RNA stability regulates differential expression of the metastasis protein, osteopontin, in hepatocellular cancer.
BACKGROUND
Osteopontin (OPN) is a potential therapeutic target in hepatocellular carcinoma (HCC), because it is a critical mediator of metastatic function. The molecular mechanisms that determine expression of OPN in HCC, however, are unknown. In this study, we examine differential OPN expression in the 2 HCC cell lines: HepG2 and Hep3B.


METHODS
OPN expression, metastatic function, OPN promoter activity, and active transcription of OPN mRNA and its decay were assessed in the 2 HCC cell lines using standard techniques. RNA gel-shift assays were performed to determine binding of cytoplasmic proteins to OPN mRNA.


RESULTS
Expression of OPN cellular/secreted protein and mRNA was greater in HepG2 than Hep3B cells (P < .01). Transient transfection of the OPN promoter construct demonstrated equivalent luciferase activities in the 2 cell lines; the rate of transcription was also equivalent as determined by chromatin immuno-precipitation assay. OPN mRNA half-life was 21 +/- 1 h and 3 +/- 1 h in HepG2 and Hep3B, respectively (P < .02). In HepG2 and Hep3B, the nucleotide sequence of OPN and its 5'-UTR, 3'-UTR, and poly (A) tail lengths were identical. A luciferase construct coupled in line with OPN-5'-UTR and OPN 3'-UTR presented greater expression in HepG2 (P < .01 vs Hep3B). Deletion of nt 10-57 of the OPN 5'-UTR restored luciferase and HA-tagged OPN protein expression in Hep3B but not in Hep G2. RNA gel-shift assays demonstrate different patterns of protein binding to OPN 5'-UTR between the 2 HCC cell lines.


CONCLUSIONS
We conclude that RNA stability is a new, previously unrecognized mechanism that regulates OPN expression in HCC to convey metastatic function.
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Sirisha
S
Emani
Jinping
J
Zhang
Lucie
L
Guo
Hongtao
H
Guo
Paul C
PC
Kuo
D020121:5' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006207:Half-Life; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020871:RNA Stability / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism
D020121:5' Untranslated Regions; D012333:RNA, Messenger; D053495:Osteopontin