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  • br We have previously reported that the


    We have previously reported that the GL21.T aptamer alone or conjugated to Let-7g miR rapidly internalizes into A549 (Axl+), getting about 60% of internal-
    ization at 2 h of incubation.16 We thus checked whether the presence of miR-137 could alter this function. To this end, high-salt washes were used to remove cell-surface-bound molecules and recover the internalized fraction.22 Total bound or internalized fractions were measured by qRT-PCR. In accordance with previous data, we found that GL21.T-137 preserves GL21.T internalization property on A549 cells and reaches about 62% of internalization after 2 h (Figure 1D). The same result was obtained by using proteinase K (PK) treatment to remove cell-surface molecules (Figure S1). These data confirm that GL21.T-137 preserves aptamer binding ability and internaliza-tion in Axl+ NSCLC cells.
    GL21.T-Mediated Functional Delivery of miR-137 in NSCLC
    As a next step, we confirmed that GL21.T-137 increases intracellular miR-137 levels at a comparable extent of a commercial miR-137 mimic upon transfection (Figure S2), thus preserving miR moiety function as well. Thus, we analyzed whether the treatment of A549 Axl-expressing cells may effectively increase the corresponding
    Molecular Therapy: Nucleic Acids
    a-tubulin (aTub) Malonyl Coenzyme A were used as a loading control. Values below the blots indicate quantization relative to untreated (“ ,” for treatment) or ctrl-miR (for transfection) normalized on the loading control signals. Molecular weights of indicated proteins are reported. 
    endogenous miR. In our previous report, we showed that 400 nmol/L treatment effectively assures a functional GL21.T-mediated miR delivery.16 Therefore, A549 cells were treated with 400 nmol/L of GL21.T-137, and the levels of miR-137 were determined by qRT-PCR. As shown, the AmiC treatment results in a significant increase of miR levels as compared with GL21.T or control aptamer (Fig-ure 2A), even though with several folds of difference in respect to miR-137 mimic transfection. Notably, the levels of miR-137 are not affected when cells are treated with a control complex (Figure 2B), thus confirming that miR-137 upregulation is dependent on the presence of the GL21.T aptamer in the complex rather than on non-specific molecular interactions. Then, we analyzed the levels of Cdk6, a validated miR-137 target.8 As shown in Figure 2C, GL21.T-137 treatment reduces Cdk6 of about 40% at 48 h compared to the control aptamer or the control complex. Of note, the extent of inhi-bition was comparable to the data obtained upon miR transfection (44% reduction compared to control miR) despite the significantly higher levels of miR-137 measured upon transfection (Figure 2A). This data suggests that the higher miR levels are not required to have an effect on the target but are likely related to off-target effects.
    Together, these experiments show that the AmiC effectively delivers functional miR-137 in NSCLC cells.
    GL21.T-137-Mediated Inhibition of NSCLC Cell Motility and Viability
    The GL21.T-137 AmiC combines two functional moieties, consisting of the GL21.T aptamer that is able to neutralize the Axl receptor activity inhibiting cell migration21 and miR-137, which has been described to be implicated in NSCLC survival and proliferation.8 Thus, we next analyzed the combined functional effects of the AmiC on NSCLC cells.
    We have previously reported that the GL21.T aptamer is able to inhibit ligand-dependent Axl tyrosine kinase activity (at 200 nmol/L concentration).21 We first investigated the ability of the AmiC to pre-serve this function. As shown in Figure 3A, inhibition of Gas6-depen-dent Axl activation is comparable between the GL21.T aptamer and the GL21.T-137 complex, confirming that the conjugation of the miR to the aptamer does not abrogate aptamer function. Conse-quently, the GL21.T-137 complex inhibits A549 (Axl+) cell migration at a level comparable to that of the GL21.T aptamer, reaching about 80% of inhibition (versus 75% for GL21.T). This effect mainly derives from the aptamer moiety, since the miR-137 transfection does not efficiently alter cell migration (Figure 3B). No reduction was detected by treating with the control aptamer or the control complex.