lncRNA TUG1 promotes proliferation and differentiation of osteoblasts by regulating the miR-545-3p/CNR2 axis

Osteoblast differentiation is an effective way to promote bone formation. Long non-coding RNA taurine upregulated 1 (TUG1) has been identified as a crucial modulator of multiple biological processes. This study was designed to investigate the function of TUG1 in the proliferation and differentiation of osteoblast precursor cells hFOB1.19. In this study, we found that TUG1 promoted hFOB1.19 cell proliferation, while TUG1 knockdown hindered cell proliferation. TUG1 and cannabinoid receptor 2 (CNR2) were upregulated, while miR-545-3p was down-regulated in hFOB1.19 cells undergoing osteoblastic differentiation. TUG1 induced osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and the expression of osteoblastic differentiation markers. TUG1 was a sponge of miR-545-3p and regulated osteoblastic differentiation by modulating miR-545-3p. Moreover, miR-545-3p directly targeted CNR2 and restored the effect of CNR2 on osteoblastic differentiation. In conclusion, TUG1 accelerated the proliferation and differentiation of osteoblasts by sponging miR-545-3p and increasing CNR2 expression, which might provide a new biomarker for bone diseases.


Introduction
Bone is an organ dynamically regulated by osteoblasts and osteoclasts, which emerge as crucial regulators in bone turnover (1). The major responsibility of osteoblasts is bone formation, while the primary function of osteoclasts is bone resorption (2). Unbalance of osteoblast and osteoclast functions causes several bone metabolic diseases, including osteoporosis (3). Osteogenic differentiation is an orderly process in which mesenchymal stem cells (MSCs) are transformed into osteoblasts (4). Accumulating evidence has shown that the induction of osteogenic differentiation is a vital therapeutic strategy for bone diseases.
Long non-coding RNAs (lncRNAs) are a type of noncoding RNAs (ncRNAs) longer than 200 nucleotides. Numerous studies have shown that lncRNAs are abnormally expressed in many diseases and participate in the occurrence and development of tumors (5). lncRNA taurine upregulated 1 (TUG1) has been documented to be aberrantly expressed in many cancers (6). For example, TUG1 contributed to cell proliferation and metastasis in melanoma by sponging microRNA-29c-3p and upregulating RGS1 (regulator of G-protein signaling 1) (7). TUG1 recruited microRNA-212-3p from FOXA1 (Forkhead box A1) to expedite osteosarcoma progression (8). Besides, TUG1 diminished cisplatin resistance in triple negative breast cancer by binding to microRNA-197 (9). In osteoblast differentiation, TUG1 silencing restrained osteoblast proliferation and differentiation via inactivating the Wnt/bcatenin pathway (10). Nevertheless, the precise mechanism of TUG1 in osteoblast differentiation remains poorly understood.
MicroRNAs (miRNAs) are highly conserved ncRNAs composed of 18-25 nucleotides. Emerging evidence has validated that miRNAs exert a crucial regulatory role in osteoblast differentiation and osteoclast-mediated bone resorption by repressing mRNA translation (11). For instance, inhibition of miR-451a facilitated osteogenic differentiation and impeded bone loss in osteoporosis by regulating Bmp6 expression (12). Down-regulation of miR-92b-5p regulated ICAM-1 (intercellular adhesion molecule 1) expression to block osteogenic differentiation stimulated by melatonin in bone marrow mesenchymal stem cells (BMSCs) (13). In addition, lncRNAs can serve as miRNA sponges to regulate gene expression (14). In the present research, bioinformatics analysis revealed that TUG1 might be a decoy for miR-545-3p. Previous research found that miR-545-3p hindered osteogenic differentiation (15). However, the relationship between TUG1 and miR-545-3p in osteogenic differentiation remains unclear.
Moreover, cannabinoid receptor 2 (CNR2) might be a target of miR-545-3p based on bioinformatics analysis. CNR2 is implicated in cancer, bone metabolism, and pain perception (16). CNR2 is more implicated in the physiological modulation of the skeleton compared to other pathological processes of the central nervous system (17,18). In renal cell carcinoma, CNR2 knockdown curbed tumor progression (19). In osteoporosis, upregulation of CNR2 accelerated the osteogenic differentiation of BMSCs (20).
In this research, we verified the role of TUG1 in hFOB1.19 cell proliferation and differentiation. More importantly, we explored the potential mechanisms of TUG1 in osteogenic differentiation.

