MicroRNA‑524‑5p regulates the proliferation and invasion of HTR‑8/SVneo trophoblasts by targeting NUMB in the Notch signaling pathway

Abstract

Preeclampsia is a pregnancy disorder that is primarily associated with maternal and neonatal or fetal morbidity and mortality. The discovery of dysregulated microRNAs (miRs) and their roles in preeclampsia has provided new insight into the mechanisms involved in pregnancy‑related disorders. In the present study, quantitative PCR demonstrated that the expression levels of miR‑524‑5p were lower in patients with preeclampsia than those in normal pregnant women. Cell Counting Kit‑8 and Transwell assays indicated that overexpression of miR‑524‑5p promoted the proliferation and invasion of HTR‑8/SVneo cells, whereas inhibition of miR‑524‑5p suppressed HTR‑8/SVneo cell proliferation and invasion. Furthermore, NUMB endocytic adaptor protein (NUMB), a negative regulator of the Notch signaling pathway and a target gene of miR‑524‑5p, limited the effects of miR‑524‑5p on HTR‑8/SVneo cell invasion and migration. The present study demonstrated that miR‑524‑5p regulated the proliferation and invasion of HTR‑8/SVneo cells at least partly by targeting NUMB to regulate the Notch signaling pathway.

Keywords: preeclampsia; proliferation; invasion; microRNA; NUMB; Notch.

Figure 1.

Relative miR-524-5p expression levels in controls and patients with preeclampsia. **P<0.001. miR, microRNA.

Figure 2.

Low expression levels of miR-524-5p impede trophoblast cell proliferation and invasion. Relative miR-524-5p expression levels in HTR-8/SVneo cells following transfection with (A) miR-524-5p mimic and (B) inhibitor. Viability of HTR-8/SVneo cells transfected with (C) miR-524-5p mimic and (D) inhibitor for 0, 24, 48 and 72 h. (E) Western blot analysis of PCNA and Ki67 protein levels in HTR-8/SVneo cells transfected with miR-145-5p mimic or NC, and miR-145-5p inhibitor or NC. Transwell invasion assay was performed to assess the invasiveness of HTR-8/SVneo cells transfected with (F) miR-145-5p mimic or NC or (G) miR-145-5p inhibitor or NC. Scale bar, 100 µm. **P<0.01; miR, microRNA; PCNA, proliferating cell nuclear antigen; NC, negative control; OD, optical density.

Figure 3.

miR-524-5p regulates the expression levels of NUMB. (A) microRNA.org database was used to predict binding sites between miR-524-5p and NUMB. (B) Fluorescence intensity was measured using a dual luciferase assay. Overexpressed miR-524-5p bound to NUMB-WT and fluorescence intensity was weakened. NUMB-MUT exhibited no significant difference in fluorescence intensity. RT-qPCR was used to detect NUMB expression in the (C) inhibitor and (D) mimic groups. Compared with the inhibitor NC group, NUMB expression was significantly upregulated in the miR-524-5p inhibitor group, while compared with the mimic NC group, NUMB expression in the miR-524-5p mimic group was significantly downregulated. Western blotting was used to detect NUMB expression in the (E) inhibitor and (F) mimic groups. Compared with the inhibitor NC group, NUMB expression was significantly upregulated in the miR-524-5p inhibitor group, while it was significantly downregulated in the miR-524-5p mimic group compared with the mimic NC group. (G) RT-qPCR was used to detect the expression levels of NUMB in 40 preeclampsia tissues and Pearson's correlation coefficient was used to analyze the correlation between NUMB and miR-524-5p expression. There was a significant negative association between NUMB and miR-524-5p expression. **P<0.01. miR, microRNA; NUMB, NUMB endocytic adaptor protein; WT, wild-type; MUT, mutant; RT-q, reverse transcription-quantitative; NC, negative control; UTR, untranslated region; ns, not significant.

Figure 4.

miR-524-5p regulates the Notch signaling pathway via NUMB. (A) Cell Counting Kit-8 was used to detect the proliferation ability of each group for 0, 24, 48 and 72 h. (B) Western blot analysis of PCNA and Ki67 protein expression. (C) Invasive ability was detected using Transwell assay (Scale bar, 100 µm) and (D) quantified. (E) Western blot was used to detect the expression levels of NUMB, Notch1, Bcl-2, Cyclin D1 and CDK6 in the Notch signaling pathway. *P<0.05; **P<0.01; ***P<0.001 vs. mimic NC+ pcDNA3.1 group; ^#P<0.001 vs. miR-524-5p mimic + pcDNA3.1 group. miR, microRNA; NUMB, NUMB endocytic adaptor protein; PCNA, proliferating cell nuclear antigen; NC, negative control; OD, optical density; EV, empty vector.