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FTVI promotes osteogenic differentiation of bone marrow mesenchymal stem cells through LncRNA HIF1A-AS2

Journal of Orthopaedic Surgery and Research volume 20, Article number: 548 (2025) Cite this article

Abstract

Objectives

The treatment of bone defects caused by trauma and pathological factors was a common problem in clinic. Extracorporeal fucosylation has been proved to promote osteogenic differentiation. Nevertheless, the biological processes by which they promote osteogenesis are currently poorly understood. Long non-coding RNAs (lncRNAs) were essential for controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This study aimed to investigate whether LncRNA HIF1A-AS2 could mediate the effects of alpha-(1,3)-fucosyltransferase VI (FTVI) on osteogenic differentiation of BMSCs.

Methods

Rat BMSCs with lncRNA HIF1A-AS2 interference plasmid or the FTVI overexpression plasmid were co-cultured in osteogenic differentiation medium. The effects of fucosylation modification of FTVI on osteogenic differentiation of BMSCs were examined, with a focus on LncRNA HIF1A-AS2.

Results

FTVI could upregulate HIF1A-AS2, and inhibition of lncRNA HIF1A-AS2 in FTVI- transfected BMSCs could decrease type I collagen (Col I), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor 165 (VEGF165) proteins expressions which were increased by FTVI. Furthermore, it was discovered that inhibiting lncRNA HIF1A-AS2 decreased the the homing ability of BMSCs demonstrating by antigen contents of HECA452 and CD15s.

Conclusions

According to these results, the effects of FTVI on osteogenic differentiation of BMSCs depend on the existence of lncRNA HIF1A-AS2. A better understanding of the pathophysiological mechanism of bone defect helped to provide theoretical basis for the reconstruction of bone damage using stem cells in clinic.

Highlights

• Overexpression of lncRNA HIF1A-AS2 can promote cell proliferation.

• FTVI could enhance the homing ability and osteogenic differentiation of BMSCs.

• The effects of FTVI on cell homing and osteogenic differentiation depend on LncRNA HIF1A-AS2.

Graphical Abstract

Introduction

Bone defects are most commonly caused by accidental bone trauma, bone tumors, bone diseases, and bone infections, which can lead to a diminished quality of life. Therefore, despite the development of techniques such as autogenous bone grafting and allogenic bone grafting, the clinical treatment of bone defects was challenging [1]. Recently, there has been increased interest in the use of tissue engineering for the treatment of bone defects [2]. Targeted treatment of refractory bone defects through tissue engineering represents a new hope in orthopedics, yet it requires further exploration and optimization.

Mesenchymal stem cells (MSCs) have been demonstrated significant potential for tissue engineering applications [3]. It has been reported that mouse bone mesenchymal stem cells (mBMSCs) were transfected with lentivirus to generate Foxq1-overexpressing BMSCs, which was shown to be essential for the osteogenic differentiation-promoting effect of FOXQ1 in the BMSCs [4]. Moreover, through FTVI transfection, BMSCs were fucosylated on N-glycans of CD44 to become HCELL positive. Engineering the glycan of CD44 on BMSCs through FTVI transfection might enhance the homing and regenerative ability of BMSCs [5].

Fucosyltransferases (FUTs) are a family of enzymes that catalyze the transfer of fucose from GDP-fucose to glycoconjugates. It has been found that the FTXI (a member of fucosyltransferases) was a direct target gene of HIF1α by bioinformatics analysis. HIF1α binds to the promoter of FTXI, thereby increasing its transcription and co-expression with FTXI [6, 7]. Fucosyltransferase VI (FTVI), another member of fucosyltransferases family, adds a fucose in an α1,3 configuration to N-acetylglucosamine, generating sialyl Lewis X (sLex) epitopes on proteins of live cells. This process enhances their ability to bind E-selectin, thus enhancing the homing ability [8].

