62. MiRNA Regulation of Oncolytic Measles Virus Infectivity

Oncolytic measles virus (MV) strains represent a promising novel oncolytic platform that is currently evaluated in several clinical trials including glioblastoma. The recently identified MV receptor, poliovirus receptor-related 4 (PVRL4), has accumulated great interest due to its strong biochemical binding affinity to MV and tumor specificity. However the regulation of PVRL4 is not understood. Immunohistochemistry and western blot analysis studies demonstrated increased PVRL4 protein levels in glioblastoma with no significant change in PVRL4 mRNA levels detected by qRT-PCR, suggesting that PVRL4 is likely post-transcriptionally regulated. Therefore, we investigated the potential role of miRNA in PVRL4 regulation and thereby MV infectivity. From miRNAs predicted, by the algorithms microRNA.org and RegRNA, to bind the 3’UTR of PVRL4, the miRNAs miR-31 and miR-128 which are reportedly down-regulated in glioblastoma, were selected as potential regulators of PVRL4. Luciferase functional binding studies showed that miR-31 and miR-128 binds to the 3’UTR of PVRL4. Similarly, over-expression of miR-31/-128 down-regulated PVRL4 protein levels while anti-miRs-31/-128 increased PVRL4 protein levels suggesting that PVRL4 is a miRNA targeted gene. Furthermore, miR-31 and miR-128 expression levels were down-regulated in glioblastoma patient samples (n= 18) compared with control gliosis (n=15) samples and showed statistically significant negative correlations with their respective PVRL4 protein levels signifying the post-transcriptional regulation of PVRL4 by miR-31 and miR-128. Gain-and-loss of function studies using a wild-type MV strain that exclusively utilizes PVRL4 as its receptor showed that over-expression of miR-31/128 decreases MV infectivity while inhibition of the respective miRNAs via anti-miRs increase MV infectivity in cell culture. In order to assess the miRNA regulation on MV infectivity in vivo, athymic nude mice were treated with 3-4 consecutive MV injections intratumorally at 1 × 106 TCID50 following establishment of subcutaneous glioblastoma (U87) tumor xenografts stably transduced with lentiviral-vectors encoding miRs alone or in combination with PVRL4 and anti-miRs. Tumor sizes were significantly decreased in tumors expressing anti-miRs-31/-128, while tumors expressing miR-31/ -128 showed increase in size after MV administration. Additionally, statistically significant correlations were observed between miR-128 levels, MV infectivity measured by qRT-PCR and tumor sizes validating the impact of miRNAs on MV infectivity and thus viral oncolysis. In addition to this regulation of MV infectivity by host miRNAs, we observed that MV infection up-regulated host miR-31expression levels in glioblastoma cells. This up-regulation of miR-31 expression resulted in down-regulation of PVRL4 suggesting that MV infection regulate host miRNA and in turn host PVRL4 levels. In conclusion, this study suggests that PVRL4 is post-transcriptionally regulated by miR-31/-128 and represent possible miRNA targets that could modulate MV infectivity to enhance MV based oncolytic therapeutic strategies, while the latter signifies the simultaneous regulation of host miRNA by MV infection.