lncRNA FGD5-AS1 Regulates Bone Marrow Stem Cell Proliferation and Apoptosis by Affecting miR-296-5p/STAT3 Axis in Steroid-Induced Osteonecrosis of the Femoral Head.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a common hip joint disease, which is more harmful and seriously affects the lives of patients. This study aims to clarify the regulatory mechanism of lncRNA FGD5-AS1 in ONFH.
METHODS: The expression of the protein and mRNA was detected by RT-qPCR and Western blot assay. The regulatory mechanism of lncRNA FGD5-AS1 was detected by the dual-luciferase reporter assay, CCK-8 assay, and flow cytometry assay.
RESULTS: Dex can inhibit cell proliferation and differentiation and induce apoptosis in hBMSCs in a dose-dependent manner. Overexpression of lncRNA FGD5-AS1 promoted cell proliferation and restrained apoptosis in Dex-treated hBMSCs. In addition, lncRNA FGD5-AS1 acts as a sponge for miR-296-5p. Also, miR-296-5p directly targets STAT3. More importantly, miR-296-5p and STAT3 can affect the function of lncRNA FGD5-AS1 in Dex-treated hBMSCs.
CONCLUSION: lncRNA FGD5-AS1 promotes cell proliferation and inhibits apoptosis in steroid-induced ONFH through acting as a sponge for miR-296-5p and upregulation of STAT3.

1. Introduction

Osteonecrosis of the femoral head (ONFH) is a disease of femoral head structural changes, femoral head collapse, and joint dysfunction caused by interruption or damage to the blood supply of the femoral head [1]. ONFH can be divided into two categories: traumatic and nontraumatic. Nontraumatic ONFH is a refractory disease in the field of orthopedics with a very high incidence [2]. It is caused by nontraumatic factors such as glucocorticoids or excessive alcohol consumption [3]. According to statistics from the World Health Organization, the age of patients with ONFH is mostly between 30 and 50 years old. Also, the incidence rate of ONFH is gradually increasing with age [4]. This disease is more harmful. If patients miss the best time for treatment, the affected limb may be limp. In severe cases, the affected limb may even be paralyzed [5], which will have a serious impact on the life and work of the patients.

In recent years, as the popularity of long noncoding RNA (lncRNA) continues to rise, its role in ONFH has been discovered. For example, reduced serum and local lncRNA MALAT1 expressions were linked with disease severity in patients with nontraumatic ONFH [6]. lncRNA AWPPH participated in the development of ONFH by upregulating Runx2 [7]. However, the effect of lncRNA FGD5-AS1 in ONFH remains unclear. lncRNA FGD5-AS1 has been reported to express abnormally and serves as an oncogene in human malignant tumors. lncRNA FGD5-AS1 accelerated cancer progression and increases cisplatin resistance in laryngeal squamous cell carcinoma [8]. Li et al. found that lncRNA FGD5-AS1 enhanced osteosarcoma cell proliferation and migration by targeting the miR-506-3p/RAB3D axis [9]. In addition, upregulation of lncRNA FGD5-AS1 was found to ameliorate myocardial ischemia/reperfusion injury via miR-106a-5p and miR-106b-5p [10]. However, the regulatory mechanism of lncRNA FGD5-AS1 in ONFH is still needed to be elucidated.

Many studies have demonstrated that lncRNAs exert effects in human diseases by miRNAs or genes. lncRNA KCNQ1OT1 has been reported to facilitate the progression of cervical cancer and tumor growth through modulating the miR-296-5p/HYOU1 axis [11]. Most studies showed that miR-296-5p functioned as a tumor suppressor in human cancers, such as renal cell carcinoma and breast cancer [12, 13]. More importantly, Cao et al. proposed that circ-E2F3 promoted cervical cancer progression by inhibiting miR-296-5p and increasing signal transducer and activator of transcription 3 (STAT3) expression [14]. miR-296-5p inhibited cell invasion and migration of esophageal squamous cell carcinoma by downregulating STAT3 [15]. These findings indicate that STAT3 serves as an oncogene in human cancers. In addition, STAT3 has been found to promote hepatic inflammation in alcohol-associated liver disease [16]. However, the effects of the miR-296-5p/STAT3 axis in ONFH have not been reported in previous studies.

Therefore, the biological effects of lncRNA FGD5-AS1/miR-296-5p/STAT3 on cell proliferation, apoptosis, and OPG/RANK/RANKL pathway were investigated in human bone marrow-derived mesenchymal stem cells (hBMSCs). This study may provide a novel target for the treatment of ONFH.

