br shown Quantification of signal
shown. Quantification of signal was normalized to cellular
GAPDH for each replicate. The dashed line represents
mock-infected values. (C) Validation of the miR-99b target
at KLF8 30 UTR. HEK293T Olivetolic Acid were co-transfected with a psiCHECK-2 reporter plasmid, containing wild-type
expression plasmid (miRVec control, miRVec-99b, or
miRVec-485). Renilla and luciferase activities were eval-
uated 72 hr post-transfection. (D and E) Relative infective
viral particle release in control PANC-1 cells (Lenticrv2)
and three CRISPR/Cas9-modified PANC-1 clones for
ELF4 (D) and KLF8 (E). Cells were seeded in triplicate and
tants were collected and titrated by viral infectious units.
The dashed line represents PANC-1 Lenticrv2 cells
at least three independent biological replicates. Signifi-
cance was assessed by comparison to mock-infected
cells using a one-sample t test and to infected cells using a
two-tailed Mann-Whitney test. For (C), data are shown as
mean ± SEM for at least three independent biological
replicates. Significance was assessed by comparison to
miRVec control-transfected cells using a one-sample t test. For (D), data are shown as mean ± SEM for at least five independent biological replicates. Significance was assessed by comparison to PANC-1 Lenticrv2 cells using a one-sample t test. *p < 0.05, **p < 0.01, ***p < 0.001.
need for novel adenoviral designs with increased potency. miRNAs have been shown to play a role in the progression of the adenoviral cycle.9–11 However, it remains unclear how adenoviral replication is affected by the extensively deregulated miRNA profile of tumor cells. We hypothesized that, by expressing specific miRNAs, it would be feasible to re-establish the levels of key cellular genes relevant for pro-ductive viral infection, leading to improved adenoviral oncolysis.
Adenoviruses have the potential of being armed with sequences of in-terest that provide additional functions. We performed an approach based on the generation of an adenoviral miRNA library encoding 243 human miRNAs, followed by a replication-based bioselection strategy that identified miR-99b and miR-485 as enhancers of the adenoviral activity in pancreatic cancer cells. A similar strategy has been previously used in the context of an in vivo alpha virus infection to identify interactions between the virus and host factors impacting viral replication.23 However, the designs of these approaches were
substantially different, since they were not expressing specific miRNAs but hairpin sequences designed to target unique murine open reading frames. Our strategy is also distinct from that of a pre-vious work studying miRNA effects on adenovirus propagation, in which the modulation of miRNA content was dissociated from adenoviral infection as miRNAs were introduced into cells 1 day before adenoviral infection.11 Here, by incorporating the miRNAs directly into the adenoviral genome, we were able to couple their expression with the infection, forcing cancer cells to parse out those factors that are important for the adenoviral replication cycle. The temporal differences in cellular miRNA expression, and probably the tumor model of pancreatic versus prostate cancer, may at least partially explain the identification of different miRNAs regulating adenoviral activity.
Our data show that miR-99b and miR-485 improved the intracellular formation and led to the earlier release of infective virions, leading to
Days post virus injection
ICOVIR15 miR miR
Days post virus injection
Figure 6. miR-99b and miR-485 Confer Increased Oncolytic Activity and Enhanced Antitumoral Response to ICOVIR15
(A) Relative infective viral particle release. PANC-1 cells were seeded in triplicate and infected with ICOVIR15, ICOVIR15 miR-99b, or ICOVIR15 miR-485 (0.5 IFU/cell). At
48 hr PI, supernatants were collected and titrated by viral infectious units. The dashed line represents ICOVIR15 values. (B) In vitro oncolytic activity in PANC-1 cells. Cells were seeded in triplicate and infected with a dose range of ICOVIR15, ICOVIR15 miR-99b, or ICOVIR15 miR-485. Cell viability was measured at 7 days PI by MTT assay and
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enhanced lytic effects. Changes in the expression of these two miRNAs in response to viral infections have been documented for other viruses. During an influenza virus infection, miR-485 expres-sion increases in order to reduce RIG-1 levels and to diminish the induction of downstream antiviral proteins.24 On the other hand, miR-99 family members facilitate hepatitis B virus (HBV) replication in hepatoma cells by increasing autophagic activity and by promoting HBV protein production.25 Thus, miR-99b and miR-485 seem to have a proviral function that supports their bioselection during our screening strategy.