Fetal growth restriction is associated with abnormal placental cell (cytotrophoblast) proliferation. Using an explant model of human first trimester placenta, we have demonstrated that the IGFI and -II stimulate proliferation in cytotrophoblast and are probably essential for normal placental growth. IGF activates signalling through both Akt and ERK, so the regulation of these pathways in placenta is important for normal pregnancy outcome. The tissue contains high levels of microRNAs (miRs) some of which have been implicated in regulating IGF signalling. The production of miRs depends on processing from non-functional (pre)-miRs to mature miRs by two enzymes, Drosha and Dicer. In this study we have used Dicer siRNA to inhibit miR biogenesis in placental explants and examined the impact on IGF-stimulated proliferation in cytotrophoblast.
Dicer-specific or non-targeting siRNA (200 nM) was delivered to first trimester villous tissue fragments by nucleofector. Following transfection, tissue was maintained in culture for 72 h, then treated with IGF1 or 2 (10 nM) for a further 24 h before immunohistochemical (IHC) or QPCR analysis. Following exposure to siRNA, Dicer protein and mRNA expression were significantly decreased. IHC analysis of cell proliferation (Ki67) revealed enhanced levels of both basal (from 17.8±2.4 to 57.8±3.2%; P<0.05, n=5) and IGF-induced proliferation (from 53.6±5.3 to 78.0±1.8% for IGF1, and from 50.4±3.2 to 75±3.5% for IGF2; P<0.05, n=5) following Dicer knockdown. To examine the potential mechanism by which Dicer-dependant miRs regulate cytotrophoblast proliferation we examined the expression of molecules within the placental IGF-axis following Dicer knockdown. IHC analysis revealed no difference in IGF receptor (IGF1R) expression. However, the expression of both Akt and ERK was enhanced in the absence of Dicer.
Dicer-dependant miRs regulate expression of components in two important pathways downstream of the IGF receptor to influence placental growth. Future investigations will define specific target genes. This will uncover potential therapeutic avenues for placental undergrowth, which may ultimately lead to treatments for problem pregnancies.