Quercetin regulates ERα mediated differentiation of BMSCs through circular RNA

Highlights

• 136 circRNA expression profiles alternated in the Lv-shERα group, and 120 in the Lv-shERα + QUE group.

• QUE moderated the osteogenic and adipogenic differentiation of BMSCs aroused by ERα deficiency.

• ERα and QUE regulate the differentiation of rBMSCs through the circRNA–miR-326-5p–mRNA axis.

Abstract

Circular RNA (circRNA) participates in regulation of gene transcription, while estrogen receptor alpha (ERα) and quercetin (QUE) positively regulate bone formation, but little is known about the correlation among circRNA, ERα and QUE. In this experiment, we created an ERα-deficient rBMSC model treated with QUE and evaluated the effects of ERα or QUE on rBMSCs, then analyzed differentially-expressed circRNAs by RNA-Seq and bioinformatics. The results showed that ERα deficiency constrained osteogenic differentiation and stimulated adipocytic differentiation of rBMSCs, while QUE abrogated those effects. We identified 136 differentially-expressed circRNAs in the Lv-shERα group and 120 differentially-expressed circRNAs in the Lv-shERα + QUE group. Thirty-two circRNAs retroregulated by ERα and QUE were involved in Rap1 and Wnt signaling, and four of them together sponged miR-326-5p, the target genes of which are osteogenic and adipogenic differentiation factors. Further study showed that over-expressed miR-326-5p could stimulate osteogenic differentiation, while attenuating adipogenic differentiation of rBMSCs. Therefore, we concluded that ERα and QUE might regulate the differentiation of rBMSCs through the circRNA–miR-326-5p–mRNA axis.

Introduction

Bone mesenchymal stem cells (BMSCs) have the characteristic of multipotential differentiation. Meanwhile, the equilibrium state between osteoblasts and adipocytes is closely related to the occurrence of bone metabolic disorders such as osteoporosis (OP) (Kolf et al., 2007). These processes are influenced by a variety of factors, including age, secretion of hormones and cytokines and so on. A decrease or deficiency in estrogen secretion is the core reason for the high risk of OP in postmenopausal women (Passos-Soares et al., 2017). Estrogen ligands mainly comprise estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Interestingly, ERα has a more important regulatory function in bone metabolism than ERβ, being essential for the strain-related response in osteoblast lineage cells (Lee et al., 2003). Iravani et al. (2017) identified ERα as the main mediator of estrogenic effects on bone growth and maturation. Furthermore, ERα is more important than ERβ in fracture healing induced by vibration (Iravani et al., 2017). Therefore, ERα plays a greater role in regulating the osteogenic differentiation of rBMSCs.

Circular RNA (circRNA) belongs to the class of non-coding RNAs, and there is emerging evidence that multifarious circRNAs participate in various essential life processes, as well as being closely related to the occurrence of bone metabolic disease (Huang et al., 2019). Long et al. (2018) demonstrated that 43 circRNAs are differentially expressed in the process of osteogenic differentiation of mouse adipose-derived stromal cells. CircRNA.33186 is differentially expressed in destabilized medial meniscus-induced osteoarthritis, and is sponged by miR-127-5p to regulate the proliferation and apoptosis of chondrocytes (Zhou et al., 2019). Yu and Liu (2019) compared the expression of circRNAs and miRNAs in postmenopausal osteoporosis patients and healthy people, and they concluded that circ_0016624 sponged miR-98 to facilitate the progression of osteoporosis. Therefore, we wondered whether circRNAs play a crucial role in the process of rBMSC differentiation.

Quercetin (QUE) is a class of flavone, which is extracted from rutin. As the structure and bioactivity of QUE is similar to estrogen, it is viewed as a phytoestrogen. The antioxidant effects of QUE have been known for many years (Kawabata et al., 2015), while its anti-osteoporotic effect was identified more recently (Rayalam et al., 2011). Experiments in vivo and in vitro both revealed that QUE stimulates angiogenesis and osteogenesis, and inhibits osteoclastogenesis under different osteoporotic conditions (Orsolic et al., 2018, Zhou et al., 2017). Furthermore, QUE influences the direction of differentiation of human MSCs via Wnt/beta-catenin signaling (Casado-Diaz et al., 2016). Pang et al. (Pang et al., 2018) proved that QUE mediates the osteogenic differentiation of rBMSCs through ER signaling. Hence, we wanted to know whether QUE could regulate differentiation in ERα-deficient rBMSCs and if so, what mechanism is involved.

In this study, we constructed an ERα-deficient rBMSC model, and treated it with QUE, to explore the effects of QUE on the direction of differentiation in ERα-deficient rBMSCs. We then compared the differentially-expressed circRNAs involved in these processes, to analyze the correlation among the ERα, circRNAs, QUE and the differentiation of rBMSCs, in an effort to identify future therapeutic targets of bone metabolism, which may provide a new approach to prevent and cure bone metabolic diseases.