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  • br GL T Mediated Functional Delivery


    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) Amiloride HCL 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.
    Then, we analyzed the complex functional effects dependent on the presence of miR-137. We found that the complex reduces A549 cell viability of approximately 30% following 72 h. On the contrary, no significant effect was detected by the GL21.T aptamer alone or in the presence of the CtrlApt or the CtrlApt-137 complex (Figure 4A). Further, in accordance with previous findings on miR-137 function in NSCLC,8 the GL21.T-137 treatment results in the downregulation of
    (A) Serum-starved A549 (Axl+) cells left untreated or
    treated for 3 h with 200 nmol/L of control aptamer,
    presence of each RNA. Cell lysates were immunoblotted
    a-tubulin antibodies. Quantization of the ratio of pAxl/Axl
    relative to untreated are indicated below the blots.
    Molecular weights of indicated proteins are reported. (B)
    was analyzed by transwell migration assay. Left panel:
    migrated Amiloride HCL cells were stained with crystal violet and pho-
    tographed. Right panel: migrated cells were quantified
    and expressed as percent of migrated cells with respect
    to untreated cells. Error bars depict mean ± SD. Statistics
    (versus untreated).
    the cyclin D1 and of the phosphorylated Rb, downstream miR-137 targets involved in cell proliferation8 (Figure 4B). We also analyzed the conjugate action in combination with the chemotherapeutic Paclitaxel and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor Erlotinib that are currently in clinical use as anti-cancer therapeutics. In our experimental conditions, A549 resulted resistant to Erlotinib, whereas a significant reduction of cell viability was found with Paclitaxel or GL21.T-137 treatment. No improvement of sensitivity was observed upon combined regimes of either Erlotinib or Paclitaxel and the complex treatment or miR-137 transfection (Figure S3).