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LncRNA HOXA‐AS2 positively regulates osteogenesis of mesenchymal stem cells through inactivating NF‐κB signalling

Abstract As is previously reported, mesenchymal stem cells have potential ability to differentiate into osteocytes. However, the underlying mechanism during this biological process is poorly understood. In the present study, we identify a novel long non‐coding RNA named HOXA‐AS2 as a critical regulator during the formation of osteogenesis. Attenuation of HOXA‐AS2 can reduce the calcium deposition and repress the alkaline phosphatase activity. Moreover, the expressions of osteogenic marker genes are markedly downregulated after HOXA‐AS2 depletion. Mechanistically, we found HOXA‐AS2 can regulate the transcriptional activity of NF‐κB, a critical inhibitor of osteogenesis. More importantly, HOXA‐AS2 knockdown could result in the transcriptional repression of the osteogenic master transcription factor SP7 by a NF‐κB/HDAC2‐coordinated H3K27 deacetylation mechanism. Based on these studies, we conclude that HOXA‐AS2 may serve as a promising therapeutic target for bone tissue repair and regeneration in the near future.

Long non-coding RNAs, a subset of non-coding transcripts with more than 200 nucleotides in length, have been shown to play important roles in numerous biological processes, for example inflammation, immunity and various cancers. [22][23][24][25] With the development of high throughput RNA sequencing approach, a variety of novel lncRNAs were identified. However, due to the tissue specific and low expression properties, functional involvement of a majority of lncRNAs remains unclear, up to date. Even so, a variety of lncRNAs were well investigated and found to play critical regulatory roles during MSC differentiation, such as a recently identified lncRNA named PU.1-as, a antisense transcript of the PU.1 gene, can prevent PU.1 mRNA translation by forming a mRNA/AS lncRNA duplex, consequently promotes adipogenesis of MSCs. 26 Another well studied lncRNA is ANCR, which could negatively regulate the osteogenic differentiation, enforced overexpression of ANCR can inhibit osteogenesis through EZH2mediated transcriptional repression of RUNX2. 27 In this study, we first identified HOXA-AS2 as a positive regulator for osteogenesis of MSCs. Attenuation of HOXA-AS2 significantly elevated the activity of NF-κB signalling which was demonstrated to have an inhibitory effect on osteogenic differentiation of MSCs. Inhibition of NF-κB dramatically enhanced the calcium deposition and alkaline phosphatase (ALP) activity. More importantly, we determined the underlying mechanism by which HOXA-AS2 regulates osteogenic differentiation of MSCs and discovered HOXA-AS2 can epigenetically mediate the expression of the osteogenic master transcription factor SP7 in a NF-κB and HDAC2-coordinated manner.

| Isolation and culture of MenSCs
The menstrual blood samples were collected from healthy female donors by menstrual cup during the first few days of menses. The samples were then mixed with equal volume PBS and subjected to standard Ficoll procedures within 72 hours. After centrifugation, the karyocytes and the deciduous endometrium suspending in buffy coat were transferred into a new tube and washed twice with PBS. Finally, the cell pellets were resuspended with complete DMEM medium supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin and the medium was changed twice a week. All the menstrual blood derived used in this study was harvested with the informed consent of the donors and this study was approved by Ethics Committee of the Xinxiang Medical University.

| Isolation and culture of UCMSCs
The umbilical cord vessels were removed in 0.9% normal saline following disinfection in 75% ethanol for 1 minute. The cord was cut into cubes of about 1 cm 3 . After removal of the supernatant fraction, the precipitate was rinsed with DMEM and then centrifuged at 250 × g for 5 minutes. The tissue was digested with collagenase II at 37°C for 1 hour and further treated with 0.25% trypsin at 37°C for 0.5 hour. To neutralize the excess trypsin, FBS was then added to the mesenchymal tissue. The dissociated umbilical cord mesenchymal stem cells (UCMSCs) were resuspended with DMEM medium and counted using a haemocytometer. The live cells were then plated in a 6-well culture plate at a density of 1 × 10 6 cells per well and the medium was changed twice a week. All the UCMSCs used in this study were harvested with the informed consent of the donors and this study was approved by Ethics Committee of the Xinxiang Medical University.