Long non-coding RNAs (lncRNAs) were the non-coding RNAs with a minimum length of 200 nucleotides in size, and exhibited diverse roles and functions in many important biological processes [9]. Recently, with more research focusing on the lncRNAs, it became evident that lncRNAs were becoming important regulators in gene expression networks, including post- transcriptional and post-translational regulation of protein, protein complex organization, signal transduction and recombination at multiple levels, thus affecting the occurrence and development of diseases [10]. At present, it has been confirmed that certain lncRNAs regulated the osteogenesis of stem cells and played essential roles in osteogenic differentiation and bone regeneration [11, 12]. Recent studies have found that overexpression of the lncRNA HIF1A-AS2 leads to increases HIF1-α accumulation, which triggers activation of angiogenesis and neovascularization [13].

However, the role of lncRNA HIF1A-AS2 in regulating FTVI-mediated osteogenic differentiation and angiogenesis in BMSCs has not been reported. Thus, in this study, the FTVI-pSV2-neo plasmid was obtained and transfected into BMSCs. Subsequently, the effects of lncRNA HIF1A-AS2 on FTVI-mediated fucosylation and osteogenic differentiation in BMSCs were assessed using an lncRNA HIF1A-AS2 interfering plasmid, aiming to provide a theoretical basis for the clinical application of BMSCs in bone defect.

Materials & methods

Cell transfection and grouping

Rat bone marrow mesenchymal stem cells (BMSCs) were provided from the Shanghai Cell Bank of Chinese Academy of Sciences. The constructions of the interference plasmid and the overexpression plasmid were synthesized by Hualianke Biotechnology Co., Ltd (Wuhan, China). Before transfection, BMSC cells were randomly assigned into a control group (without transfection), a lncRNA HIF1A-AS2-NC group (transfected with lncRNA HIF1A-AS2-NC plasmid), a lncRNA HIF1A-AS2 overexpression group (transfected with lncRNA HIF1A-AS2 overexpression plasmid), and lncRNA HIF1A-AS2 interference groups (transfected with HIF1A-AS2-shRNA1 plasmid, HIF1A-AS2-shRNA2 plasmid or HIF1A-AS2-shRNA3 plasmid). BMSCs were cultured in α-MEM medium at 37℃ and 5% CO2 in an incubator. When the cells reached 80–90% confluence, they were digested with 0.25% trypsin and subcultured at a ratio of 1:2.

We used liposome transfection to transiently transfect overexpressing or interfering plasmids into rat bone marrow mesenchymal stem cells (BMSCs). After plasmids were stably transfected into BMSC cells, the transfection efficiency was detected by RT-PCR.

Then the experiment cells was divided into another six groups: Control group, osteogenic group, lncRNA HIF1A-AS2 NC1 group, lncRNA HIF1A-AS2 overexpression group, lncRNA HIF1A-AS2 NC2 group, and lncRNA HIF1A-AS2 interference group. The culture solution in control group was brought to 2 mL with normal medium, while other five groups were brought to 2mL with osteoblast differentiation medium. Then the six groups were incubated in the incubator for 7 d. Cell viability was detected by cell counting kit-8 (CCK-8).

After the constructions of the PSV2-neo-FTVI plasmid and the lncRNA HIF1A-AS2 interference plasmid synthesized by Hualianke Biotechnology Co., Ltd (Wuhan, China), the overexpressing or interfering plasmid was transiently transfected into BMSCs by lipofectamine transfection. Then BMSCs cells were divided into the following 4 groups: control group (untreated), NC Group (transfected with empty plasmid), FTVI group (transfected with PSV2-neo-FTVI), and FTVI + HIF1A-AS2 group (transfected with PSV2-neo-FTVI first, then transfected with lncRNA HIF1A-AS2 interfering plasmid).

All BMSCs were cultured in a culture chamber at 37℃ with 5% CO2. When the cell fusion degree reaches 90%, they were digested with 0.25% trypsin. The digested BMSCs were inoculated into six-well plates coated with 1 x polylysine, and 2 mL complete medium was added to each well. Then the cells were placed in the culture chamber again. Once the cell fusion degree reached 65%, the complete medium in the hole was carefully removed, and 2 mL complete osteogenic induction differentiation medium for BMSCs were added to the six-well plate for 7 d.

Alkaline phosphatase and Alizarin red staining

The cells were stained with alkaline phosphatase (ALP) and alizarin red, according to the instructions of staining kit. The cells were observed under inverted fluorescence microscope and photographed.

