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  • br By applying a stick based approach this


    By applying a stick-based approach, this aptamer was recently linked to miR-137, generating a complex (named GL21.T-137) to target glioblastoma cancer stem-like cells.18
    Given the promising role of miR-137 in NSCLC, in this paper we analyzed the functional effect of GL21.T-137 aptamer-miR complex (AmiC) on lung cells.
    Our results show that GL21.T-137 treatment leads to inhibiting NSCLC migration and survival by combining both the inhibitory function of GL21.T aptamer on Axl receptor and the reduction of miR-137 targets. In addition, GL21.T-137 complex demonstrated to effectively reduce tumor growth in vivo in NSCLC mouse xenografts.
    The described complex has a broad applicability to cancer treatment and represents a potential tool for NSCLC treatment.
    8These authors contributed equally to this work.
    Correspondence: Carla Lucia Esposito, Istituto di Endocrinologia ed Oncologia
    Sperimentale, Consiglio Nazionale delle Ricerche (CNR), Via T. de Amicis, 80131
    Naples, Italy.
    This is an open access article under the CC BY-NC-ND license (
    sticky bridge GL
    -st ladder
    Figure 1. GL21.T-137 Preparation, Binding, and Internalization
    (A) Scheme of GL21.T-137 AmiC based on stick-end annealing. (B) The annealing efficacy was confirmed by loading each component or annealed conjugate on a 12% non-denaturing polyacrylamide gel followed by staining with ethidium bromide. GL21.T-st, GL21.T sticky; 137-pass-st, miR-137 passenger strand sticky; 137-guide, miR-137 guide strand. (C) Binding of
    control complex (CtrlApt-137) on A549 (Axl+) versus MCF-7 (Axl ) Pimozide measured by qRT-PCR after 30 min of incubation. Statistics were calculated using Student’s t test, **p < 0.01. (D) Internalization of 200 nmol/L GL21.T-137 was monitored by qRT-PCR (see Materials and Methods for details). The percentage of internalization is expressed as the amount of internalized RNA relative to total bound RNA.
    Ctrl Apt
    Time (min)
    GL21.T-137 Conjugate Binding and Internalization in NSCLC
    We have recently designed a multifunctional complex (GL21.T-137) in which the GL21.T aptamer, an Axl receptor antagonist, is used as a
    delivery carrier for miR-137.18 For the complex generation, we used a stick-based strategy (Figure 1A). As we previously reported,16,19 we
    derived the miR mimetic portion from the distal stem of the human miR-137 precursor using 29 bases of the 50 strand and 28 of the 30 strand, in order to produce an internal partial complementarity and
    a more effective Dicer substrate.20 The annealing efficiency was monitored by the presence of a shifted band of migration on a non-denaturing gel (Figure 1B). Considering that it has been shown that
    miR-137 functions as an oncosuppressor in NSCLC and that high miR-137 levels correlate with a higher survival rate,6–9 we analyzed GL21.T-137 complex on NSCLC cells.
    As a first attempt, we analyzed whether in the context of the AmiC the aptamer preserves a good binding ability on A549 (Axl+) NSCLC cells. We used as negative control MCF-7 (Axl-) cells, on which we  have already found no detectable binding of the GL21.T aptamer.16,17,21 As shown in Fig-
    ure 1C, the GL21.T-137 complex preferentially binds target A549 (Axl+) cells compared to the MCF-7 (Axl ) cells. No discrimination was de-tected by treating either with a control aptamer (CtrlApt) or with a control complex containing the CtrlApt linked to miR-137 (CtrlApt-137), supporting that the GL21.T-137 complex spe-cifically targets Axl-expressing cells. This result is in good agreement with data obtained for the GL21.T aptamer21 or GL21.T complexes con-taining other therapeutic RNA cargoes.6,17
    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.