Multidrug resistance is the main obstacle for hepatocellular carcinoma (HCC) treatment. Through bioinformatics prediction, literature review, and real-time PCR, we found that elevated miR-32-5p was associated with tumorigenesis in different cancer types, including HCC. miR-32-5p also contributes to castration resistance, radioresistance and chemoresistance in prostate cancer, but its function in multidrug resistance is still unclear. Exosomes are the most abundant type of extracellular vehicles (EVs), containing RNAs (especially miRs), proteins and other bioactive molecules. Recently, exosomes generated from chemoresistant cells have been proven to deliver miRs and transfer malignant phenotype to sensitive cells. Here we aim to find out the function of miR-32-5p in inducing multidrug resistance and the possible underlying mechanisms.
We detected the expression of miR-32-5p and PTEN in the cells and exosomes from both the multidrug-resistant and the sensitive cell lines, HCC and para-carcinoma liver tissues through real-time PCR. Dual-luciferase reporter assay verified PTEN is the target of miR-32-5p. Exosomes were obtained and confirmed through ultracentrifuge and Nano Analyzer. Gain- and loss-of-function experiments, rescue experiments, an exosome biogenesis inhibitor, and nude mice xenograft models were used to determine the underlying mechanisms of miR-32-5p and PTEN, as well as exosomal miR-32-5p in inducing multidrug resistance in vitro and in vivo.
miR-32-5p was significantly elevated in multidrug-resistant HCC cell line, Bel/5-FU as well as the exosomes derived from Bel/5-FU. An inverse correlation between miR-32-5p and PTEN was confirmed in HCC cell lines and patients; moreover, high expression of miR-32-5p and low expression of PTEN were positively associated with poor prognosis. Both in vitro and in vivo expriments reveal that exosomal miR-32-5p leads to multidrug resistance by targeting PTEN and activating the PI3K/Akt pathway through promoting EMT and angiogenesis.
Our study demonstrated that the multidrug-resistant cell, Bel/5-FU delivers miR-32-5p to sensitive cell, Bel7402 by exosome and activates the PI3K/Akt pathway to further induce multidrug resistance by modulating angiogenesis and EMT.
Clinical trial identification
Legal entity responsible for the study
The First Affiliated Hospital of Xi'an Jiaotong University.
National Natural Science Foundation of China (No. 81301909 and 81672810). International cooperation project in science and technology of Shaanxi province (No. 2016KW-017). Natural Science Foundation of Shaanxi Province (No.2017JM8019).
All authors have declared no conflicts of interest.