Real-time fluorescence quantitative PCR (RT-PCR)

The total RNA was extracted using Trizol method, and the first strand cDNA was obtained by reverse transcription, 40 cycles. GAPDH was used as the internal reference gene. The primer sequences were shown in Table 1.

Table 1 The primer sequences of RT-PCR
Full size table

Western blot

The antibodies involved in the experiment were BMP2 Polyclonal Antibody, VEGF Polyclonal Antibody, FTVI Polyclonal Antibody, GAPDH Polyclonal Antibody and Goat Anti-Rabbit IgG. These antibodies were all obtained from Bioswamp (Osaka Fu, Japan).

The protein concentration was determined after full lysis of the treated cells. The gel was concentrated at 80v for 40 min and separated at 120v for 50 min, then the membrane was wet transferred. After that, the 5% skim milk powder was blocked overnight at room temperature. The first antibody (1:1000 dilution) was incubated for 1 h. Excess antibody was then removed by washing the membranes in PBS for three times, and the membranes were incubated in secondary antibodies (1:10000 dilution) for 1 h. After being washed in PBS again, ECL luminescent solution was added, and the bands were detected in an automatic chemiluminescence analyzer. Then the density of the individual bands was read by Tanon GIS software.

Flow cytometry

After cells were resuspended with 100 µl PBS, 2 µl HECA452 antibody was added to cells and incubated for 30 min at 4℃ without light. The supernatant was washed once with 2 ml PBS, and centrifuged at 400 g for 5 min at 4℃. Cells were resuspended with 400 ml PBS and analyzed by NovoCyte software.

Cells were washed once with 2 ml PBS, and centrifuged at 400 g for 5 min at 4℃. After the supernatant was discarded, cells were fixed with 1 ml 4% paraformaldehyde for 30 min at room temperature. 200 µl CD15s antibody (diluted 1:500) was added to cells and incubated for 1 h at room temperature. After being washed twice with PBS, 200 µl secondary antibody (diluted 1:200) was added and incubated for 1 h at 37℃. Cells were washed twice with PBS, resuspended with 400 µl PBS, and stored at 4℃ without light.

Statistical analysis

All data were presented as mean ± standard deviation (\(\bar X \pm S\)). ANOVA was used for statistical analysis to compare values among all groups, followed by the Student-Newman-Keuls Q test and Dunnett’s T3 test to measure the differences between any two groups. P values of less than 0.05 were considered statistically significant.

Results

The transfection efficiency of LncRNA HIF1A-AS2 overexpression vector and interference vector

In order to identify the transfection efficiency of lncRNA HIF1A-AS2 overexpression vector and interference vector, the expression level of HIF1A-AS2 mRNA was detected by RT-PCR. The findings indicated that there was no significant difference in the expression level of HIF1A-AS2 mRNA between the lncRNA HIF1A-AS2 NC group and the control group post-transfection (P > 0.05). However, the expression level of HIF1A-AS2 mRNA in lncRNA HIF1A-AS2 overexpression group was significantly higher than that in control group (P < 0.01). Conversely, the expression level of HIF1A-AS2 mRNA was reduced in HIF1A-AS2-shRNA1, HIF1A-AS2-shRNA2, and HIF1A-AS2-shRNA3 groups (P < 0.01), suggesting that the transfections of both the lncRNA HIF1A-AS2 overexpression vector and interference vectors were effective. The results were shown in Fig. 1A. In summary, the data indicated that the expression of HIF1A-AS2 mRNA was effectively suppressed in the lncRNA HIF1A-AS2 knockdown groups, providing a good basis for the following experiment.

