Human Umbilical Cord Mesenchymal Stem Cells Improve Premature Ovarian Failure through Cell Apoptosis of miR-100-5p/NOX4/NLRP3

Abstract

Premature ovarian failure refers to a series of symptoms of perimenopausal hot flashes, night sweats, decreased libido, vaginal dryness, insomnia, reduced menstruation, sparse hair, even amenorrhea, and even infertility before the age of 40 due to the decline of ovarian function. Premature ovarian failure is a common and difficult disease in gynecology. Its prevalence is increasing gradually, and the trend is younger. The aim of this experiment was to elucidate the role of human umbilical cord mesenchymal stem cells (HUCMSCs) in premature ovarian failure and its mechanism. HUCMSCs, KGN cells, and HEK293T cells were used in this experiment. Quantitative PCR and microarray analysis, ELISA inflammation and oxidative stress kits, RNA pull-down assay, luciferase reporter assay, proliferation assay, EDU staining, and Western blot analysis were used. In an in vitro model of premature ovarian failure, HUCMSCs attenuated inflammatory response, oxidative stress, and apoptosis. HUCMSCs ameliorated the premature ovarian failure model. The miR-100-5p expression was induced by HUCMSCs through methylation. miR-100-5p regulation influenced the role of HUCMSCs in an in vitro model of premature ovarian failure. HUCMSCs inhibited the in vitro expression of NOX4, NLRP3, and GSDMD proteins in the model. NOX4/NLRP3 signaling pathway affects the role of HUCMSCs in an in vitro model of premature ovarian failure through miR-100-5p. This experiment elucidated the role of HUCMSCs in premature ovarian failure and its mechanism, with a view to providing a clinical reference.

Figure 1

Human umbilical cord mesenchymal stem cells improved premature ovarian failure in model cell growth (a), the number of EdU cell (b), cell metastasis (c), and caspase-3/9 activity levels (d, e). Control: control group; low/med/high: low/med/high of the HUCMSC group. ^∗∗p < 0.01 compared with the control group.

Figure 2

HUCMSCs reduced inflammation, oxidative stress, and cell pyroptosis in vitro model of premature ovarian failure IL-1β and IL-1α levels (a, b), ROS production levels (c), MDA level (d), SOD (e), CAT (f) and GSH-px (j) levels, cell growth (g), LDH activity levels (h), JC-1 disaggregation (i), PI levels (k), and calcein-AM/CoCl2 (l). Control: control group; low/med/high: low/med/high of the HUCMSC group. ^∗∗p < 0.01 compared with the control group.

Figure 3

HUCMSCs induced the miR-100-5p expression by methylation miR-100-5p expression (a), METTL3 and METTL14 mRNA expression (b, c), RIP-PCR assays performed by using m6A anti-body (d), depletion either METTL3 or METTL14 led to m6A modification level (e), and si-METTL3 or si-METTL14 resulted in downregulation of miR-100-5p level (f). Control: control group; low/med/high: low/med/high of the HUCMSC group. METTL3 or METTL14: METTL3 upregulation or METTL14 upregulation group. ^∗∗p < 0.01 compared with control or negative control group.

Figure 4

The regulation of miR-100-5p affected the effects of the HUCMSC in vitro model of premature ovarian failure MiR-100-5p (a), the number of EdU cell (b), cell metastasis (c), cell growth in vitro model by overexpression of miR-100-5p (d), MiR-100-5p (e), the number of EdU cell (f), cell metastasis (g), and cell growth in vitro model by downregulation of miR-100-5p (h). Vector: negative control group; miR-100-5p: overexpression of miR-100-5p group; sh-nc: sh-negative control group; sh-miR-100-5p: downregulation of miR-100-5p group; HUCMSCs: HUCMSC group; ^∗∗p < 0.01 compared with vector or sh-negative control group.

Figure 5

The regulation of miR-100-5p affected the effects of HUCMSCs on inflammation, oxidative stress, and cell pyroptosis in vitro model of premature ovarian failure IL-1β and IL-1α levels (a, b), ROS production levels (c), MDA level (d), SOD (e), CAT (f), and GSH-px (j) levels, cell growth (g), LDH activity levels (h), JC-1 disaggregation (i), PI levels (k), and calcein-AM/CoCl2 (l). Vector: negative control + HUCMSC group; miR-100-5p: overexpression of miR-100-5p + HUCMSC group; sh-nc: sh-negative control + HUCMSC group; sh-miR-100-5p: downregulation of miR-100-5p + HUCMSC group; ^∗∗p < 0.01 compared with vector or sh-negative control group.

Figure 6

NOX4/NLRP3 signaling pathway affected the effects of the HUCMSC in vitro model of premature ovarian failure by miR-100-5p heat map and combine sample correlation (a), volcanic map (b), result analysis (c), NOX4 mRNA (d, e), luciferase reporter levels and KLF13 constructed targeting miR-100-5p (f), and NOX4 protein expression (g, h). Control: control group; low/med/high: low/med/high of the HUCMSC group. Vector: negative control group; miR-100-5p: overexpression of miR-100-5p group; sh-nc: sh-negative control group; sh-miR-100-5p: downregulation of miR-100-5p group; HUCMSCs: HUCMSC group; ^∗∗p < 0.01 compared with vector or sh-negative control group.

Figure 7

NOX4/NLRP3 protein expression in NOX4, NLRP3, and GSDMD protein expression levels in the in vitro model by HUCMSCs; HUCMSCs promoted NOX4, NLRP3, and GSDMD protein expression (a)–(c), NOX4, NLRP3, and GSDMD protein expression levels in the in vitro model by overexpression of miR-100-5p + HUCMSCs (d)–(f), and NOX4, NLRP3, and GSDMD protein expression levels in the in vitro model by downregulation of miR-100-5p + HUCMSCs (g)–(i). Control: control group; low/med/high: low/med/high of the HUCMSC group. Vector: negative control group; miR-100-5p: overexpression of the miR-100-5p group; sh-nc: sh-negative control group; sh-miR-100-5p: downregulation of the miR-100-5p group; HUCMSCs: HUCMSC group; ^∗∗p < 0.01 compared with vector or sh-negative control group.