Published online Aug 18, 2016. doi: 10.4254/wjh.v8.i23.976
Peer-review started: March 7, 2016
First decision: May 17, 2016
Revised: June 1, 2016
Accepted: July 11, 2016
Article in press: July 13, 2016
Published online: August 18, 2016
AIM: To investigate the effect of microRNA on insulin-like growth factor binding protein-3 (IGFBP-3) and hence on insulin-like growth factor-II (IGF-II) bioavailability in hepatocellular carcinoma (HCC).
METHODS: Bioinformatic analysis was performed using microrna.org, DIANA lab and Segal lab softwares. Total RNA was extracted from 23 HCC and 10 healthy liver tissues using mirVana miRNA Isolation Kit. microRNA-17-5p (miR-17-5p) expression was mimicked and antagonized in HuH-7 cell lines using HiPerFect Transfection Reagent, then total RNA was extracted using Biozol reagent then reverse transcribed into cDNA followed by quantification of miR-17-5p and IGFBP-3 expression using TaqMan real-time quantitative PCR. Luciferase reporter assay was performed to validate the binding of miR-17-5p to the 3’UTR of IGFBP-3. Free IGF-II protein was measured in transfected HuH-7 cells using IGF-II ELISA kit.
RESULTS: Bioinformatic analysis revealed IGFBP-3 as a potential target for miR-17-5p. Screening of miR-17-5p and IGFBP-3 revealed a moderate negative correlation in HCC patients, where miR-17-5p was extensively underexpressed in HCC tissues (P = 0.0012), while IGFBP-3 showed significant upregulation in the same set of patients (P = 0.0041) compared to healthy donors. Forcing miR-17-5p expression in HuH-7 cell lines showed a significant downregulation of IGFBP-3 mRNA expression (P = 0.0267) and a significant increase in free IGF-II protein (P = 0.0339) compared to mock untransfected cells using unpaired t-test. Luciferase assay validated IGFBP-3 as a direct target of miR-17-5p; luciferase activity was inhibited by 27.5% in cells co-transfected with miR-17-5p mimics and the construct harboring the wild-type binding region 2 of IGFBP-3 compared to cells transfected with this construct alone (P = 0.0474).
CONCLUSION: These data suggest that regulating IGF-II bioavailability and hence HCC progression can be achieved through targeting IGFBP-3 via manipulating the expression of miRNAs.
Core tip: microRNA-17-5p (miR-17-5p) was extensively underexpressed in hepatocellular carcinoma tissues, while insulin-like growth factor binding protein-3 (IGFBP-3) mRNA showed significant upregulation in the same set of patients. In HuH-7 cell line, miR-17-5p directly targets and downregulates IGFBP-3, consequently elevating the level of free insulin-like growth factor-II (IGF-II). Thus, manipulation of microRNAs can potentially control the activation of the oncogenic IGF axis.