Fig. 1
figure 1

The transfection efficiency of LncRNA HIF1A-AS2 vector A: The expression level of HIF1A-AS2 mRNA after transfection. Compared with Control group, **P < 0.01. There was no significant difference in HIF1A-AS2 mRNA expression level between lncRNA HIF1A-AS2 NC group and control group. Compared with control group, HIF1A-AS2 mRNA expression level in lncRNA HIF1A-AS2 overexpression group was significantly higher, and HIF1A-AS2 mRNA expression levels decreased in HIF1A-AS2-shRNA1, HIF1A-AS2-shRNA2 and HIF1A-AS2-shRNA3 groups. B: Cell viability was measured by CCK-8. Compared with Control group, ##P < 0.01; Compared with Osteogenic group, **P < 0.01. The OD value of osteogenic group was significantly higher than that of control group, and the OD value of lncRNA HIF1A-AS2 overexpression group was significantly higher than that of osteogenic group. However, the OD value of lncRNA HIF1A-AS2 interference group was significantly lower compared to osteogenic group

Full size image

As shown in Fig. 1B, cell proliferation was detected by CCK-8. CCK-8 test of cell viability was performed, and the proliferation of untreated BMSCs was elevated by osteogenic differentiation. Concurrently, overexpression of lncRNA HIF1A-AS2 enhanced proliferation activity. Conversely, interference with lncRNA HIF1A-AS2 reduced the proliferation of differentiated BMSCs. In short, overexpression of lncRNA HIF1A-AS2 could promote cell proliferation, which may promote the differentiation of BMSC cells into osteoblasts.

Identification of overexpression and interfering plasmids

To up-regulate the expression of FTVI gene, FTVI-pSV2-neo plasmid with enhanced GFP was transfected into BMSCs. After transfection of FTVI-pSV2-neo plasmid for 48 h, the figures showed that the number of GFP positive cells in FTVI overexpression group was more than that in control group (Fig. 2A). RT-PCR results also suggested that FTVI-pSV2-neo plasmid was successfully transfected into BMSCs.

Fig. 2
figure 2

Identification of overexpression and interference plasmids. A: Overexpression of FTVI; B: Interference of LncRNA HIF1A-AS2. Compared with Control group/NC group, *P < 0.05. Based on above results, in order to investigate the effects of lncRNA HIF1A-AS2 on FTVI-mediated fucosylation and osteogenic differentiation in BMSCs, FTVI-pSV2-neo plasmid and lncRNA HIF1A-AS2 interfering plasmid were obtained and transfected into BMSCs. As shown in figures, transfection with fluorescent plasmid was observed in fluorescence microscope cells. RT-PCR results showed that both FTVI overexpression and lncRNA HIF1A-AS2 interfering plasmids were successfully expressed in cells

Full size image

In addition, for the purpose of further understanding the role and mechanism of FTVI mediating homing ability and osteogenic differentiation in BMSCs, and investigating the effects of lncRNA HIF1A-AS2 on FTVI-mediated cell changes in BMSCs, lncRNA HIF1A-AS2 interfering plasmid with enhanced GFP was also transfected into BMSCs. It has been revealed that lncRNA HIF1A-AS2 interfering plasmids were successfully expressed with green fluorescent reporter gene in cells (Fig. 2B).

The effect of FTVI overexpression and LncRNA HIF1A-AS2 interference on cell viability and the expression levels of FTVI and HIF1A-AS2 mRNA

To investigate whether the down-regulation of lncRNA HIF1A-AS2 affected cell proliferation mediated by FTVI in BMSCs, CCK-8 test was performed to detect the proliferation of BMSCs (Fig. 3A). The CCK-8 assay showed that compared with control group/NC Group, the cell viability of FTVI group increased significantly. However, cell growth was dramatically inhibited in FTVI + HIF1A-AS2 group relative to FTVI group.

Fig. 3
figure 3

The effect of FTVI overexpression and LncRNA HIF1A-AS2 interference on cell viability and the expression levels of FTVI and HIF1A-AS2. Compared with Control group/NC group, *P < 0.05; Compared with FTVI group, #P < 0.05. A: Cell viability after FTVI overexpression and LncRNA HIF1A-AS2 interference, compared with control group/NC group, the cell viability of FTVI group increased significantly, while that of FTVI + HIF1A-AS2 group decreased significantly; B: The expression level of HIF1A-AS2 mRNA after FTVI overexpression and HIF1A-AS2 interference, compared with control group/NC group, HIF1A-AS2 mRNA of FTVI group increased significantly, while that of FTVI + HIF1A-AS2 group decreased significantly; C: The expression level of FTVI protein after FTVI overexpression and HIF1A-AS2 interference, compared with control group/NC group, FTVI protein expression of FTVI group increased significantly, while that of FTVI + HIF1A-AS2 group decreased significantly

