EphrinA1 inhibits malignant mesothelioma tumor growth via let-7 microRNA-mediated repression of the RAS oncogene

Abstract

EphrinA1 binding with receptor EphA2 suppresses malignant mesothelioma (MM) growth. The mechanisms whereby EphrinA1 attenuates the MM cell (MMC) growth are not clear. In this study, we report that the activation of MMCs with EphrinA1 leads to an induction of let-7 microRNA (miRNA) expression, repression of RAS proto-oncogene and the attenuation of MM tumor growth. The expression of miRNAs was determined by reverse transcription-quantitative polymerase chain reaction and in situ hybridization. RAS expression was determined by q-PCR, western blotting and immunofluorescence. MMC proliferation and tumor growth were determined by WST-1 and Matrigel assay, respectively. EphrinA1 activation induced several fold increases in let-7a1, let-7a3, let-7f1 and let-7f2 miRNA expression in MMCs. In contrast, EphrinA1 activation significantly downregulated H-RAS, K-RAS and N-RAS expression and inhibited MMC proliferation and tumor growth. In MMCs transfected with 2 0 -O-methyl antisense oligonucleotides to let-7 miRNA, EphrinA1 activation failed to inhibit the proliferative response and tumor growth. In mismatch antisense oligonucleotidetreated MMCs, the proliferation and tumor growth were comparable to untreated proliferating cells. Furthermore, the transfection of MMCs with let-7a miRNA precursor inhibited RAS expression and attenuated MMC tumor growth. Our data revealed that EphrinA1 signaling induces let-7 miRNA expression and attenuates MM tumor growth by targeting RAS proto-oncogene in MMCs.

Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer

“…4,24 In brief, cells were incubated at 37°C in 5% CO 2 and 95% air and were cultured to 80% confluence for 3-4 days and subcultured in 60 mm cell culture dishes (5 × 10 5 cells/dish) for various treatments accordingly. MPM and NSCLC in near confluence cultures were treated with or without 3.5 µg/mL recombinant mouse Ephrin-A1/ Fc chimera (R&D Systems, Minneapolis, MN, USA) in serum-free RPMI 1640, for the indicated times as reported earlier.…”

“…MPM and NSCLC in near confluence cultures were treated with or without 3.5 µg/mL recombinant mouse Ephrin-A1/ Fc chimera (R&D Systems, Minneapolis, MN, USA) in serum-free RPMI 1640, for the indicated times as reported earlier. 4 The cells were also treated with ephrin-A1 conjugated LNP (ephrin-A1-LNP) or let-7a miR encapsulated LNP (miR-LNP) or miR-control-LNP or only LNP. The cells were processed for total lysates and total RNA for immunoblotting and quantitative real-time polymerase chain reaction (qPCR), respectively.…”

“…4 Protein was estimated by using the Pierce ® BCA Protein Assay Kit (Thermo Fisher Scientific Inc), and equal amounts of protein (20 mg/lane) were loaded. Proteins in the sample were separated in denaturing sodium dodecyl sulphate polyacrylamide gels (Bio-Rad Laboratories) and transferred electrophoretically onto polyvinylidene difluoride membrane (Immobilon-P; EMD Millipore, Billerica, MA, USA).…”

“…4,37 Two thousand cells were seeded in each well of the 96-well plate and incubated in media containing LNP complexes. Cells were treated with free and LNP-encapsulated let-7a miR (miR-LNP, 20 nM), ephrin-A1 (ephrin-A1-LNP, 1 µg/mL-2 µg/mL) and the miR-ephrin-A1-LNP combination (20 nM-2 µg/mL-4 µg/mL).…”

“…Matrigel (BD, Franklin Lakes, NJ, USA) was diluted with serum-free media in the ratio (1:1) as reported earlier; 4,37 200 µL of diluted Matrigel solution were added into each well of 48-well plate and then allowed to gel at 37°C for at least 1 hour. After gelation, cells at a density of 3,000 per well were plated in 200 µL of serum-free media.…”