Regulation of HBEGF by Micro-RNA for Survival of Developing Human Trophoblast CellsReport as inadecuate




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Introduction

The growth factor HBEGF is upregulated post-transcriptionally in the low O2 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 O2 in human first trimester trophoblast.

Methods

HTR-8-SVneo trophoblast cells were cultured at 2% or 20% O2. 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% O2 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% O2 than 2% O2, mediated by the proteasome. However, proteasome inhibition failed to initiate HBEGF accumulation at 20% O2. 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% O2. Nevertheless, HBEGF upregulation at 2% O2 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% O2, possibly through a miRNA-mediated mechanism.



Author: Chandni V. Jain, Philip Jessmon, Brian A. Kilburn, Meritxell Jodar, Edward Sendler, Stephen A. Krawetz, D. Randall Armant

Source: http://plos.srce.hr/



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