Full size image

To comprehend the role of lncRNA HIF1A-AS2 in FTVI-mediated changes, lncRNA HIF1A-AS2 interfering plasmid was transfected into BMSCs, and the expression levels of HIF1A-AS2 mRNA and FTVI protein were measured by western blot and RT-PCR methods respectively (Fig. 3B and C). Compared with control group/NC Group, the expression levels of HIF1A-AS2 and FTVI protein in FTVI group were significantly increased. Furthermore, compared with FTVI group, the expression levels of HIF1A-AS2 and FTVI protein in FTVI + HIF1A-AS2 group were significantly lower, suggesting that LncRNA HIF1A-AS2 interfering plasmid may exert its succeeding effects through inhibiting FTVI expression.

The effects of FTVI overexpression and LncRNA HIF1A-AS2 interference on HECA452 and CD15s

To study the relationship between lncRNA HIF1A-AS2 and FTVI, it was necessary to simultaneously compare the effects of FTVI over-expression and inhibition of lncRNA HIF1A-AS2 on the homing ability of BMSCs. Flow cytometric analysis was performed to detect the antigen contents of HECA452 and CD15s. In Fig. 4, the results showed that HECA452 and CD15s expressed significantly higher in FTVI group than those in control group/NC group. However, the contents of HECA452 and CD15s antigen in FTVI + HIF1A-AS2 group were significantly lower than those in FTVI group.

Fig. 4
figure 4

The antigen content of HECA452 and CD15s after FTVI overexpression and LncRNA HIF1A-AS2 interference. A: HECA452; B: CD15s. Compared with Control group/NC group, *P < 0.05; Compared with FTVI group, #P < 0.05. Compared with control group/NC group, the antigen contents of HECA452 and CD15s in FTVI group were significantly increased, while that of FTVI + HIF1A-AS2 group decreased significantly

Full size image

The effects of FTVI overexpression and LncRNA HIF1A-AS2 interference on osteoblastic differentiation

To explore changes in the osteogenic differentiation potential after lncRNA HIF1A-AS2 interfering plasmid tranfection in FTVI-stimulated cells, alkaline phosphatase staining, alizarin red staining, western blot and RT-PCR methods were performed.

The alkaline phosphatase staining results, which are phenotypic markers of osteogenic differentiation, showed that in control and NC groups, there were grayish brown deposits and a small amount of dark brown deposits in the cytoplasm. After overexpression of FTVI, there were a lot of brown-black granular precipitates in the cytoplasm, and some were filled with dark black masses. In the FTVI + HIF1A-AS2 group, there were only a small amount of brown-brown precipitates in the cytoplasm (Fig. 5A).

Fig. 5
figure 5

Effects of FTVI overexpression and LncRNA HIF1A-AS2 interference on osteogenic differentiation of cells. Compared with Control group/NC group, *P < 0.05; Compared with FTVI group, #P < 0.05. A: Alkaline phosphatase & Alizarin red staining. ALP staining showed more alkaline phosphatase produced by the FTVI group than that by control group/NC group. Alizarin red staining also revealed more mineralized nodules in the FTVI group than in control group/NC group. These results suggested that FTVI over-expression increased the osteogenic differentiation potential of BMSCs, which could be inhibited after lncRNA HIF1A-AS2 interfering plasmid tranfection; B: The expression levels of osteogenic differentiation related genes, compared with control group/NC group, Col I and RUNX2 genes expressions of FTVI group increased significantly, while after lncRNA HIF1A-AS2 interference, the expression levels of Col I, OCN and RUNX2 genes were significantly decreased; C: The expression levels of BMP2 and VEGF165 protein, compared with control group/NC group, BMP2 and VEGF165 proteins expressions of FTVI group increased significantly, while that of FTVI + HIF1A-AS2 group decreased significantly

Full size image

Extracellular matrix mineralization was considered to be one of the important markers of late maturation of osteoblast differentiation. Alizarin red was used to stain the mineralized region of osteoblasts. Alizarin red staining (Fig. 5A) showed that orange-red complex increased after FTVI overexpression, but decreased after lncRNA HIF1A-AS2 interference, indicating that FTVI could promote the formation of calcium nodules in BMSCs osteoblasts and thus promote osteogenic differentiation, which could be inhibited after LncRNA HIF1A-AS2 interfering plasmid tranfection.

