Exosome-transmitted microRNA-19a promotes drug resistance in breastcancer by targeting phosphatase and tensin homolog/protein kinase B/p53 axis

Abstract

Objective To investigate the specific effect and underlying molecular mechansim of microRNA (miRNA, miR)-19a through MCF-7/A secrete dexosomes in drug resistance transfer. Methods Exosomes were extracted from the supernatant of cultured MCF-7/A cells by fractionation ultracentrifugation and were identified by transmission electron microscopy and Nanosight. GFP-MCF-7/S, a breast cancer sensitive cell line stably expressing green fluorescent protein (GFP), was constructed by recombinant lentiviral vector with GFP. The co-cultured experiments of cell-cell and cell-exosomewere designed to observe thephenomenon of drug resistance transfer. Real-time quantitative polymerase chain reaction(Real-time PCR), Western blotting and cell transfection techniquewere used to determine the specific effects of miR-19a and phosphatase and tensin homologue deleted on chromosometen (PTEN)/protein kinase B (Akt)/p53 axis in the progress of drug resistance. Results Compared to MCF-7/S, the expression of miR-19a was significantly increased in MCF-7/A cells (1.03±0.16 vs. 3.56±0.58, P=0.002), down-regulaion of miR-19a apparently increased sensitivity to Adriamycin (ADM) in MCF-7/A cells [(41.17±10.65)% vs. (23.45±8.89)%, P=0.011]. Transmission electron microscopy showed that the exosomes cells had adiameter of 40-100 nm and exhibited round or elliptic shape secreted by both two type cancer cells. The cell inhibition ratio of ADM detected by the intensity of GFP and cell counting kit-8 (CCK-8) assay showed that cell inhibition rate of MCF-7/S+ MCF-7/A and MCF-7/S+ MCF-7/A exos group were significantly lower than the MCF-7/S+ MCF-7/S [(31.50±6.68)% vs. (51.20±12.44)%, P=0.006] and MCF-7/S+ MCF-7/S exos group [(27.67±6.02)% vs. (49.65±10.52)%, P=0.000], which companied with the increasing expression miR-19a (1.01±0.13 vs. 2.67±0.62, P=0.000). Western blotting assay showed that miR-19 in exosomes secreted by drug resistant cancer cells significantly inhibited the expression of PTEN (1.15±0.18 vs. 0.67±0.13, P=0.000) and p53 protein (0.99±0.18 vs. 0.49±0.11, P=0.005), whereas increases phosphorylation of Akt (1.15±0.18 vs. 0.67±0.13, P=0.000), which promoted the occurrence of drug resistance in receptor cells. Conclusion MiR-19a transfers drug resistance between breast cancer cells by exosome secreted from drug-resistance cancer cells through the regulation of PTEN/Akt/p53 axis in receptor cancer cells. Key words: Exosome; MicroRNA-19a; Breast cancer; Drug assistant; Phosphatase and tensin homologue deleted on chromosometen/protein kinase B/p53 axis