2. Materials and Methods

2.1. Isolation and Culture of hBMSCs

During conventional orthopedic surgery, bone marrow aspirate (10 ml) was obtained from the patient's proximal femur. Next, the aspirate was resuspended by phosphate buffered saline (PBS). The cell suspension was poured into a centrifuge tube with lymphocyte separation fluid. After centrifugation, the white layer mononuclear cells were cultured in the DMEM medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (GIBCO-BRL, Thermo Fisher Scientific, USA) and low sugar at 37°C with 5% CO2.

2.2. Cell Treatment and Transfection

Different concentrations of dexamethasone (Dex; 10−6, 10−7, and 10−8 M) were used to treat hBMSCs. Untreated hBMSCs were used as the control. FGD5-AS1 siRNA, FGD5-AS1 and STAT3 vector, or miR-296-5p mimics and inhibitor (GenePharma, Shanghai, China) were transfected into Dex-treated hBMSCs cells by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

2.3. RT-qPCR

Total RNA was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Next, total RNA was reverse transcript into cDNA using a PrimeScript RT reagent kit (TaKaRa, Dalian, China). PCR amplification was performed with a SYBR® Premix Ex Taq™ kit (Takara). U6 and GAPDH were used as internal controls. Relative expression of lncRNA FGD5-AS1, miR-296-5p, and STAT3 was analyzed by the 2−ΔΔCq method. Primer sequences were as follows: miR-296-5p: F (5′-GTA TCC AGT GCA GGG TCC GA-3′) and R (5′-CGA CGA GGG CCC CCC CT-3′); STAT3: F (5′-CTC AAC TTC AGA CCC GTC AAC A-3′) and R (5′-GCT CCA CGA TTC TCT CCT CCA-3′); lncRNA FGD5-AS1: F (5′-AAC AGT GCC TAT GTG GAC GG-3′) and R (5′-CCC ATC ACA GAG GTC CAC AC-3′); U6: F (5′-TGC GGG TGC TCG CTT CGG CAG C-3′) and R (5′-CCA GTG CAG GGT CCG AGG T-3′); and GAPDH: F (5′-CAA GAT CAT CAG CAA TGC CT-3′) and R (5′-ATG GAC TGT GGT CAT GAG T-3′).

2.4. CCK-8 Assay

The hBMSCs (2 × 104 cells/well) with different treatments were cultured in 96-well plates for 24 h. Then, the cells were incubated for 0, 24, 48, and 72 h, respectively. After that, 10 μl CCK-8 solution was added to incubate these cells for 2 h. Finally, a microplate reader (BioTek, Winooski, USA) was employed to measure the absorbance at 490 nm.

2.5. Flow Cytometry Assay

First, the cell suspension was centrifuged for 5 minutes. Then, 300 μL of 1 × binding buffer was added to suspend the cells. Next, the hBMSCs cells were incubated with 5 μL of annexin V-FITC and PI at room temperature for 30 minutes in the dark. Apoptotic rates were detected by flow cytometry.

2.6. Dual-Luciferase Reporter Assay

The 3′UTR of wild-type and mutant FGD5-AS1 or STAT3 was inserted into psiCHECK-2 vectors (Promega, Madison, WI, USA). Then, the abovementioned vectors were transfected into hBMSCs cells with miR-296-5p mimics. The cells were incubated for 24 h. A dual-luciferase reporter gene assay kit (Beyotime) was used to assess relative luciferase activity.

2.7. Western Blot Assay

Protein was extracted by using RIPA buffer (Beyotime, Shanghai, China). 10% SDS-PAGE was used to separate the protein samples. Then, the protein was transferred into PVDF membrane. After blocking with 5% nonfat milk, the protein was incubated with OPG (ab73400, 1 : 1000, Abcam), RANK (ab200369, 1 : 1000, Abcam), RANKL (ab9957, 1 : 1000, Abcam), STAT3 (ab119352, 1 : 1000, Abcam), and GAPDH (ab8245, 1 : 2000, Abcam) primary antibodies at 4°C overnight. Then, peroxidase-conjugated secondary antibody (ab7090, 1 : 2000, Abcam) was added to incubate the protein for 1 h at room temperature. The protein bands were developed by using the ECL system (Pierce, Rockford, IL, USA). The density of the bands was analyzed utilizing the Quantity One software.

