H19 sense and antisense transgenes modify insulin-like growth factor-II mRNA levels

The oppositely-imprinted genes insulin-like growth factor-II (IGF2) and H19 , a putative tumor suppressor, often show coordinate, reciprocal regulation and are believed to play a role in carcinogenesis. To explore the possible interactions between these genes, we stably transfected diHepG2 cells with a plasmid containing either the sense or the antisense H19 cDNA sequences and verified their expression by Northern analysis and by RNase protection analysis. Levels of H19, IGF2 and gamma-actin mRNA were quantified by compe titive RT-PCR analysis. Although H19 sense transgene overexpression (n = 24 clones) did not decrease the low, basal levels of IGF2 mRNA compared to co ntrol cells, levels of IGF2 mRNA were positively correlated with the levels of H19 antisense mRNA (P < 0.0001, n = 40 clones). Furthermore, the increa se in IGF2 mRNA level was accompanied by an elevation of IGF-II peptide in conditioned media. To see if H19 mRNA had a specific effect on transcriptio n, we also performed transient transfections with reporter gene constructs containing IGF2 promoter 3 in the presence of sense or antisense H19 cDNA s equences under control of a cytomegalovirus promoter. We show a lower repor ter gene activity from reporter gene constructs in the presence of sense H1 9 cDNA than from those with antisense or neomycin. Our results suggest that H19 participates in the repression of IGF2, at least in part through effec ts on IGF2 transcription, an effect which may contribute to its action as a tumor suppressor.