FOS, CDKN1A(p21), TNFRSF12A(TWEAKR), RAD21 and RBL2 mRNAs, whose regulation had previously been validated at the level of protein expression. A list summarizing the recovery of previously published targets of the KSHV and EBV miRNAs is presented in Table S9. NIHMS333942-supplement-01 Table S9 Recovery of previously published targets for KSHV or EBV miRNAs.
