ObjectiveTo investigate the regulation of microRNA (miRNA)–155 expression under hypoxia and its effects on migration of trophoblast cells.

Method(1) Cobalt chloride (CoCl ₂) (100 μmol/L) was used to induce hypoxia in cultured human–trophoblast–derived HTR–8/SVneo cells, quantitative real–time polymerase chain reaction (PCR) was used to detect the expression of miRNA–155, JunB and FosB protein were then evaluated using Western blot, and wound healing assays were performed to assess cell migration. (2) SP600125 and/or PDTC were added to inhibit activation of the active protein–1 (AP–1) and nuclear factor–κB (NF–κB) pathways, and miRNA–155 was tested by quantitative real–time PCR. (3) HTR–8/SVneo cells were co–transfected with plasmids containing pEGFP–miRNA–155/pEGFP–C1 and pGL3–pro–cyclin D1 3'UTR, and luciferase reporter assays were used to assess the regulatoin of cyclin D1 '3 untranslated region (3'UTR) by miRNA–155. (4) HTR–8/SVneo cells were co–transfected with plasmids containing pEGFP–miRNA–155/pEGFP–C1 and pFLAG–CMV–cyclin D1/pFLAG–CMV–2 to induce overexpression of miRNA–155 and/or cyclin D1, and wound healing assays were used to assess cell migration. The two independent–samples  t test, one–way analysis of variance and LSD test were used for statistical analysis.

Results(1) Compared to cells cultured without CoCl ₂, hypoxia, induced by CoCl ₂ (100 μmol/L) for 24 and 48 h, induced enhanced expression of miRNA–155 [(1.40±0.28) fold and (1.74±0.14) fold,  t=3.302 and 8.578,  P=0.030 and 0.001, respectively], JunB and FosB protein were upregulated by hypoxia induced by 100 μmol/L CoCl ₂. However, migration rate decreased at 12, 24 and 48 h, especially at 48 h [(52.98±3.77)% vs (64.68±3.92)%,  t=5.259,  P=0.000].(2) In the AP–1 inhibited subgroup, NF–κB inhibited subgroup and AP–1+NF–κB inhibited subgroup, miRNA–155 was downregulated to (50.45±3.53)%, (47.18±2.14)% and (66.79±3.92)% of the non–inhibited subgroup ( t was 3.630, 4.100 and 3.392, all  P<0.05, respectively). (3)The luoiferase activity of cyclin D1 3'UTR was significatly decreased in the overexpression miRNA–155 subgroup than in the normal expression miRNA–155 subgroup ( t=46.682,  P=0.000). (4) The migration rates of HTR–8/SVneo cells in the overexpression miRNA–155 subgroup were lower than in the normal–expression miRNA–155 subgroup [at 48 h, (33.31±6.19)% vs (47.20±2.82)%, LSD test,  P=0.002, respectively], and in the overexpression miRNA–155 + cyclin D1 subgroup was higher than the overexpression miRNA–155 subgroup [at 48 h, (43.04±1.44)% vs (33.31±6.19)%, LSD test,  P=0.002, respectively].

ConclusionsHypoxia induces the expression of miRNA–155 via activation of the AP–1 and NF–κB pathways. Overexpression of miRNA–155 inhibits trophoblast migration by down–regulating cyclin D1.