miR-373-3p Regulates the Proliferative and Migratory Properties of Human HTR8 Cells via SLC38A1 Modulation

Abstract

The genetic pathogenesis of selective intrauterine growth restriction (sIUGR) remains elusive, with evidence suggesting an important role of epigenetic factors such as microRNAs. In this study, we explored the relevance of miR-373-3p to the occurrence of sIUGR. Hypoxia enhanced the levels of miR-373-3p and hypoxia-inducible factor (HIF)-1α, while HIF-1α knockdown not only boosted the migration and proliferation of HTR8 cells but also suppressed the hypoxia-induced upregulation of miR-373-3p and SLC38A1. By contrast, HIF-1α overexpression induced miR-373-3p downregulation and SLC38A1 upregulation, reducing cell growth and migration, which could be reversed by a miR-373-3p inhibitor. Importantly, the miR-373-3p inhibitor and mimic reproduced phenomena similar to those induced by HIF-1α downregulation and overexpression, respectively (including altered SLC38A1 expression, mTOR activation, cell growth, and migration). Mechanistically, the miRNA regulated cell behaviors and related mTOR signaling by targeting SLC38A1 expression through an interaction with the 3'-untranslated region of SLC38A1. The placental tissues of smaller sIUGR fetuses exhibited miR-373-3p and HIF-1α upregulation, SLC38A1 downregulation, and activated mTOR. Overall, miR-373-3p appears to restrict the growth and migration of HTR8 trophoblast cells by targeting SLC38A1, as observed in the placental tissues associated with smaller sIUGR fetuses, and it could have utility in the diagnosis and treatment of this disorder.

Figure 1

HIF-1α knockdown increased HTR8 cell migration and proliferation under hypoxic conditions. (a, b) qRT-PCR and western blot of the relative mRNA and protein levels of HIF-1α under normal and hypoxic conditions in human HTR8 cells. ^∗∗∗p < 0.001 vs Normal. (c, d) HIF-1α shRNAs significantly suppressed HIF-1α expression in HTR8 cells. ^∗∗∗p < 0.001 vs shNC. (e) Transwell assay of cell migration. ^∗∗∗p < 0.001 vs shNC. ^!!!p < 0.001 vs shNC. (f) CCK8 assay of the proliferation of HTR8 cells after transfecting shNC and shHIF-1α under hypoxia conditions. ^∗p < 0.05 vs control, ^∗∗∗p < 0.001 vs control; ^!!!p < 0.001 vs hypoxia + shNC. (g) qRT-PCR of the relative mRNA levels of SLC38A1 and miR-373-3p in HTR8 cells after transfecting with shNC or shHIF-1α under hypoxic conditions. ^∗∗∗p < 0.001 vs control; ^!!!p < 0.001 vs hypoxia + shNC. (h) Western blot of SLC38A1 protein expression in HTR8 cells after transfecting with shNC or shHIF-1α under hypoxic conditions.

Figure 2

HIF-1α overexpression abolished miR-373-3p inhibitor function in HTR8 cells. (a, b) qRT-PCR and western blot of the relative HIF-1α mRNA and protein levels in HTR8 after transfecting with oeNC and oeHIF-1α. ^∗∗∗p < 0.001 vs oeNC. (c) qRT-PCR of SCL38A1 protein levels in HTR8 cells treated with miR-373-3p miNC or inhibitor after transfecting with oeHIF-1α. ^∗p < 0.05 vs oeNC, ^∗∗p < 0.01 vs oeNC, ^∗∗∗p < 0.001 vs oeNC. ^!!!p < 0.001 vs miNC + oeHIF-1α. (d) Western blot of the SCL38A1 protein levels in HTR8 cells treated with miR-373-3p miNC or inhibitor after transfecting with oeHIF-1α. (e, f) The miR-373-3p inhibitor significantly promoted the proliferation and migration of HTR8 cells after transfecting with oeHIF-1α. ^∗p < 0.05 vs oeNC, ^∗∗∗p < 0.001 vs oeNC. ^!!!p < 0.001 vs miNC + oeHIF-1α.

Figure 3

miR-373-3p suppressed HTR8 cell proliferation and migration. (a) miR-373-3p silencing and overexpression induced by the corresponding mimic and inhibitor. ^∗∗∗p < 0.001 vs miNC. (b) qRT-PCR of the relative mRNA levels of SLC38A1 in HTR8 cells treated with miNC, miR-373-3p, and inhibitor. ^∗∗∗p < 0.001 vs miNC. (c) Western blot of the protein levels of SLC38A1, mTOR, and p-mTOR in HTR8 cells treated with miNC, miR-373-3p, and inhibitor. (d) CCK8 assay of HTR8 cell proliferation after treatment with the miR-373-3p inhibitor or mimic. ^∗p < 0.05 vs miNC, ^∗∗∗p < 0.001 vs miNC. (e) Transwell assay of HTR8 cell migration after treatment with the miR-373-3p inhibitor or mimic. ^∗∗∗p < 0.001 vs miNC; ^!!!p < 0.001 vs inhibitor.

Figure 4

miR-373-3p inhibited SLC38A1 function by binding to its 3′-UTR. (a) WT and mutant binding sites of miR-373-3p in the 3′-UTR of SLC38A1. (b) Mutant 3′-UTR abolished the interaction between miR-373-3p and SLC38A1. ^∗∗∗p < 0.001 vs miNC. (c, d) Lentiviral-mediated vector in HTR8 cells induced SLC38A1 overexpression. ^∗∗∗p < 0.001 vs oeNC. (e) SLC38A1 overexpression significantly upregulated the proliferation of mimic treated cells. ^∗p < 0.05 vs oeNC + miNC, ^∗∗∗p < 0.05 vs oeNC + miNC, ^!p < 0.05 vs oeSLC38A1 + miNC, ^!!!p < 0.001 vs oeSLC38A1 + miNC. (f) The miR-373-3p mimic inhibited the migration of oeSLC38A1 transfected cells. ^∗p < 0.05 vs oeNC + miNC, ^∗∗p < 0.01 vs oeNC + miNC, ^∗∗∗p < 0.001 vs oeNC + miNC. ^!p < 0.05 vs oeSLC38A1 + miNC, ^!!!p < 0.001 vs oeSLC38A1 + miNC. (g) Western blot of SCL38A1, mTOR, and p-mTOR protein levels in cells.