2.8. Statistical Analysis

All experiments were repeated 3 times. GraphPad Prism 6.0 was used to perform statistical analysis. Data are shown as mean ± SD. The differences between groups were analyzed by Student's t-test or one-way ANOVA followed by Tukey's post hoc test. P < 0.05 indicates statistically significant.

3. Results

3.1. Dex Inhibits Proliferation and Induces Apoptosis in hBMSCs

First, the effects of Dex (0, 10−6, 10−7, and 10−8 M) on cell proliferation, apoptosis, and OPG/RANK/RANKL pathway were investigated in hBMSCs. We found that cell proliferation was inhibited by Dex. Also, the inhibitory effect of Dex on cell proliferation was increased as the concentration increases (P < 0.05, Figure 1(a)). The western blot assay and RT-qPCR showed the decreased expression of OPG and increased expression of RANK and RANKL in hBMSCs treated with different concentrations of Dex (P < 0.05, Figure 1(b)). In addition, Dex induced apoptosis of hBMSCs, and the apoptosis rate was also increased as the concentration of Dex increases (P < 0.05, Figure 1(c)). All these results indicate that Dex can inhibit proliferation, differentiation, and induce apoptosis in hBMSCs in a dose-dependent manner.

Figure 1

Dex inhibits proliferation and induces apoptosis in hBMSCs. (a) Dex inhibited cell viability in hBMSCs. (b) Dex regulated OPG, RANK, and RANKL proteins in hBMSCs. (c) Dex induced apoptosis of hBMSCs. P < 0.05 and P < 0.01.

3.2. Overexpression of lncRNA FGD5-AS1 Promotes Cell Proliferation and Restrains Apoptosis in Dex-Treated hBMSCs

Next, the expression of lncRNA FGD5-AS1 was detected in Dex-treated hBMSCs. RT-qPCR showed that Dex reduced FGD5-AS1 expression in a dose-dependent manner (P < 0.01, Figure 2(a)). Based on the results, hBMSCs treated by 10−6 M was selected to perform the functional experiment. Next, the FGD5-AS1 vector was transfected to Dex-treated hBMSCs to investigate its effects on osteoblastic differentiation, proliferation, and apoptosis. FGD5-AS1 expression was found to be increased in Dex-treated hBMSCs with its vector (P < 0.01, Figure 2(b)). The CCK-8 assay showed that Dex-induced proliferation reduction was attenuated by upregulation of FGD5-AS1 (P < 0.01, Figure 2(c)). Moreover, the increased apoptotic rate induced by Dex was also weakened by FGD5-AS1 upregulation (P < 0.01, Figure 2(d)). We also found that the Dex-induced decreased expression of OPG was recovered in Dex-treated hBMSCs with the FGD5-AS1 vector. Also, the expression of RANK and RANKL which was increased by Dex was reduced by the FGD5-AS1 vector (P < 0.01, Figure 2(e)). These results imply that the upregulation of FGD5-AS1 can block the progression of ONFH.

Figure 2

Overexpression of lncRNA FGD5-AS1 promotes cell proliferation and restrains apoptosis in Dex-treated hBMSCs. (a) The FGD5-AS1 expression was detected in different concentrations of Dex-treated hBMSCs. (b) The expression of FGD5-AS1 was detected in hBMSCs with Dex or Dex + FGD5-AS1 vector. (c, d) Cell proliferation and apoptosis were detected in the control, Dex, or Dex + FGD5-AS1 vector groups. (e) The expression of OPG, RANK, and RANKL was examined in the control, Dex, or Dex + FGD5-AS1 vector groups. P < 0.05 and P < 0.01.

3.3. lncRNA FGD5-AS1 Acts as a Sponge of miR-296-5p

Then, the downstream target of lncRNA FGD5-AS1 was searched on starBase2.0 (http://starbase.sysu.edu.cn/). We found that miR-296-5p has a binding site with lncRNA FGD5-AS1 (Figure 3(a)). In order to confirm their relationship, the dual-luciferase reporter assay was performed. The results indicated that miR-296-5p mimics reduced the Wt-FGD5-AS1 luciferase activity, but did not affect the Mut-FGD5-AS1 luciferase activity in hBMSCs (P < 0.01, Figure 3(b)). Moreover, miR-296-5p expression was reduced by the FGD5-AS1 vector and enhanced by si-FGD5-AS1 in Dex-treated hBMSCs (P < 0.01, Figure 3(c)). Similarly, miR-296-5p mimics also reduced FGD5-AS1 expression, while miR-296-5p inhibitor increased FGD5-AS1 expression in Dex-treated hBMSCs (P < 0.01, Figure 3(d)). Briefly, lncRNA FGD5-AS1 acts as a sponge of miR-296-5p in ONFH.