| Osteogenic induction of MSCs
To induce osteogenic differentiation of MenSCs and UCMSCs, the culture medium was removed when the cells grow to 60% confluency and then replaced by the osteogenic medium (Sigma-Aldrich, Beijing, China) containing α-MEM with 10% FBS, 10 mmol/L βglycerol phosphate, 10 nmol/L dexamethasone and 50 μg/mL ascorbic acid phosphate. The medium was changed every 2 days.

| Alizarin Red S staining and ALP activity detection
For Alizarin Red staining, cells were fixed in 70% ethanol for 30 minutes and then subjected to 1% Alizarin-Red solution for 1 minute.
Images of the stained cells were scanned and captured. For ALP staining, cells were fixed with 70% ethanol for 30 minutes and then

Highlights
• HOXA-AS2 functions as a positive regulator for osteogenesis of mesenchymal stem cells.
• Attenuation of HOXA-AS2 epigenetically regulates the expression of SP7, a critical master transcription factor of osteogenic differentiation, through a NF-κB/HDAC2coordinated H3K27 deacetylation mechanism. incubated with the BCIP/NBT liquid substrate system at 37°C for 30 minutes. Images of the stained cells were scanned and captured.

| Quantitative RT-PCR
Total RNAs were isolated from cells using Trizol reagent according to

| Chromatin immunoprecipitation
Briefly, 10 7 HUVEC cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature and then quenched with glycine at the concentration of 125 mmol/L. After washing two times with PBS, the cells were lysed by a lysis buffer (50 mmol/L Tris-HCl pH7.5, 150 mmol/ L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.1%SDS, 0.5 mmol/L PMSF), subsequently sonicated with a sonicator so that the chromatin DNA was sheared into fragments from 100 to 500 bp. The sonicated lysates were then cleared and incubated with indicated antibodies for 6 hours at 4°C. Thirty microlitre protein A beads were added and incubated for 1 hour at 4°C with gentle rotation. Immunoprecipitates were washed three times with the lysis buffer and one time with a high salt buffer (50 mmol/L Tris-HCl pH 7.5, 300 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, 0.5 mmol/L PMSF). DNA was eluted in elution buffer (100 mmol/L NaHCO3, 1% SDS) and cross-links were reversed overnight. RNA and protein were digested using RNase A and Proteinase K respectively. The immunoprecipitated DNAs were determined by qRT-PCR. The SP7 primer sequences used in chromatin immunoprecipitation (ChIP) assays were F: 5′-GTTGATGGGAAGCT-CAGGTC-3′, R: 5′-AGA TAGGCAAGGGGACCCTC-3′.

| Statistical analysis
Three different MSC batches were used to do individual experiments in this study. Student's t test was used for comparisons between two groups. P < 0.05 was considered statistically significant. All data are representative of at least three independent experiments and presented as mean ± SD.

| HOXA-AS2 expression is significantly increased during osteogenic induction
It is well known that inflammation is critically associated with osteogenesis. Moreover, recent studies showed the lncRNA HOXA-AS2 plays a regulatory role in inflammation-linked cancers. 29-31 Therefore, we suggested HOXA-AS2 is involved in the regulation of osteogenic differentiation. To test this hypothesis, we monitored the dynamic change of HOXA-AS2 expression in menstrual blood-derived mesenchymal stem cells (MenSCs) and UCMSCs during osteogenic induction by qRT-PCR analysis. As a consequence, we found the expression of HOXA-AS2 is dramatically reduced in both MenSCs and UCMSCs upon osteogenic media induction ( Figure 1A,B), indicating HOXA-AS2 may act as a crucial regulator participating in the osteogenic regulation. To verify the osteogenic differentiation after specific induction, we also measured the RNA expression levels of osteogenesis-associated marker genes and we found that RUNX2, SP7 and SPP1 all displayed increased expressions after osteogenic induction ( Figure 1C,D), which is consistent with the prior findings.

