Regulation of HBEGF by Micro-RNA for Survival of Developing Human Trophoblast Cells

INTRODUCTION: The growth factor HBEGF is upregulated post-transcriptionally in the low O(2) environment of the human placenta during the first 10 weeks of pregnancy. We have examined the possible roles of HBEGF turnover and micro-RNA (miRNA) in its regulation by O(2) in human first trimester trophoblast. METHODS: HTR-8/SVneo trophoblast cells were cultured at 2% or 20% O(2). The cells were transfected with a dual luciferase reporter construct (psiCHECK-2) containing no insert (control), the HBEGF 3’ untranslated region (3’UTR), or sub-regions of the 3’UTR, as well as with siRNA for DGCR8. RNA was extracted from trophoblast cells cultured at 2% O(2) for 0–4 h for next-generation sequencing. HBEGF was quantified by ELISA. HBEGF, DGCR8, and β–actin were examined by western blotting. RESULTS: Protein turnover studies, using 10 μg/ml cyclohexamide, 1 μg/ml lactocystin, or 100 μg/ml MG132, demonstrated faster HBEGF degradation at 20% O(2) than 2% O(2), mediated by the proteasome. However, proteasome inhibition failed to initiate HBEGF accumulation at 20% O(2). Reporter assays, comparing to empty vector, demonstrated that the intact HBEGF 3’ UTR inhibited expression (0.26), while fragments containing only its flanking regions increased reporter activity (3.15; 3.43). No differential expression of miRNAs was found in trophoblast cells cultured at 2% and 20% O(2). Nevertheless, HBEGF upregulation at 2% O(2) was blocked when the miRNA-processing protein DGCR8 was silenced, suggesting a role for miRNA. CONCLUSION: Our findings suggest involvement of flanking regions of the 3’UTR in activating HBEGF protein synthesis in response to 2% O(2), possibly through a miRNA-mediated mechanism.