PNA-a221 with the argininocalix[4]arene 1 (Fig. 3a). To further confirm that the effect is sequence-specific the effects of
argininocalix[4]arene 1 delivered PNA-a221 on other miRNAs should be determined. The expected result is that hybridization specific for other miRNAs expressed in U251 cells (for
instance, miR-96-5p, miR-155-5p, and miR-210-3p) was
found unchanged following delivery of the PNA-a221 with
the argininocalix[4]arene 1 (Fig. 3b). In fact, the representative experiments shown in Figure 3b demonstrate no effect of
argininocalix[4]arene 1 delivered PNA-a221 on miR-96-5p,
miR-155-5p, and miR-210-3p. Altogether these experiments
support the concept that the effects of argininocalix[4]arene
1 delivered PNA-a221 are sequence-specific (see Note 20 for a
further discussion on the effects of PNAs on the hybridization
efficiency of targeted miRNA). Of course, the effects of PNA
against microRNAs should also be analyzed on the expression
of miRNA-regulated functions and/or target mRNAs.
20. Explanation of the effects of PNAs delivered by the argininocalix[4]arene 1 on microRNA accumulation. PNAs might
interact very stably with target mRNAs. Therefore, before
performing RT-qPCR, it is necessary to demonstrate that
PNAs are not co-purified with target mRNAs. High-quality
RNA preparation is necessary. Complementary analyses (for
instance, Northern blotting) might also be considered. Studies
suggesting a PNA-mediated effect on microRNA content
should be in any case accompanied by studies on the effects
on the expression of mRNAs demonstrated to be target of the
studied microRNAs [46].
21. The effects of PNA against microRNAs should also be analyzed
on the expression of miRNA-regulated functions. In our example, several biological functions have been firmly established to
be target molecules of miR-221-3p, such as apoptosis [65–
72]. In the context of the development of anti-tumor
approaches, the pro-apoptotic activity following delivery of
PNA-a221 by argininocalix[4]arene 1 to U251 cells is of
great interest. This is described in Fig. 5. First of all, apoptosis
is not induced by a mutated R8-PNA-a221 (Fig. 5a) or by the
PNA-a221 lacking the R8 peptide (Fig. 5b). By contrast, high
levels of apoptosis were obtained when 1/PNA-a221 formulation is employed (Fig. 5b–d).
22. The results shown in Fig. 6 were performed in order to verify
whether argininocalix[4]arene 1 was to some extent cytotoxic.
Figure 6a, b shows that argininocalix[4]arene 1 employed at
concentrations equal or lower than 5 μM did not reduce the
extent of viable cells (panel a) and did not exhibit antiproliferative effects (panel b). Moreover, argininocalix[4]arene 1 did
not induce alteration of morphology of U251 cells.
138
Alessia Finotti et al.
argininocalix[4]arene 1 delivered PNA-a221 on other miRNAs should be determined. The expected result is that hybridization specific for other miRNAs expressed in U251 cells (for
instance, miR-96-5p, miR-155-5p, and miR-210-3p) was
found unchanged following delivery of the PNA-a221 with
the argininocalix[4]arene 1 (Fig. 3b). In fact, the representative experiments shown in Figure 3b demonstrate no effect of
argininocalix[4]arene 1 delivered PNA-a221 on miR-96-5p,
miR-155-5p, and miR-210-3p. Altogether these experiments
support the concept that the effects of argininocalix[4]arene
1 delivered PNA-a221 are sequence-specific (see Note 20 for a
further discussion on the effects of PNAs on the hybridization
efficiency of targeted miRNA). Of course, the effects of PNA
against microRNAs should also be analyzed on the expression
of miRNA-regulated functions and/or target mRNAs.
20. Explanation of the effects of PNAs delivered by the argininocalix[4]arene 1 on microRNA accumulation. PNAs might
interact very stably with target mRNAs. Therefore, before
performing RT-qPCR, it is necessary to demonstrate that
PNAs are not co-purified with target mRNAs. High-quality
RNA preparation is necessary. Complementary analyses (for
instance, Northern blotting) might also be considered. Studies
suggesting a PNA-mediated effect on microRNA content
should be in any case accompanied by studies on the effects
on the expression of mRNAs demonstrated to be target of the
studied microRNAs [46].
21. The effects of PNA against microRNAs should also be analyzed
on the expression of miRNA-regulated functions. In our example, several biological functions have been firmly established to
be target molecules of miR-221-3p, such as apoptosis [65–
72]. In the context of the development of anti-tumor
approaches, the pro-apoptotic activity following delivery of
PNA-a221 by argininocalix[4]arene 1 to U251 cells is of
great interest. This is described in Fig. 5. First of all, apoptosis
is not induced by a mutated R8-PNA-a221 (Fig. 5a) or by the
PNA-a221 lacking the R8 peptide (Fig. 5b). By contrast, high
levels of apoptosis were obtained when 1/PNA-a221 formulation is employed (Fig. 5b–d).
22. The results shown in Fig. 6 were performed in order to verify
whether argininocalix[4]arene 1 was to some extent cytotoxic.
Figure 6a, b shows that argininocalix[4]arene 1 employed at
concentrations equal or lower than 5 μM did not reduce the
extent of viable cells (panel a) and did not exhibit antiproliferative effects (panel b). Moreover, argininocalix[4]arene 1 did
not induce alteration of morphology of U251 cells.
138
Alessia Finotti et al.