Archives

  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-08
  • br By applying a stick based approach this

    2020-08-18


    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.
    E-mail: [email protected]
    This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
    www.moleculartherapy.org
    A
    B
    sticky bridge GL
    C
    -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 ) Propidium iodide 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
    D
    Internalization
    Time (min)
    RESULTS
    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.