LncRNA‐PCAT1 targeting miR‐145‐5p promotes TLR4‐associated osteogenic differentiation of adipose‐derived stem cells

Abstract This study was aimed to explore the differential expression of long noncoding RNAs (lncRNA)‐PCAT1, miR‐145‐5p and TLR4 in osteogenic differentiation via the Toll‐like receptor (TLR) signalling pathway and consequently determine the potential molecular mechanism. The mRNAs and pathways related to the osteogenic differentiation in human adipose‐derived stem cells (hADSCs) were analysed by bioinformatics. The MiRanda and TargetScan database were employed to detect the potential binding sites of miRNAs on lncRNAs and mRNAs. The differential expression of lncRNA‐PCAT1, miR‐145‐5p and TLR4 were detected by qRT‐PCR. Rrelated protein expression was analysed by Western blot. The targeted relationships between lncRNA‐PCAT1, miR‐145‐5p and TLR4 were verified by dual‐luciferase reporter assay. Alkaline phosphatase (ALP) activity and ARS staining assays were used to measure the impacts exerted by lncRNA PCAT1, miR‐145‐5p and TLR4 mRNA on osteogenic differentiation. After the induction of osteoblast differentiation, the expression of lncRNA‐PCAT1 and TLR4 increased, while the expression of miR‐145‐5p decreased. Dual‐luciferase reporter assay confirmed the targeted relationship between lncRNA‐PCAT1, miR‐145‐5p, and TLR4. LncRNA‐PCAT1 negatively regulated miR‐145‐5p and positively regulated TLR4. Knockdown of lncRNA‐PCAT1 or TLR4 decreased the expression of osteogenic differentiation‐related proteins, reduced the ALP and ARS levels and the activity of the TLR signalling pathway. MiR‐145‐5p could reverse the effects of PCAT1 and TLR4 in hADSCs osteogenic differentiation. LncRNA‐PCAT1 negatively regulated miR‐145‐5p, which promoted TLR4 expression to promote osteogenic differentiation by activating the TLR signalling pathway.


| INTRODUCTION
Human adipose-derived stem cells (hADSCs) are regenerative cells locating in adipose tissue, with multilineage differentiation potentials. 1 hADSCs can facilitate the metabolism and maintenance of tissues through adipocytes replacement and promote angiogenesis. 2,3 As an important cell type, they possess great potential to be applied to cell-based therapies. Recent studies have indicated that hADSCs can serve as an alternative cell source to birth bone marrow mesenchymal stem cells (BMSCs), the cell source for bone regeneration. 4 The proliferative abilities and osteogenic differentiation capabilities of BMSCs decreased with age and osteoporosis, whereas hADSCs showed consistent osteogenic differentiation capability despite of old age and osteoporotic conditions. 5,6 Therefore, the molecular mechanism related to the osteogenic differentiation of hADSCs has aroused more and more attentions.
Accumulated studies have demonstrated a significant role for long noncoding RNAs (lncRNAs) in regulating gene expression, including a significant influence on biological activities in the skeletal system, such as in osteoporosis and osteoarthritis. 7 For example, lncRNA TUG1 promoted the differentiation of osteoblast by sponging miR-204-5p, which led to the up-regulation of Runx2. 8 LncRNA MEG3 restrained the osteogenic differentiation of BMSCs by targeting miR-133a-3p. 9 Some studies also revealed that lncRNAs acted as epigenetic regulators in the osteo-adipogenic lineage commitment because of the reciprocal relationship between osteogenic and adipogenic differentiation. 10 LncRNA MIAT knockdown promoted the osteogenic differentiation of hADSCs and reversed the negative effects of inflammation. 11 PCAT1 is a lncRNA related to the proliferation and metastasis of osteosarcomas. 12 It acts as an oncogene with extremely high expression in osteosarcoma tissues. 13 However, its association with the osteogenic differentiation of hADSCs remains unclear.
MiR-145 is a microRNA (miRNA) that is broadly expressed in germline and mesoderm-derived tissues. 14 Several studies have indicated that it is closely related to the bone formation and differentiation. The stable expression of miR-145 inhibits osteoblastogenesis and bone regeneration because it decreases the levels of Cbfb, a Runx2 co-transcription factor. 15 Osteogenic differentiation was suppressed by miR-145 as it negatively regulates the expression of Sp7, which is a transcription factor in the coordinated network essential for osteogenesis. 16 The expression of miR-145 was also down-regulated in osteosarcoma compared with normal osteoblast cell lines. 17 MiR-145 mainly exhibited a negative effect on osteogenic differentiation.
Toll-like receptors (TLRs) are one of the largest and best studied families of pattern recognition receptors and are related to the Toll proteins. 18 A previous study showed that osteoblasts can express the mRNAs encoding TLR2 and TLR4. 19 TLR4 activation can enhance both angiogenesis and osteogenesis through the coculture system of outgrowth endothelial cells and primary osteoblasts. 20 The dysregulation of TLR4 stands a good chance of affecting osteogenic differentiation. The associations between the TLR signalling pathway and osteogenic differentiation also remain unclear and need to be further studied.
In this study, we investigated the differential expression of mRNAs, lncRNAs and pathways in osteogenesis through bioinformatics analysis. LncRNA PCAT1 and TLR4 were up-regulated, and miR-145-5p was the common target miRNA of them. PCAT1 was found to promote the osteogenic differentiation of hADSCs by sponging miR-145-5p, which indirectly up-regulated TLR4 and activated the TLR pathway. Our discoveries indicated that lncRNA PCAT1 plays a crucial role in facilitating the osteogenic differentiation of hADSCs.

