MALAT1/miR-7-5p/TCF4 Axis Regulating Menstrual Blood Mesenchymal Stem Cells Improve Thin Endometrium Fertility by the Wnt Signaling Pathway

Abstract

Thin endometrium (TE) is a significant factor contributing to fertility challenges, and addressing this condition remains a central challenge in reproductive medicine. Menstrual blood-derived mesenchymal stem cells (MenSCs) play a crucial role in tissue repair and regeneration, including that of TE. The Wnt signaling pathway, which is highly conserved and prevalent in eukaryotes, is essential for cell proliferation, tissue development, and reproductive functions. MALAT1 is implicated in various transcriptional and molecular functions, including cell proliferation and metastasis. However, the combined effects of the Wnt signaling pathway and the long non-coding RNA (lncRNA) MALAT1 on the regulation of MenSCs' regenerative capabilities in tissue engineering have not yet been explored. To elucidate the regulatory mechanism of MALAT1 in TE, we analyzed its expression levels in normal endometrium and TE tissues, finding that low expression of MALAT1 was associated with poor clinical prognosis. In addition, we conducted both in vitro and in vivo functional assays to examine the role of the MALAT1/miR-7-5p/TCF4 axis in cell proliferation and migration. Techniques such as dual-luciferase reporter assay, fluorescent in situ hybridization, and immunoblot experiments were utilized to clarify the molecular mechanism. To corroborate these findings, we established a TE model and conducted pregnancy experiments, demonstrating a strong association between MALAT1 expression and endometrial fertility. In conclusion, our comprehensive study provides strong evidence supporting that lncRNA MALAT1 modulates TCF4 expression in the Wnt signaling pathway through interaction with miR-7-5p, thus enhancing MenSCs-mediated improvement of TE and improving fertility.

Keywords: Wnt signaling pathway; endometrial fertility; long non-coding RNA; menstrual blood–derived mesenchymal stem cells; thin endometrium.

Figure 1.

Isolation and identification of MenSCs. (A) Extraction process and brightfield morphology of primary MenSCs. Scale bar: 210 μm. (B) Flow cytometry analyses of MenSCs at passage 3 revealed the percentages of cells expressing CD73 (90.02%), CD90 (98.55%), CD105 (94.61%), and being negative for CD11b/CD34/CD79a (<1%). (C) Immunofluorescence staining demonstrated the differentiation potential of MenSCs into adipogenesis (FABP-4), osteogenesis (Osteocalcin), and chondrogenic (Aggrecan) lineages. Scale bar: 170 μm.

Figure 2.

Investigation of the MALAT1 and its knockdown. (A) MALAT1 expression in normal and TE (n = 5/group). (B) Expression of MALAT1 in the MenSCs group, si-MALAT1 group, and NC group. (C) CCK8 assay showing the proliferation of MenSCs and si-MALAT1 groups from day 1 to day 4; the MenSCs group is represented by a black line, and the si-MALAT1 group by a red line. (D) Wound-healing assays for MenSCs and si-MALAT1 groups (scale bar: 500 μm) at 0 h (top) and 24 h (bottom). (E) Quantitative analysis of the migration area for both groups. Data are presented as mean ± SEM, ****P < 0.0001, **P < 0.01, *P < 0.05 (Student’s t-test, n = 3).

Figure 3.

Relationship between MALAT1 and the Wnt signaling pathway. (A) Changes in RNA expression levels of CTNNB1, CD44, CCND1, C-MYC, GSK3B, and TCF4 in the Wnt signaling pathway following MALAT1 knockdown in MenSCs. (B and C) Alterations in protein expression of β-catenin, CD44, Cyclin D1, and GSK3B in the Wnt signaling pathway post-MALAT1 knockdown in MenSCs, accompanied by relative quantitative analysis. Data are presented as mean ± SEM, ****P < 0.0001, **P < 0.01, *P < 0.05 (Student’s t-test, n = 3).

Figure 4.

Localization of MALAT1 and its function as a sponge of miR-7-5p. (A) Localization of MALAT1 in MenSCs confirmed via FISH assay, with MALAT1 marked in green and the nuclear marker (DAPI) in blue. Scale bar: 100 μm (top) or 50 μm (bottom). (B) Comparison of wild-type (WT) and mutant-type (MT) complementary binding sequences between MALAT1 and miR-7-5p. (C) Variations in relative fluorescein activity between MALAT1-WT and MALAT1-MT upon altering miR-7-5p levels. Data are presented as mean ± SEM, ****P < 0.0001 (Student’s t-test, n = 3).

Figure 5.

miR-7-5p directly regulates TCF4 stability and influences endometrial regeneration. (A) Analysis of the binding sites between miR-7-5p and TCF4. (B) Variations in relative fluorescein activity between TCF4-WT and TCF4-MT in response to miR-7-5p levels. (C and D) qPCR and Western blot (WB) analyses were used to assess the expression changes in endometrial receptivity-related genes LIF and HOXA10 following MALAT1 knockdown in MenSCs, along with relative quantitative assessments. Data are presented as mean ± SEM, ****P < 0.0001, **P < 0.01, *P < 0.05 (Student’s t-test, n = 3).

Figure 6.

The effects of MALAT1 on endometrium and fertility were verified in vivo. (A) Overview of animal experimental procedures. (B) Comparative analysis of endometrial thickness across various treatment groups (n = 4). (C) Statistical evaluation of Gland numbers in different treatment groups (n = 4). (D) Comparative diagrams and statistical analysis of pregnancy sac counts in the Control group versus sham group, sham group versus TE group, sham group versus TE-MenSCs group, and sham group versus TE-si-MALAT1-MenSCs group (n = 3). Values are presented as mean ± SEM, ****P < 0.0001, **P < 0.01, ***P < 0.001, *P < 0.05 (one-way ANOVA).