At the same time, the expression levels of osteoblast differentiation-associated proteins and genes were measured using western blot and RT-PCR. It has been found that bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor 165 (VEGF165) protein expressions, as well as the expression levels of type I collagen (Col I) and runt-related transcription factor 2 (RUNX2) were significantly increased after FTVI overexpression. However, after lncRNA HIF1A-AS2 interference, the expression levels of Col I, osteocalcin (OCN) and RUNX2 genes and BMP2, VEGF165 proteins expressions were significantly decreased (Fig. 5B and C). The results showed that FTVI overexpression could promote osteoblast differentiation, while lncRNA HIF1A-AS2 interference could inhibit osteoblast differentiation.

Discussion

The repair of bone defects has been a hotspot of orthopedic research. Damaged or diseased bone can be treated using autografts or a range of different bone grafting biomaterials; however limitations with such approaches have motivated increased interest in bone tissue engineering strategies [14]. Bone marrow stem cells (BMSCs) have multipotential differentiation into osteoblasts, which was considered as a promising therapeutic approach for bone defect repair [15]. The ability of BMSCs to self-renew and their therapeutic potential play important roles in regulating the healing of bone defects [16]. There was substantial evidence to support the theory that MSCs can home to tissues, and the mechanism by which MSCs home to tissues was not yet fully understood [17]. However, it was likely that the functions and effectiveness of BMSCs may still be impaired due to deficiency of homing ability. Homing was a process that relied on intracellular signaling and interaction between chemokines, chemokine receptors, adhesion molecules, and proteases, all of which promote HSC adhesion to microvessels. Nowadays, the use of viral vector delivery methods as potential carriers for modified genes has proven to be more effective in generating HCELL-positive BMSCs. In this study, to promote the aggregation and rolling of BMSCs, thereby enhancing their homing ability, FTVI-pSV2-neo plasmid was used to increase the homing ability of BMSCs through HCELL expression. Compared with the control group and NC group, the contents of HECA452 and CD15s in FTVI group were significantly increased, demonstrating that FTVI overexpression can increase HCELL expression and cell homing ability. However, the interference of lncRNA HIF1A-AS2 reversed this change.

LncRNA HIF1A-AS2, also known as HIF1A-AS2, is the endogenous antisense transcript of hypoxia-inducible factor 1α (HIF1α). Lin et al. demonstrated upregulated expression of HIF1A-AS2 in 60 osteosarcoma (OS) tissues compared with the adjacent healthy tissues. HIF1A-AS2 may serve as a promising target for bone repair [18]. The expression of LncRNA HIF1A-AS2 was significantly increased, thus promoting angiogenesis in diabetic patients [19]. Some findings support the role of lncRNA HIF1A-AS2 in activation of angiogenesis and neovascularization [13]. Furthermore, knockdown of HIF1A-AS2 suppressed the proliferation and migration of smooth muscle cells [20]. In this study, lncRNA HIF1A-AS2 overexpression increased the proliferation activity, while lncRNA HIF1A-AS2 interference declined the proliferation activity of differentiated BMSCs, suggesting that lncRNA HIF1A-AS2 can promote cell proliferation, which may promote the differentiation of BMSC cells into osteoblasts.

The fucosyltransferase (FUT) family was a group of glycosyltransferase molecules involved in the synthesis of glycoproteins and glycolipid chains on the cell surface, which focused on cell adhesion, lymphocyte homing, leukocyte transport, blood group formation and embryonic development. According to the activity of each enzyme, the fucosyltransferase family could be divided into four subfamilies: α1,2, α1,3, α1,6, and PO-fucosyltransferase, respectively [21]. FTVI (α1,3 fucosyltransferase VI) was the most abundant subfamily and played a key role in the synthesis of sialylated Louis oligosaccharide x (SLeX). Previous studies have shown that the abnormal expression of these enzymes is significantly related to development, invasion and metastasis [22]. The results of this study showed that after FTVI overexpression, the expression levels of Col I, RUNX2 as well as BMP2 and VEGF165 were increased, suggesting that FTVI could promote osteogenic differentiation.