| Clinical samples
The study group consisted of eight osteoporosis patients who had undergone an iliac bone graft procedure during surgery at Peking

| Microarray analysis
Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) provided the gene expression data derived from four different donors (GSE89330). To ascertain those genes that had higher chances of being differentially expressed in one group compared with another, we used the statistical test in the R Limma package. Those genes with at least an absolute value of 1 for log 2 (FC) and adj. P < 0.05 cut-off were identified. The overall profile was normalized for each array to correct for systematic data bias and remove nonbiological impact.

| Pathway analysis
Gene set enrichment analysis (GSEA) tested whether a set of predefined genes, usually united by shared association, showed enriched in expression levels. In our study, the uploaded gene set consisted of normalized expression data of mRNAs expression data and was sorted by the mean log 2 signal ratios. The aggregated distribution of gene expression levels in pathways was then determined with a normalized enrichment score, which represented the statistical significance through enrichment analysis. Pathways that were significantly biased in the undifferentiated or osteogenesis group were identified. The GSEA enrichment plot offered us with visualized results. YU ET AL. | 6135

| DEG correlation network analysis
By evaluating the correlation between gene expression patterns, we hoped to discover potential drug targets and candidate biomarkers.
The "Pearson" approach in the R package Psych was harnessed to identify linear correlations among DEGs in a specific pathway, this was adjusted by the "BH" method. Then, the networks were visualized using Cytoscape software. Nodes in the network represented DEGs while the edges indicated the presence of coexpression.

| Cell proliferation dynamics analysis
Passage 4 cells were seeded into 96-well plates at 6.25 × 10 4 , 1.25 × 10 5 , 2.5 × 10 5 , 5 × 10 5 , 1 × 10 6 and 2 × 10 6 cells/well cell concentration, respectively. Taking the cell density as the X-axis, the absorbance as the Y-axis, draw the standard curve. Cell proliferation was detected by using the Cell Counting Kit-8 kit (Beyotime, Shanghai, China). Cells were seeded into 96-well plates at 2 × 10 3 cells/ well cell concentration. After cell culture for 1-21 days, 10 μL CCK-8 solution was added to each well and incubated at 37°C for 2 hours.  experiments were repeated three times, the results are presented as fold change ± SD, and P < 0.05 was considered significantly different.

| QRT-PCR
After osteoblast differentiation was induced in cells for 14 days, the cells were dissociated with 0.25% trypsin with EDTA and washed twice with cold PBS. The cell suspension was centrifuged at 111.9 × g for 5 minutes to get the precipitation. RNA extraction was performed using the Takara Minibest universal RNA extraction kit (Takara, Katsushika, Tokyo) according to the manufacturer's protocol.
In total 500 ng of total RNA was reverse-transcribed to cDNA using the Takara PrimeScript RT master mix kit (Takara) according to the  Table   S1. The results were evaluated by the 2 −ΔΔCt method. with TBST, specific staining was detected using the chemiluminescence (ECL) system (VWR International GmbH, Darmstadt, Germany). All bands were densitometrically analysed with ImageJ.

