miRNA-210-3p regulates trophoblast proliferation and invasiveness through fibroblast growth factor 1 in selective intrauterine growth restriction

Abstract

Selective intrauterine growth restriction (sIUGR), which affects approximately 10%-15% of monochorionic (MC) twin pregnancies, is highly associated with intrauterine foetal death and neurological impairment in both twins. Data suggest that unequal sharing of the single placenta is the main contributor to birth weight discordance. While MC twins and their placenta derive from a single zygote and harbour almost identical genetic material, the underlying mechanisms of phenotypic discrepancies in MC twins remain unclear. MicroRNAs are small non-coding RNA molecules that regulate gene expression but do not change the DNA sequence. Our preliminary study showed that microRNA-210-3p (miR-210-3p) was significantly upregulated in the placental share of the smaller sIUGR twin. Here, we investigate the potential role of miR-210-3p in placental dysplasia, which generally results from dysfunction of trophoblast cells. Functional analysis revealed that miR-210-3p, induced by hypoxia-inducible factor 1α (HIF1α) under hypoxic conditions, suppressed the proliferation and invasiveness of trophoblast cell lines. Further RNA sequencing analysis and luciferase reporter assays were performed, revealing that fibroblast growth factor 1 (FGF1) is an influential target gene of miR-210-3p. Moreover, correlations among miR-210-3p levels, HIF1α and FGF1 expression and the smaller placental share were validated in sIUGR specimens. These findings suggest that upregulation of miR-210-3p may contribute to impaired placentation of the smaller twin by decreasing FGF1 expression in sIUGR.

Keywords: fibroblast growth factor 1; miRNA-210-3p; monochorionic twins; selective intrauterine growth restriction; trophoblast.

Figure 1

Expression of miR‐210‐3p in the placenta. A, Six paired (P1‐P6) selective intrauterine growth restriction (sIUGR) placental tissues were subjected to microRNA sequencing, and differentially expressed microRNAs between placental shares of the smaller twins and larger twins are shown on the heatmap. The scale colour bar represents log2 (fold change) between the smaller twins and larger twins. B, qRT‐PCR results showing miR‐210‐3p levels in the placental share of the smaller foetus compared to those of the larger foetus in the sIUGR group (n = 18) and control group (n = 18). C, miR‐210‐3p levels in the placental share of the smaller foetus compared to those of the larger foetus in three types of sIUGR twins

Figure 2

miR‐210‐3p inhibited HTR‐8/SVneo cell proliferation and invasiveness. HTR‐8/SVneo cells were stably transduced with miR‐210‐3p mimic, miR‐210‐3p sponge, or corresponding vector controls through lentivirus infection and antibiotic selection. A, Cell growth curves for the indicated cells were measured via an MTT assay. B, Representative images and quantification of invaded cells in the Matrigel‐coated Transwell assay are shown as indicated. (*P < 0.05, **P < 0.01)

Figure 3

miR‐210‐3p directly targets fibroblast growth factor 1 (FGF1) in HTR‐8/SVneo cells. (A,B) HTR‐8/SVneo cells stably overexpressing miR‐210‐3p and corresponding vector control cells were subjected to mRNA sequencing. The differentially expressed genes were plotted, and the top 20 upregulated and downregulated genes are shown on the heatmap. The scale colour bar represents the normalized expression level in each sample. C, Differentially expressed genes were subjected to Gene Ontology (GO) biological process analysis, and the top five enriched gene sets are listed. D, qRT‐PCR analysis of the relative expression of FGF1 in the indicated cells. E, Western blotting analysis of the protein levels of FGF1 in the indicated cells. F, Schematic representation of the putative miR‐210‐3p binding site in the 3′‐untranslated region (3′‐UTR) of FGF1 and the corresponding mutant sequence are shown as indicated. These sequences were ligated into a luciferase reporter construct, and the results of the luciferase assay with reporters driven by the wild‐type and mutated FGF1 3′‐UTR in the indicated cells are shown. (*P < 0.05, **P < 0.01)

Figure 4

Fibroblast growth factor 1 (FGF1) is required for miR‐210‐3p‐mediated regulation of HTR‐8/SVneo cell proliferation and invasiveness. (A,B) Fibroblast growth factor 1 expression was restored in miR‐210‐3p‐overexpressing cells (A), and FGF1 expression was further silenced in cells transfected with the miR‐210‐3p sponge (B). Growth curves for the indicated cells were measured via MTT assay. (C,D) Fibroblast growth factor 1 expression was restored in miR‐210‐3p‐overexpressing cells (C), and FGF1 expression was further silenced in cells transfected with a miR‐210‐3p sponge (D). Representative images and quantification of invading cells in the Matrigel‐coated Transwell assay are shown as indicated. (*P < 0.05, **P < 0.01)

Figure 5

Hypoxia regulates HTR‐8/SVneo cell proliferation and invasiveness through the HIF1α/miR‐210‐3p/FGF1 axis. (A,B) HTR‐8/SVneo cells were incubated under normoxic conditions with 21% oxygen and under hypoxic conditions with 10%, 5% and 2% oxygen for 48 h. qRT‐PCR analysis of the relative expression of miR‐210‐3p (A), and Western blotting analysis of the protein levels of FGF1 and HIF1α (B) at the four different oxygen concentrations. (C) HIF1α was silenced by siRNAs in HTR‐8/SVneo cells. qRT‐PCR analysis of the relative expression of miR‐210‐3p in the indicated cells at 21% or 2% oxygen concentrations. (D,E) HTR‐8/SVneo cells containing the miR‐210‐3p sponge and corresponding vector were incubated under normoxic conditions with 21% oxygen and under hypoxic conditions with 10%, 5% and 2% oxygen for 48 h. Growth curves for the indicated cells at different oxygen concentrations were measured via MTT assay (D). Representative images and quantification of invading cells at different oxygen concentrations in the Matrigel‐coated Transwell assay are shown (E). (*P < 0.05, **P < 0.01). HIF1α, hypoxia‐inducible factor 1α; FGF1, fibroblast growth factor 1

Figure 6

Correlation of miR‐210‐3p expression with hypoxia‐inducible factor 1α (HIF1α) and fibroblast growth factor 1 (FGF1) expression in placental tissues. A, qRT‐PCR analysis of the relative expression of miR‐210‐3p in placental tissues from eight pairs of selective intrauterine growth restriction (sIUGR) twins (P1‐P8). B, Western blotting analysis of the protein levels of FGF1 and HIF1α in placental tissues from eight pairs of sIUGR twins (P1‐P8). C, Correlation analysis of miR‐210‐3p expression with FGF1 and HIF1α expression. L, larger twin; S, smaller twin. (*P < 0.05, **P < 0.01)