Figure 3

lncRNA FGD5-AS1 acts as a sponge of miR-296-5p. (a) The binding sites between FGD5-AS1 and miR-296-5p. (b) The relationship between FGD5-AS1 and miR-296-5p was verified by the dual-luciferase reporter assay. (c) miR-296-5p expression was detected in Dex-treated hBMSCs with FGD5-AS1 siRNA and vector. (d) FGD5-AS1 expression was measured in Dex-treated hBMSCs containing miR-296-5p mimics or inhibitor. P < 0.01.

3.4. STAT3 Is a Direct Target of miR-296-5p

Furthermore, the TargetScan database (http://www.targetscan.org) shows that STAT3 is a potential target of miR-296-5p (Figure 4(a)). The luciferase activity of Wt-STAT3 was significantly reduced by miR-296-5p mimics (P < 0.01, Figure 4(b)), indicating that miR-296-5p directly targets STAT3. Also, the mRNA and protein expression of STAT3 was found to be inhibited by miR-296-5p mimics and promoted by miR-296-5p inhibitor in Dex-treated hBMSCs (P < 0.01, Figure 4(c)). At the same time, upregulation of FGD5-AS1 increased STAT3 expression, whereas FGD5-AS1 downregulation decreased STAT3 expression in Dex-treated hBMSCs (P < 0.01, Figure 4(d)). These results demonstrate that STAT3 is a direct target of miR-296-5p and can be positively regulated by lncRNA FGD5-AS1 in ONFH.

Figure 4

STAT3 is a direct target of miR-296-5p. (a) The binding sites between miR-296-5p and STAT3. (b) The relationship between miR-296-5p and STAT3 was confirmed by the dual-luciferase reporter assay. (c) STAT3 expression was measured in Dex-treated hBMSCs containing miR-296-5p mimics or inhibitor. (d) STAT3 expression was assessed in Dex-treated hBMSCs with FGD5-AS1 siRNA and vector. P < 0.01.

3.5. Knockdown of FGD5-AS1 Inhibits Cell Proliferation and Promotes Apoptosis in Dex-Treated hBMSCs by Regulating miR-296-5p/STAT3 Axis

In order to explain the regulatory mechanism of the lncRNA FGD5-AS1/miR-296-5p/STAT3 axis, the STAT3 vector or miR-296-5p inhibitor was transfected into Dex-treated hBMSCs with si-FGD5-AS1. RT-qPCR showed that si-FGD5-AS1 reduced its expression in Dex-treated hBMSCs (P < 0.01, Figure 5(a)). However, the decreased expression was recovered by the STAT3 vector or miR-296-5p inhibitor (Figure 5(a)). Functionally, the knockdown of FGD5-AS1 inhibited the proliferation of Dex-treated hBMSCs (P < 0.01, Figure 5(b)). The inhibitory effect of FGD5-AS1 knockdown on cell proliferation was weakened by STAT3 upregulation and miR-296-5p downregulation (Figure 5(b)). We also found that FGD5-AS1 downregulation reduced the OPG expression and increased the RANK and RANKL expression in Dex-treated hBMSCs (P < 0.01, Figure 5(c)). However, the effect of FGD5-AS1 on the OPG/RANK/RANKL pathway was also reversed by STAT3 and miR-296-5p (Figure 5(c)). In addition, FGD5-AS1 downregulation-stimulated cell apoptosis was also eliminated by the STAT3 vector or miR-296-5p inhibitor (P < 0.01, Figure 5(d)). Taken together, knockdown of FGD5-AS1 inhibits cell proliferation and promotes apoptosis in Dex-treated hBMSCs by regulating the miR-296-5p/STAT3 axis.

Figure 5

Knockdown of FGD5-AS1 inhibits cell proliferation and promotes apoptosis in Dex-treated hBMSCs by regulating the miR-296-5p/STAT3 axis. (a) FGD5-AS1 expression was detected in Dex-treated hBMSCs with si-FGD5-AS1, si-FGD5-AS1 + miR-296 inhibitor, or si-FGD5-AS1 + STAT3 vector. (b, c) Cell proliferation and apoptosis were detected in Dex-treated hBMSCs with si-FGD5-AS1, si-FGD5-AS1 + miR-296 inhibitor, or si-FGD5-AS1+STAT3 vector. (d) OPG, RANK, and RANKL expression was measured in Dex-treated hBMSCs with si-FGD5-AS1, si-FGD5-AS1 + miR-296 inhibitor, or si-FGD5-AS1 + STAT3 vector. P < 0.05 and P < 0.01.