| Alkaline phosphatase staining
The expression of alkaline phosphatase (ALP) in the cell layers was assessed using a kit according to the manufacturer's instructions (Yeasen, Shanghai, China). Fourteen days after osteogenic induction, the hADSCs were rinsed three times with PBS three times and fixed with 70% alcohol for 30 minutes. The fixed cells were soaked in BCIP/NBT solution, washed with ddH2O, and then observed with a scanner (ImageScanner III, GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Each assay condition was repeated in triplicate.
All the results were repeated in three independent experiments.

| Alizarin red S staining
Alizarin red S staining was used to detect matrix mineralization.
Fourteen days after osteogenic differentiation, the cultured cells were treated with 75% ethanol for 20 minutes, and then stained with 1% Alizarin Red S pH 4.2 (ShangHai Haoran Biological Technology, Shanghai, China) for 1 hour at 37°C for the semi-quantitative evaluation of the degree of mineralization.

| Dual-luciferase reporter assay
The PCAT1 and TRL4 sequences containing the predicted potential miR-145-5p binding sites were amplified. The PCAT1-WT and TRL4-WT plasmids were constructed using a PCR method. PCAT1-MUT and TLR4-MUT were generated by site-directed mutagenesis, replac-

| Statistical analysis
Numerical data were expressed as the mean ± SD. Student's t test rendered statistical comparisons. The criterion for significance was set to as P < 0.05. Figure 1A and B showed 20 selected mRNAs and lncRNAs, respectively, that demonstrated the most significant differences in their expression profiles during osteogenic differentiation, with 10 ranked at the top and 10 at the bottom sorted by their fold-change value.

| The profiling of dysregulated mRNAs, lncRNAs, and activated toll like receptor signalling pathway during osteogenic differentiation
As shown in Figure 1C and D, the pathways which were significantly biased were demonstrated by ridgeplot and dotplot based on the GSEA analysis results. The Gseaplot result showcased that the TLR signalling pathway was activated ( Figure 1F). Furthermore, TLR4 was highly expressed in TLR signalling pathway, which was shown in Figure 1E. All results demonstrated that TLR signalling pathway was activated in osteogenic differentiation. Therefore, these results provided us with insights that led us to focus on TLR signalling pathway in the next study.

| The putative targeted relationship between lncRNA PCAT1, miR-145-5p, and TLR4
An enrichment map was implemented to indicate those pathways containing cross-referencing genes. The TLR signalling pathway was marked with dark red, which denoted it was highly enriched. The connections showed overlapping parts, indicating that the TLR signalling pathway contained genes that cross-referenced with four other pathways (Figure 2A were visualized via Cytoscape 3.6.5. Considering all the miRNAs that targeted TLR4, PCAT1, and the TLR signalling pathway together, 13 miRNAs were identified by the Veen diagram. We choose miR-145-5p, which was included among these thirteen miR-NAs, for further study ( Figure 2C). The PCAT1, miR-145 and TLR4 binding sites as well as the putative binding relationship were illustrated in Figure 2D.

| The morphology, propagation, and characterization of passage 4hADSCs
The seeded hADSCs began to grew by static adherence in 24 hours.
The primary cells were relatively single, short shuttle-like, or round.
While after three times of passage, cell grew into spindle-shaped, fibroblast-like appearance cells with spherical or orbicular-ovate nucleus under high power lens (×100), with rapid proliferation and swirl distribution ( Figure 3A). A linear relationship between absorbance and density of live cells in growth period was displayed (Figure 3B). Furthermore, CCK-8 assay was performed to detect the YU ET AL.
| 6137 proliferation of hADSCs, and the growth curve was draw for population doublings calculation. As shown in Figure 3C (D) The expression of TLR4 protein was detected by Western-blot, and also increased after osteoblast differentiation induction, especially after 14 and 21 days (*P < 0.05 compared with 0 day) expressed and that the surface markers CD34 and CD45 were minimally expressed in hADSCs ( Figure 3D).
As shown in Figure 4A, following the induction of osteoblast differentiation, lncRNA PCAT1 expression was up-regulated compared with 0 day, exhibiting a significant increase until 14 and 21 days after the induction of osteogenic differentiation. However, miR-145-5p expression showed the opposite tendency ( Figure 4B); after induced, its expression decreased. Additionally, after induction for 14 and 21 days, the decrease was notable. The results for TLR4 mRNA were similar to lncRNA-PCAT1. Until 14 and 21 days after induced osteogenic, the expression of TLR4 was remarkable (Figure 4C). TLR4 protein expression was also detected by Western blot ( Figure 4D). TLR4 protein expression was positively correlated with mRNA expression.

