Induction of high levels of apoptosis of temozolomide-resistant T98G glioma cells by co-administration of peptide nucleic acids targeting miR-221 and miR-222

Gliomas express at high levels miR-221, promoting malignant progression through activation of the Akt pathway and inhibition of p27Kip1; in addition, miR-221 mediated down-regulation of other genes such as PUMA, ICAM-1, TIMP3 and PTEN might be associated with glioma onset and progression. Therefore, miR-221 appears to be a specific target for treatments. We have previously reported that a Peptide Nucleic Acid (PNA) carrying an Arg(8) peptide and targeting the miR-221 was able to increase the expression of p27Kip1 in a breast cancer cell line (Brognara et al., 2012). The major conclusion of the present study is that a PNA against miR-221 can be internalized by glioma cells when it is linked to a Arg(8) tail (R8), leading to inhibition of miR-221. No inhibitory effects were observed on miR-222; moreover the mutant R8-PNA-a221-MUT was inactive in inhibiting miR-221, using RT-qPCR as the validation assay. Interestingly, the inhibitory effect of R8-PNA-a221 on miR-221 was associated with increased expression of p27Kip1 in U251 and T98G cells. The expression of another miR-221 target gene, TIMP3 was upregulated following R8-PNA-a221 treatment of T98G cells. Treatment of all the glioma cells lines with R8-PNA-a221 (but not with PNA-a221 or R8-PNA-a221-MUT) induces the activation of the early apoptotic pathway. In addition, our results demonstrated that inhibition of miR-221 activity might lead to sensitization of T98G glioma cells to temozolomide, as recently suggested. These data support the concept that targeting miR-221 with antagomiR molecules might bring novel options in developing protocols for treatment of gliomas.