Alkaline phosphatase (ALP) activity determination
ALP activity, a hallmark enzyme of mature osteoblasts, was examined using the ALP activity colorimetric assay kit (BioViSion, USA). First, the cells were washed twice with PBS and then extracted with RIPA buffer (Solarbio). The optical density at 405 nm was measured using Varioskant LUX multimode microplate reader (Thermo Fisher Scientific).

RNA immunoprecipitation (RIP) assay
RIP assay was performed using Magna RNA immunoprecipitation kit (Millipore). In brief, hFOB1.19 cells were transfected with miR-545-3p or NC. After collecting cell lysates, they were incubated with magnetic beads containing anti-Ago2 or anti-IgG. Finally, the enrichment of TUG1 and miR-545-3p was measured by qRT-PCR.

Statistical analysis
Graphpad Prism 7.0 software (USA) was utilized to analyze the data. Data are reported as means ± SD with three independent experiments. The difference was analyzed by Student's t-test and one-way analysis of variance. Po0.05 was considered statistically significant.

cell proliferation
To investigate the effect of TUG1 on cell proliferation, hFOB1.19 cells were transfected with TUG1 or si-TUG1. First, transfection efficiency was evaluated using qRT-PCR. TUG1 expression in the TUG1 group was significantly higher than that in the vector group, and TUG1 expression in the si-TUG1 group was significantly lower than that in the si-NC group ( Figure 1A). In addition, CCK-8 analysis showed that overexpression of TUG1 significantly expedited the viability of hFOB1.19 cells, while down-regulation of TUG1 suppressed hFOB1.19 cell viability ( Figure 1B). Moreover, western blot assay revealed that PCNA expression was elevated after transfection with TUG1, whereas PCNA expression was decreased after the introduction of si-TUG1 ( Figure 1C). These data indicated that TUG1 promoted hFOB1.19 cell proliferation.
TUG1 and CNR2 were upregulated, while miR-545-3p was down-regulated in osteogenic differentiated hFOB1.19 cells TUG1 expression was significantly increased in hFOB1.19 cells cultured with OM in a time-dependent manner ( Figure 2A). qRT-PCR suggested a significant reduction in miR-545-3p expression at 7, 14, and 21 days in hFOB1.19 cells stimulated with OM ( Figure 2B). Furthermore, western blot assay indicated a marked increase in CNR2 expression at 4, 7, 14, and 21 days in hFOB1. 19 cells cultured with OM ( Figure 2C). These data suggested that TUG1 might play a vital role in osteogenic differentiation.

TUG1 facilitated hFOB1.19 cell differentiation
ALP activity was increased in hFOB1.19-OM cells by overexpressing TUG1 relative to the vector group, whereas knockdown of TUG1 restrained the activity of ALP ( Figure 4A). Moreover, the protein levels of ALP, Runx2, OCN, and OPN were significantly increased in hFOB1.19-OM cells transfected with TUG1 compared with the vector group, while the levels were decreased in osteogenic differentiated hFOB1.19 cells introduced with si-TUG1 ( Figure 4B). These data indicated that TUG1 facilitated hFOB1.19 cell differentiation.