4. Discussion

Changes in the osteogenic differentiation ability of mesenchymal stem cells (MSCs) can cause necrosis and bone regeneration imbalance, which is a key factor in the pathogenesis of nontraumatic ONFH [17]. Osteoblasts, chondroblasts, and adipocytes derived from MSCs can secrete growth factors to promote tissue regeneration [18]. Previous studies have shown that the osteogenic differentiation of BMSCs is associated with the progression of orthopedic diseases. For example, lncRNA RP11-84C13.1 promoted the osteogenic differentiation of BMSCs and alleviated osteoporosis progression via the miR-23b-3p/RUNX2 axis [19]. Huang et al. reported that Circ_0067680 expedited the osteogenic differentiation of hBMSCs through the miR-4429/CTNNB1/Wnt/β-catenin pathway [20]. In our study, Dex reduced the FGD5-AS1 expression and inhibited the cell viability in a dose-dependent manner. Similarly, Zhou et al. found that steroid could inhibit the osteogenic differentiation of hBMSCs [21]. In addition, we also found that the FGD5-AS1 overexpression promoted cell proliferation and differentiation and restrains apoptosis in Dex-treated hBMSCs, while FGD5-AS1 downregulation had the opposite results. Yang et al. reported that FGD5-AS1 was downregulated in cartilage tissues of osteoarthritis (OA) patients. Also, FGD5-AS1 upregulation promoted the viability of C20/A4 cells but repressed apoptosis and ECM degradation [22]. All these findings reveal that overexpression of FGD5-AS1 can alleviate the progression of steroid-induced ONFH.

In mechanism, lncRNA FGD5-AS1 improved Dex-induced inhibition of proliferation and differentiation and promotion of apoptosis by regulating the miR-296-5p/STAT3 axis in hBMSCs. Han et al. found that miR-296-5p was upregulated in ONFH [23]. Here, miR-296-5p weakened the protective effect of FGD5-AS1 in steroid-induced ONFH, indicating that miR-296-5p upregulation can inhibit cell proliferation and differentiation and promote apoptosis in Dex-treated hBMSCs. On the contrary, Yu et al. proposed that miR-296 promoted osteoblast differentiation by upregulating Cbfal [24]. The reason for this difference may be that the cells we selected and the target genes of miR-296 are different. In present study, STAT3 was confirmed to be a direct target of miR-296-5p in ONFH. Previous studies have shown that miR-296-5p also directly targeted STAT3 in colorectal cancer and non-small cell lung cancer [25, 26]. STAT3 has been reported to promote proliferation and osteogenic differentiation of hBMSCs [27]. Consistent with our results, STAT3 was also found to promote cell proliferation and differentiation and restrains apoptosis in Dex-treated hBMSCs. These results indicate that lncRNA FGD5-AS1 promotes cell proliferation and inhibits apoptosis in steroid-induced ONFH by downregulating miR-296-5p and upregulating STAT3.

It has been reported that the dysregulation of the OPG/RANK/RANKL signaling pathway is a key step in the occurrence of steroid-induced ONFH [28]. miR-34a has been found to alleviate steroid-induced ONFH through regulating the OPG/RANK/RANKL signaling pathway [29]. In this study, Dex reduced the OPG expression and increased RANK and RANKL expression in hBMSCs in a dose-dependent manner. However, FGD5-AS1 upregulation reversed the effect of Dex on the OPG/RANK/RANKL signaling pathway. Fu et al. demonstrated that lncRNA NORAD promoted BMSCs differentiation and proliferation by regulating the OPG/RANK/RANKL signaling pathway in steroid-induced ONFH [30]. Here, we also found that lncRNA FGD5-AS1 promoted hBMSCs proliferation and repressed apoptosis by affecting the OPG/RANK/RANKL signaling pathway in steroid-induced ONFH. However, the conclusion has not been verified by an in vivo experiment. Thus, an animal experiment will be performed in the future.

5. Conclusion

lncRNA FGD5-AS1 expression was downregulated in Dex-treated hBMSCs. Functionally, the overexpression of lncRNA FGD5-AS1 promoted cell proliferation and restrained apoptosis in Dex-treated hBMSCs. In mechanism, lncRNA FGD5-AS1 can sponge miR-296-5p to upregulate STAT3 in hBMSCs. This study may provide a new idea for the diagnosis and treatment of ONFH.