| Inhibition of HOXA-AS2 induces osteogenesis
To determine the regulatory role of HOXA-AS2 in osteogenic differentiation, we performed Alizarin Red S staining in both MenSCs and UCMSCs, with or without HOXA-AS2 knockdown, to observe the effect of HOXA-AS2 on calcium deposition. The Alizarin Red S staining analysis showed that attenuation of HOXA-AS2 can markedly repress the osteogenic media-induced calcium deposition in both of the two cell lines (Figure 2A and Figure S1A). To further confirm this data, we measured the activity of ALP in control and HOXA-AS2-depleted MenSCs, respectively, and found HOXA-AS2 depletion has an inhibitory effect on ALP activity, compared to the control sample (Figure 2B). Furthermore, we examined the expressions of osteogenesis-ZHU ET AL. | 1327 associated marker genes when HOXA-AS2 was silenced. Consequently, the expressions of the osteogenic marker genes RUNX2, SP7 and SPP1 were significantly downregulated in both MenSCs and UCMSCs with HOXA-AS2 knockdown ( Figure 2C and Figure S1B).

| HOXA-AS2 regulates the transcriptional activity of NF-κB
Prior studies reported the primary inflammatory signalling NF-κB is involved in the regulation of osteogenesis. 18,32 Moreover, HOXA-AS2 was shown to play a regulatory role in inflammation-linked cancers.
Based on these investigations, we suggested that HOXA-AS2 regulates the osteogenic differentiation, at least in part, through controlling the activity of NF-κB signalling. To prove this hypothesis, we first assayed the role of NF-κB in the regulation of mesenchymal stem cell (MSC) osteogenesis. We found the NF-κB inhibitor, Bay 11-7082 markedly induced the calcium deposition and ALP activity of MenSCs ( Figure 3A,B). To confirm the inhibitory effect of NF-κB on osteogenesis, we employed lentivirus-mediated shRNA knockdown of RelA, a trans-activating subunit of NF-κB, and then determined the

| HOXA-AS2 epigenetically regulates SP7 expression by a NF-κB and HDAC2-coordinated mechanism
Given the crucial role of HOXA-AS2 in the regulation of osteogenic differentiation, we sought to determine the underlying mechanism by which HOXA-AS2 regulate osteogenesis. As shown in Figure 3, HOXA-AS2 can modulate NF-κB activity. Moreover, NF-κB can mediate the expressions of osteogenesis-associated markers. Thus, we speculate whether HOXA-AS2 could mediate expressions of the master transcription factor RUNX2 or SP7 in a NF-κB-dependent manner. To prove this hypothesis, we first performed ChIP assay, with an antibody against RelA, after osteogenic induction in MenSCs, to observe the binding ability of NF-κB at RUNX2 and SP7 promoter regions respectively. The ChIP results revealed the distribution of NF-κB at SP7 promoter was markedly abolished upon osteogenic induction ( Figure 4A). However, the RelA occupancy at RUNX2 promoter region almost had no alteration when exposed to osteogenic induction media. Furthermore, the NF-κB inhibitor Bay 11-7082-treated MenSCs showed reduced RelA distribution at SP7 promoter, compared with a PBS-treated control sample ( Figure 4B).
These data indicate NF-κB can specially bind to SP7 promoter region and contribute to its transcription regulation.
To test this notion, we first measured the binding capacity of HDAC2 at SP7 promoter, using anti-HDAC2 ChIP assay. As a consequence, HDAC2 distribution at SP7 promoter region was found to be significantly impaired after osteogenic induction ( Figure 4C). Next, we asked whether the recruitment of HDAC2 to SP7 promoter depends on NF-κB. Anti-HDAC2 ChIP was carried out in MenSCs with or without Bay 11-7082 treatment. The result showed Bay 11-7082-treated cells obviously abrogated HDAC2 occupancy at SP7 promoter, relative to PBS-treated control sample ( Figure 4D). More importantly, we conducted HOXA-AS2 knockdown in MenSCs and then examined the effect of HOXA-AS2 on NF-κB and HDAC2 binding affinity with SP7 promoter, using anti-NF-κB and anti-HDAC2 ChIP assays respectively. Interestingly, the ChIP results revealed inhibition of HOXA-AS2 dramatically augmented both the NF-κB and HDAC2 occupancies at SP7 promoter ( Figure 4E). Furthermore, we analysed the H3 lysine 27 acetylation status which can be regulated by HDAC2 in control and HOXA-AS2-depleted MenSCs by ChIP assay, with an antibody against H3K27ac. Consequently, attenuation of HOXA-AS2 displayed reduced acetylation level of histone H3 at K27 site ( Figure 4F). These observations suggest that HOXA-AS2 can regulate SP7 transcription through NF-κB and HDAC2-dependent H3K27ac regulation mechanism.

