Micro-RNA-181a regulates osteopontin-dependent metastatic function in hepatocellular cancer cell lines.
BACKGROUND
Osteopontin (OPN) is a variably expressed, secreted glycophosphoprotein that mediates the growth and metastases of hepatocellular cancer (HCC). MicroRNAs (miRNAs) may be responsible for variant OPN expression, interrupting translation by binding OPN messenger RNA (mRNA) in 3'-untranslated regions (UTRs).


METHODS
A microarray analysis identified miRNAs of interest. Plasmid constructs using a luciferase reporter with variable OPN 3'UTR mutations were transfected into 2 HCC cell lines to determine miRNA regulation of OPN expression. Western blot analyses confirmed variable OPN expression in both cell lines. Invasion, adhesion, and migration evaluated metastatic behavior in Hep G2 and Hep 3B with modified miRNA and OPN expression.


RESULTS
Hep 3B produces 36 x miRNA 181a compared with Hep G2. Luciferase activity after transfection with miRNA 181a precursor was decreased in both cell lines (P < .01); luciferase activity increased with miRNA 181a inhibitor transfection in both cell lines (P < .01). Hep 3B transfected with mutated OPN 3'UTR increased luciferase activity 108% (P < .01). Hep G2 transfected with miRNA precursor decreased OPN expression 5 x (P < .01) in Western blot analyses. Hep 3B transfection with miRNA precursor increased OPN expression 3 x (P < .01) in Western blot analyses. In vitro metastatic correlates increased in Hep 3B lines after transfection with siOPN and/or miRNA 181a inhibitor (P < .01).


CONCLUSIONS
MiRNA 181a decreases OPN expression in HCC cell lines. This previously undescribed mechanism may confer metastatic characteristics to HCC.
Bhattacharya
Syamal D
SD
Department of Surgery, Duke University Medical Center, Durham, NC, USA.
Garrison
Juline
J
Guo
Hongtao
H
Mi
Zhiyong
Z
Markovic
Jovan
J
Kim
Victoria M
VM
Kuo
Paul C
PC
D020413:3' Untranslated Regions; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000503:physiopathology / Q000556:secondary*; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000503:physiopathology; D035683:MicroRNAs / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000503:physiopathology; D020411:Oligonucleotide Array Sequence Analysis; D053495:Osteopontin / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology*; D012334:RNA, Neoplasm / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
D020413:3' Untranslated Regions; D017931:DNA Primers; C540193:MIrn181 microRNA, human; D035683:MicroRNAs; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D053495:Osteopontin