miR-6881-3p contributes to diminished ovarian reserve by regulating granulosa cell apoptosis by targeting SMAD4.

BACKGROUND: In our previous investigation, we revealed a significant increase in the expression of microRNA-6881-3p (miR-6881-3p) in follicular fluid granulosa cells (GCs) from women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR). However, the role of miR-6881-3p in the development of DOR remains poorly understood. OBJECTIVE: This study aimed to elucidate the involvement of miR-6881-3p in the regulation of granulosa cells (GCs) function and the pathogenesis of DOR. MATERIALS AND METHODS: Initially, we assessed the expression levels of miR-6881-3p in GCs obtained from human follicular fluid in both NOR and DOR cases and explored the correlation between miR-6881-3p expression and clinical outcomes in assisted reproduction technology (ART). Bioinformatic predictions and dual-luciferase reporter assays were employed to identify the target gene of miR-6881-3p. Manipulation of miR-6881-3p expression was achieved through the transfection of KGN cells with miR-6881-3p mimics, inhibitor, and miRNA negative control (NC). Following transfection, we assessed granulosa cell apoptosis and cell cycle progression via flow cytometry and quantified target gene expression through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Finally, we examined the correlation between target gene expression levels in GCs from NOR and DOR patients and their association with ART outcomes. RESULTS: Our findings revealed elevated miR-6881-3p levels in GCs from DOR patients, which negatively correlated with ovarian reserve function and ART outcomes. We identified a direct binding interaction between miR-6881-3p and the 3'-untranslated region of the SMAD4. Transfection with miR-6881-3p mimics induced apoptosis in KGN cell. Furthermore, miR-6881-3p expression negatively correlated with both mRNA and protein levels of the SMAD4. The mRNA and protein levels of SMAD4 were notably reduced in GCs from DOR patients, and SMAD4 mRNA expression positively correlated with ART outcomes. In addition, the mRNA levels of FSHR, CYP11A1 were notably reduced after transfection with miR-6881-3p mimics in KGN cell, while LHCGR notably increased. The mRNA and protein levels of FSHR, CYP11A1 were notably reduced in GCs from DOR patients, while LHCGR notably increased. CONCLUSION: This study underscores the role of miR-6881-3p in directly targeting SMAD4 mRNA, subsequently diminishing granulosa cell viability and promoting apoptosis, and may affect steroid hormone regulation and gonadotropin signal reception in GCs. These findings contribute to our understanding of the pathogenesis of DOR.

Exosomes from LSD1 knockdown breast cancer cells activate osteoclastogenesis and inhibit osteoblastogenesis.

Bone metastasis is a common and incurable complication of breast cancer. Lysine-specific demethylase 1 (LSD1), a histone demethylase, plays an important role in the metastasis of breast cancer. However, the role of LSD1 in bone metastasis of breast cancer is unclear. We hypothesized that exosomes from LSD1 knockdown breast cancer cells promote bone metastasis by remodeling bone microenvironment. To verify this hypothesis, exosomes from LSD1 knockdown Estrogen receptor-positive cancer cell lines, MCF7 and T47D, were isolated, and the effects of these exosomes on osteoblast and osteoclast differentiation were investigated. Interestingly, exosomes from LSD1 knockdown breast cancer cells inhibited osteoblast differentiation and promoted osteoclast differentiation. Mechanistically, miR-6881-3p was decreased in the exosomes from LSD1 knockdown cells, and miR-6881-3p suppressed the expression of pre-B-cell leukemia homeobox 1 (PBX1) and additional sex combs like-2 (ASXL2), two genes with essential functions in osteoblast and osteoclast differentiations respectively. Transfection of miR-6881-3p into LSD1 knockdown cells reversed the effects of the exosomes on osteoblast and osteoclast differentiations. Our study reveals important roles of LSD1 on the regulation of exosomal miRNAs and the formation of favorable bone microenvironment for metastasis.

Circ-EEF2 facilitated autophagy via interaction with mir-6881-3p and ANXA2 in EOC.

Circular RNAs, a special class of non-coding RNA with closed circular structure, have been increasingly proven to be involved in the progression of various tumors. However, the biological functions of circular RNAs in epithelial ovarian cancer (EOC) tissues remain a mystery. In this study, we detected the function of circEEF2 (has-circ-0048559) in EOC tissues. Firstly, the basic characteristics including closed circular structure and spliced mature sequence length of circEEF2 were confirmed. The location and expression in EOC tissues was detected by fluorescence in situ hybridization (FISH). The regulatory effect of circEEF2 on autophagy, proliferation, and invasion were investigated in SKOV3 and A2780 cells. The relationship between circEEF2 and mir-6881-3p was confirmed using dual-luciferase reporter gene assay. The binding of circEEF2 with ANXA2 was confirmed using RNA-pulldown assay and MALDI-TOF-MS. We found that the expression level of circEEF2 was higher in EOC tissue than in normal tissue. CircEEF2 promoted autophagy, proliferation, and invasion. CircEEF2-regulated EOC proliferation and invasion are closely related to the occurrence of autophagy. Mechanistically, circEEF2 harbor miR-6881-3p to upregulate the latter's targets ATG5 and ATG7. Moreover, circEEF2 could directly bind with ANXA2 to inhibit the expression of p-mTOR. In conclusion, findings of the current study illustrate that circEEF2 promoted autophagy, proliferation, and invasion of EOC by interacting with miR-6881-3p and ANXA2.

CircPOMT1 and circMCM3AP inhibit osteogenic differentiation of human adipose-derived stem cells by targeting miR-6881-3p.

Circular RNAs (circRNAs), novel endogenous non-coding RNAs with the special circular structure, have been found to play critical roles in various development of tissues and diseases. However, few studies have focused on the functions and mechanisms of circRNAs in the osteogenesis of human adipose-derived stem cells (hASCs). Here, we performed the circRNAs sequencing and bioinformatic analysis to investigate the expression profiles of hASCs during osteogenic differentiation. There were 150 upregulated circRNAs and 60 downregulated circRNAs expressed differentially. Among them, the expression of circPOMT1 and circMCM3AP were downregulated during the osteogenesis of hASCs. hsa-miR-6881-3p could promote the osteogenic differentiation of hASCs, while the expression of circPOMT1 and circMCM3AP were negatively correlated with it. Smad6 and Chordin, critical inhibitors of the BMPs signaling pathway, were predicted to be the targets of hsa-miR-6881-3p. Therefore, circPOMT1 and circMCM3AP might influence the osteogenic differentiation of hASCs by targeting hsa-miR-6881-3p via BMPs signaling pathway. CircPOMT1 and circMCM3AP are potential novel targets for the repairment of bone defects.