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miR-99b and miR-485 Increase the Expression of Viral Genes
To further characterize AdwtE miR-99b and AdwtE miR-485, we analyzed the mRNA levels of late (hexon and fiber) and immediate early (E1A) genes at 24 or 8 hr PI, respectively, in PANC-1 and MIA PaCa-2 cells. Increased mRNA levels of the three genes were observed in AdwtE miR-infected Paxilline (Figures 4A and 4B). Consis-tent with these results, higher protein contents of hexon, penton, fiber, and E1A were also detected (Figures 4C and 4D). These results suggest that miR-99b and miR-485 may modulate cellular pathways, affecting both early and late transcription of viral genes.
miR-99b and miR-485 Inhibit the Expression of Genes Related to Transcription Regulation
We next performed a bioinformatics study to elucidate the mecha-nisms by which miR-99b and miR-485 regulate the expression of viral genes (Supplemental Materials and Methods; Figure S4A). The list of potential target genes for miR-99b and miR-485, generated by different miRwalk prediction algorithms, was classified by the biolog-ical process subontology of gene ontology (GO) terms. One of the en-riched GO terms that was common to both miRNAs and interesting
in light of the experimental data was “regulation of gene expression.” Among the genes belonging to this term, we selected those with better prediction scores as targets for miR-99b and miR-485 (Table S2), which we could infer from published data also possessed some activity modulating viral infections or the adenoviral cycle. Thus, we selected the transcriptional regulators E74-like ETS tran-scription factor 4 (ELF4) and Kruppel-like factor 8 (KLF8), both of which have predicted target sites for miR-99b and miR-485, and MDM2 proto-oncogene (MDM2), a miR-485-validated target gene20 (Figures S4B and S5).
We first investigated whether ELF4, MDM2, and KLF8 gene expres-sion was regulated by the candidate miRNAs. PANC-1 cells were mock infected or infected with control or AdwtE miR viruses, and both mRNA and protein levels were analyzed. Reduction in the ELF4 mRNA and protein levels was observed only in AdwtE miR-99b-infected cells (Figures 5A and 5B). Thus, although ELF4 had a miR-485 prediction site (Figure S4B), we could not validate any effect in our experimental setting. MDM2 expression was significantly reduced in cells infected with either AdwtE miR-485 or AdwtE miR-99b (Figures 5A and 5B). The reduced expression observed after AdwtE miR-99b was unexpected, as MDM2 is not a direct target of miR-99b (Figure S4B). However, MDM2 downregulation could be mediated by limited transcription due to the decrease in its transcrip-tional regulator ELF4 or by other indirect mechanisms.21 KLF8 expression was significantly reduced in cells infected with either of the miR candidate viruses, as predicted, although it could be detected only at the protein level (Figures 5A and 5B).
MDM2 is a known miR-485 target.20 To validate the miR-99b target site for ELF4 and the miR-99b and miR-485 target sites for KLF8, we performed reporter assays. A fragment of the ELF4 or KLF8 30 UTR, which contains predicted wild-type or mutated miR-binding sites (Figure S5), was cloned into a dual luciferase re-porter construct and co-transfected into HEK293T cells with miRVec-99b or miRVec-485 expression plasmids. As shown in Fig-ure 5C, Renilla activity was significantly decreased for ELF4 wild-type 30 UTR with miR-99b and for KLF8 wild-type 30 UTR with miR-99b and miR-485. These effects were not observed when miR-binding sites were mutated (Figure 5C; Figure S5). These data confirmed the regulation of ELF4 by miR-99b and of KLF8 by both miR-99b and miR-485.
These results suggest that miR-99b and/or miR-485 modulation of ELF4 and KLF8 could affect adenoviral replication. To test this, we generated ELF4 and KLF8 knockdown PANC-1 cells and infected them with AdwtE. Indeed, AdwtE-infected cells released higher levels of infective particles in all clones with reduced expression of ELF4 or KLF8 (Figures 5D and 5E; Figure S6), thus validating our hypothesis.
genomes were quantified by qPCR. (E) In vitro oncolytic activity in PANC-1 and MIA PaCa-2 cells. Cells were seeded in triplicate and infected with a dose range of AdwtE, AdwtE miR-99b, or AdwtE miR-485. Cell viability was measured 7 days PI by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and IC50 values were determined. Data are shown as mean ± SEM for at least five independent biological replicates. Significance was assessed by comparison to AdwtE-infected cells using a two-tailed Mann-Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001.
AdwtE Molecular Therapy
AdwtE miR-99b B