| DISCUSSION
Mesenchymal stem cells, a source of adult stem/progenitor cells, are widely investigated as a critical cell model. More importantly, F I G U R E 5 Schematic representation of a critical molecular mechanism by which HOXA-AS2 regulates osteogenesis. HOXA-AS2 plays an inhibitory effect on NF-κB activity by repressing the K310 acetylation status of RelA. Once HOXA-AS2 becomes dysfunctional, the NF-κB will be activated and recruited to the promoter of master transcription factor SP7, co-activating the deacetylase HDAC2. The HDAC2 will then contribute to the deacetylation of H3K27, consequently leading to the transcriptional repression of SP7 mesenchymal stem cells can serve as an extremely useful medical therapeutic to regenerate or repair a variety of mature tissues, especially the bone tissue, due to its multipotential differentiation capacities. However, the potential molecular mechanism underlying osteogenic differentiation remains largely unknown, which blocks the clinical treatment of tissue repair and regeneration. In this study, we characterized the lncRNA HOXA-AS2 as a critical regulator for osteogenesis of mesenchymal stem cells. Inhibition of HOXA-AS2 significantly impaired the calcium deposition and alkaline phosphatase activity. Consistently, the expressions of osteogenesis-associated markers were repressed when HOXA-AS2 was depleted. These findings emphasize the positive role of HOXA-AS2 in the development of osteogenesis and offer us a significant therapeutic target for clinical investigation of the tissue repair and regeneration.
Previous studies have shown inflammation has a significant correlation with osteogenic differentiation. [18][19][20] Nevertheless, the precise molecular basis has not been presented up to date. In the current study, we first examined the regulatory role of the primary inflammatory signalling NF-κB in osteogenesis, using gain or loss of NF-κB function experiments. Notably, we discovered inhibition of NF-κB not only can induce the calcium deposition and alkaline phosphatase activity of MenSCs but also enhanced the expressions of osteogenic marker genes, suggesting the important inhibitory effect of NF-κB on osteogenesis.
Western blot analysis showed HOXA-AS2 could suppress the transcriptional activity of NF-κB by inhibiting K310 acetylation of RelA, which identified HOXA-AS2 as a crucial regulator of NF-κB activation. In the following investigations, we characterized the osteogenic master transcription factor SP7 as a transcriptional target of NF-κB and found both NF-κB and the deacetylase HDAC2 are enriched at the SP7 promoter region ( Figure 5). Moreover, the ChIP assay suggested NF-κB was able to help recruit HDAC2 to the SP7 promoter and facilitate the deacetylation of H3K27, eventually lead to the transcriptional repression of SP7 ( Figure 5). Collectively, these findings suggest that the lncRNA HOXA-AS2 could positively regulate the osteogenesis of MSCs by epigenetically mediating the master transcription factor SP7 expression. The functional importance of HOXA-AS2 in osteogenic differentiation reveals it may become a promising therapeutic target for clinical bone tissue repair and regeneration.