TUG1 directly targeted miR-545-3p
LncBase Predicted v.2 online database (http://carolina. imis.athena-innovation.gr/diana_tools/web/index.php?r= lncbasev2/index-predicted) predicted that TUG1 contained the complementary binding sites of miR-545-3p ( Figure 5A). Dual-luciferase reporter assay disclosed that the luciferase activity of TUG1-wt reporter was significantly decreased after transfection with miR-545-3p mimic, but the luciferase activity of TUG1-mut reporter was not affected when the binding sites were mutated ( Figure 5B). Furthermore, RIP assay was performed to verify whether miR-545-3p was a target of TUG1. The results showed that TUG1 and miR-545-3p were enriched in Ago2 antibody complex compared with the anti-IgG group ( Figure 5C). In addition, miR-545-3p expression was detected in hFOB1.19 cells introduced with vector, TUG1, si-NC, or si-TUG1. The results showed that upregulation of TUG1 resulted in a distinct decrease in miR-545-3p expression, while down-regulation of TUG1 induced a significant increase in miR-545-3p expression ( Figure 5D). These data demonstrated that miR-545-3p was a direct target of TUG1 in hFOB1.19 cells.
TUG1 restored the effect of miR-545-3p on osteogenic differentiation To investigate the role of miR-545-3p in TUG1-mediated osteogenic differentiation, ALP activity and osteoblastic differentiation markers were detected in osteogenic differentiated hFOB1.19 cells after transfection. The results of qRT-PCR showed that co-transfection of miR-545-3p and TUG1 recovered the increase in miR-545-3p expression induced by transfection of miR-545-3p, and the reduction in miR-545-3p expression triggered by inhibition of miR-545-3p was restored after introduction of anti-miR-545-3p and si-TUG1 ( Figure 6A). Moreover, the activity of ALP was significantly restrained in miR-545-3p-treated osteogenic differentiated hFOB1.19 cells, while TUG1 upregulation abrogated this effect ( Figure 6B). Similarly, inhibition of miR-545-3p increased ALP activity, which was weakened by TUG1 knockdown (Figure 6C). In addition, overexpression of miR-545-3p resulted in a decrease in the levels of ALP, Runx2, OCN, and OPN, whereas the levels were reverted by reintroduction of TUG1 ( Figure 6D). Consistently, transfection with anti-miR-545-3p significantly increased the expression of ALP, Runx2, OCN, and OPN, while the effect was relieved after transfection with si-TUG1 ( Figure 6E). These results showed that TUG1 restored the inhibitory effect of miR-545-3p on osteogenic differentiation in hFOB1.19 cells.

Discussion
Osteoblast-induced bone formation plays a vital role in bone turnover (21). During bone remodeling, a series of markers of bone turnover are released (22). Runx2 binds to osteoblast-specific cis-acting element 2 to function as a key transcriptional regulator for osteoblast differentiation (23). Osteoblasts produce a variety of extracellular matrix proteins, including ALP, collagen type I, OCN, and OPN (24). Inducing osteogenic differentiation has become a vital therapeutic strategy for bone loss-related diseases (25).
Accumulating evidence has demonstrated that lncRNAs participate in bone remodeling to regulate metabolic bone disease, such as osteoporosis (26). For example, Yu et al. (27) reported that lncRNA PCAT1 positively regulated osteoblast differentiation in human adipose-derived stem cells (hADSCs) via binding to miR-145-5p and increasing TLR4 expression. Xiao et al. (28) discovered that MALAT1 accelerated osteogenic differentiation in human aortic valve interstitial cells (hAVICs) by modulating the miR-204/Smad4 axis. Moreover, Yu et al. (29) revealed that TUG1 facilitated osteoblast differentiation in human aortic valves via sponging miR-204-5p and increasing Runx2 expression. The findings of this study were consistent with previous studies. TUG1 promoted osteoblast proliferation and differentiation.
Investigations have corroborated that lncRNAs could function as competing endogenous RNAs (ceRNAs) to down-regulate miRNAs (14). In the current study, we verified that TUG1 was a sponge of miR-545-3p. Furthermore, miR-545-3p acted as a modulator in a variety of diseases. For example, miR-545-3p targeted MT1M to facilitate the progression of human hepatocellular carcinoma (30). Li et al. (15) reported that SP1-triggered miR-545-3p suppressed the expression of osteogenic differentiation markers and facilitated apoptosis in differentiated MC3T3-E1 cells through inactivation of the LRP5-induced Wnt/b-catenin signaling pathway, thereby inhibiting osteoblast differentiation. Similar to previous research, miR-545-3p expression was decreased in a time-dependent manner in hFOB1.19 cells undergoing osteogenic differentiation. In addition, TUG1 inversed the inhibitory effect of miR-545-3p on osteogenic differentiation, suggesting that TUG1 regulated osteogenic differentiation via sponging miR-545-3p.
Additionally, our research demonstrated that CNR2 was a target of miR-545-3p. A previous report suggested that deficiency of CNR2 expedited age-related bone loss in mice and CNR2 was related to low bone mineral density in females (17). Besides, the absence of CNR1 and CNR2 receptors suppressed osteoclasts, thereby preventing age-related bone loss (31). Furthermore, recent studies have verified that miRNAs could repress the expression of target genes by binding to their mRNAs (32). Xu et al.