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Figure 3. Scheme outlining the strategy to identify dysregulated microRNAs (a) in tumor patients and design possible therapeutic
approaches based on anti-miRNA therapy and miRNA-replacement. The activity of down-regulated miRNA might be replaced with the
use of miRNA mimicking molecules (b); the activity of up-regulated miRNAs might be counteracted with antagomiRNA molecules (c). A
partial list of dysregulated miRNAs in gliomas is depicted in Supplementary Table S1

level of miR-155, supporting the concept that characterization of the GBM patients is required before the
activation of a miRNA-155 targeting approach. Therefore, the analysis of the miRNA pattern appears to be
a required step in the road of personalized therapy on precision medicine based on miRNA therapeutics.

Figure 3 indicates an overall strategy for determining miRNA targets in cancer patients, on the road of
personalized therapy in precision oncology. The first step (step “a”) is the characterization of the miRNA
profile in tumor tissues, performed with either surgery-based biopsy or liquid biopsy. The characterization
of dysregulated miRNAs (down-regulated, “b” and up-regulated, “c”) will be helpful for staging, prognosis
of the neoplasia as well as for determining the response of the patients to drugs. In addition, the analysis
of dysregulated miRNAs might allow stratifying patients with respect to expression of specific miRNAs,
with the final objective to propose a therapeutic intervention (either based on anti-miRNA or miRNA-
replacement approach, as outlined in Figure 1). Liquid biopsy will be the best choice to monitor the outcome
of this tailored therapeutic approach .
[110]

PEPTIDE NUCLEIC ACID-MEDIATED TARGETING OF MICRORNAS IN GBM CELL LINES
The studies focusing on the possible use of PNAs targeting microRNAs are few. The first report was published
by Brognara et al. , who found that a PNA targeting miR-221 (R8-PNA-a221), bearing an oligoarginine
[62]
peptide (R8) enabling efficient uptake by glioma cells [58,59,62] , strongly inhibited miR-221-3p in U251, U373
and T98G glioma cells. This inhibition of miR-221-3p activity was associated with increased expression
of the miR-221 target p27Kip1, analyzed by RT-qPCR and by Western blotting [62,66] . In a second study,
Bertucci et al. reported the efficient combined delivery of temozolomide and the same anti-miR221 PNA
[111]
using mesoporous silica nanoparticles . More recently, high levels of apoptosis on glioma cell lines were
[111]
obtained following co-treatment with two PNAs, one targeting miR-221-3p, the other targeting miR-222-3p.
In addition, Seo et al. [113] showed the use of a PNA targeting miR-21 as in vivo inhibitor of glioma U87 cells.

A further example is a PNA against miR-155-5p. The possibility of obtaining clinically-relevant effects
following targeting miR-155-5p with PNA-based molecules is shown in Figure 4 and elsewhere reported in
the study by Milani et al. , describing the synthesis and validation of a PNA targeting miR-155-5p on the
[109]
temozolomide-resistant T98G glioma cell line .
[66]