Interplay between microRNA-17-5p, insulin-like growth factor-II through binding protein-3 in hepatocellular carcinoma.
OBJECTIVE
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).


CONCLUSIONS
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.
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt.
Danira Ashraf
DA
Habashy
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt.
Hend Mohamed
HM
El Tayebi
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt.
Injie Omar
IO
Fawzy
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt.
Karim Adel
KA
Hosny
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt.
Gamal
G
Esmat
Danira Ashraf Habashy, Hend Mohamed El Tayebi, Injie Omar Fawzy, Department of Pharmacology and Toxicology, German University in Cairo, Main Entrance Al Tagamoa Al Khames, Cairo 11835, Egypt.
Ahmed Ihab
AI
Abdelaziz
Hepatocellular carcinoma; Insulin-like growth factor binding protein-3; Insulin-like growth factor signaling pathway; Insulin-like growth factor-II; MicroRNA