The effects of lncRNA HIF1A-AS2 on FTVI-mediated osteogenic differentiation in BMSCs were evaluated using lncRNA HIF1A-AS2 interfering plasmid transfection. It has been found that transfection of lncRNA HIF1A-AS2 interfering plasmid significantly reduced the expression level of genes and proteins related to osteogenic differentiation. This demonstrated that interfering HIF1A-AS2 could inhibit the osteogenic differentiation of BMSCs induced by FTVI overexpression. Reports indicate that the methylation levels of sites within the HIF1A transcription factor are closely associated with the mRNA expression of α-1,3-fucosyltransferase, suggesting a potential relationship between lncRNA HIF1A-AS2 and FTVI [23]. This study also showed that interfering lncRNA HIF1A-AS2 may be involved in the inhibitory effect of osteogenic differentiation through decreasing FTVI expression.

More recently, new research has suggested that beyond lncRNAs, small non-coding RNAs (sncRNAs) also significantly contribute to regulating BMSCs osteogenic differentiation [24]. It has been reported that among sncRNAs, MicroRNAs (miRNAs) and small interfering RNA (siRNA) function by controlling gene expression [25]. MiRNA can act on BMSCs by inhibiting their differentiation, while siRNAs selectively target a gene and silence it, inhibiting the expression of the gene [26, 27]. They can be useful for identifying molecules that participate in osteogenic differentiation processes, which provides a new perspective for further study of BMSC osteogenic differentiation and for enhancing the homing and regenerative ability of BMSCs in clinical settings [28].

This study had some limitations. Though it has been demonstrated that lncRNA HIF1A-AS2 had effects on FTVI-mediated osteogenic differentiation in BMSCs, it was not clear whether the protocol was effective for humans. A large number of animal and clinical studies were still required to verify this. Moreover, the mechanism of osteogenic differentiation was complex and needed further exploration.

Conclusions

Overexpression of FTVI can promote osteogenic differentiation. The impact of FTVI on the homing ability and osteogenic differentiation of BMSCs is contingent upon the presence of lncRNA HIF1A-AS2. A deeper comprehension of the molecular mechanisms of HIF1A-AS2 and FTVI in osteogenic differentiation could provide a theoretical basis for the clinical application of BMSCs in bone tissue engineering.

Data availability

Data is provided within the manuscript or supplementary information files.

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Acknowledgements

This work was supported by the Hubei Provincial Natural Science Foundation [grant number NO.2025AFC094].

Funding

This work was supported by the Hubei Provincial Natural Science Foundation [grant number NO.2025AFC094].

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Authors and Affiliations

  1. Department of Orthopedics, Wuhan Fourth Hospital, 473 Hanzheng Street, Qiaokou District, Wuhan, Hubei, 430033, China

    Fei Xiao, Keke Cheng, Haiyuan Xing, Tianrun Lei & Junwen Wang

  2. Wuhan University, Wuhan, Hubei, People’s Republic of China

    Zidan Wang

Authors
  1. Fei Xiao
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  2. Zidan Wang
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  3. Keke Cheng
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  4. Haiyuan Xing
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  5. Tianrun Lei
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Contributions

Fei Xiao contributed to writing original draft of the manuscript or figures. Zidan Wang, Keke Cheng, Haiyuan Xing, Tianrun Lei contributed to the acquisition and analysis of data. Junwen Wang contributed to the conception and design of the study as well as reviewing the manuscript.

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Correspondence to Fei Xiao or Junwen Wang.

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Xiao, F., Wang, Z., Cheng, K. et al. FTVI promotes osteogenic differentiation of bone marrow mesenchymal stem cells through LncRNA HIF1A-AS2. J Orthop Surg Res 20, 548 (2025). https://doi.org/10.1186/s13018-025-05952-4

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  • DOI: https://doi.org/10.1186/s13018-025-05952-4

Keywords

Journal of Orthopaedic Surgery and Research

ISSN: 1749-799X