| The targeted relationship between lncRNA-PCAT1, miR-145-5p, and TLR4
The luciferase reporter gene assay was conducted in HEK293T cells for verifying their binding relationship. The result revealed that the F I G U R E 6 The relationship between lncRNA PCAT1, miR-145-5p and TLR4. (A) Dual luciferase reporter gene assay results demonstrated that the luciferase activity in the miR-145-5p mimics+PCAT1-WT group was significantly weaker than in the miR-NC+PCAT1-WT group. However, there were no significant changes between miR-145-5p mimics+PCAT1-MUT group and miR-NC+ PCAT1-MUT group (*P < 0.05 compared with the miR-NC group). (B) The luciferase activity in the miR-145-5p mimics+TLR4-WT group was much lower than in the miR-NC group. There were no significant changes between the miR-145-5p mimics group and NC group after TLR4 mutation (*P < 0.05 compared with miR-NC group). (C) LncRNA PCAT1 expression was decreased after PCAT1 knockdown (*P < 0.05 compared with the mock group). (D) The expression of miR-145-5p was increased after PCAT1 knockdown (*P < 0.05 compared with mock group). (E) The expression of TLR4 was decreased after PCAT1 knockdown (*P < 0.05 compared with the mock group). (F) MiR-145-5p expression was decreased after miR-145-5p knockdown (*P < 0.05 compared with the mock group). (G) TLR4 expression was increased after miR-145-5p knockdown (*P < 0.05 compared with mock group). (H) LncRNA PCAT1 expression showed no change after miR-145-5p knockdown (P > 0.05 compared with the mock group) luciferase activity of cells cotransfected with miR-145-5p and PCAT1-WT was significantly inhibited, whereas miR-145-5p had no such impact on the PCAT1-MUT fluorescence ( Figure 6A). Similar results are shown for the relationship between miR-145-5p and TLR4 mRNA ( Figure 6B). The luciferase activity of cells cotransfected with miR-145-5p and TLR4-WT was significantly reduced, while the luciferase activity of cells cotransfected with miR-145-5p and TLR4-MUT was practically similar. These results indicated that lncRNA-PCAT1 and miR-145-5p as well as miR-145-5p and TLR4 mRNA directly regulated one another.

affected osteogenic differentiation
The transfection of si-PCAT1 and si-TLR4 into hADSCs led to a simultaneous in TLR4 mRNA expression level and TLR4 protein levels ( Figures 7A and B). PCAT1 and TLR4 knockdown remarkably inhibited ALP levels, while miR-145-5p inhibitors increased ALP levels. Mix1 and mix2 recovered ALP levels to normal. The relative ARS levels were similar to ALP levels ( Figures 7C-F), Western blot detected the expression of OCN, OPN and RUNX2, which are related to osteogenic differentiation. As shown in Figures 7G and H, compared with the noninduced group, OCN, OPN, and RUNX2 expression levels decreased significantly in the si-PCAT1 and si-TLR4 groups, but were up-regulated in the miR-145-5p inhibitor group. LncRNA PCAT1, miR-145-5p, and mRNA TLR4 affected Toll like receptor signalling pathway

| DISCUSSION
In the present study, we investigated the osteogenic differentiation of hADSCs, showing that lncRNA PCAT1 and TLR4 were up-regulated in ASC osteogenic differentiation and that the TLR signalling pathway that was activated. MiR-145-5p is the common PCAT1 and TLR4 target miRNA. LncRNA PCAT1 acted as a competing endogenous RNA (ceRNA) of miR-145-5p in ASC to promote the osteogenic differentiation by up-regulating TLR4 and activating the TLR pathway.
The differentially expressed lncRNAs, mRNAs and pathways in hADSCs osteogenic differentiation were screened by microarray analysis and using the GSEA software. The expressions of lncRNA PCAT1, TLR4 mRNA and the TLR pathway were remarkably increased in hADSCs osteogenic differentiation. PCAT1 is a lncRNA that has been extensively studied in terms of its correlation with tumour progression and prognosis. 21 It was found to show significant biased expression in various kinds of human cancers, such as prostate cancer, 22 glioblastoma, 23 and gastric cancer. 24 However, its function in the human skeletal system has not been previously emphasized. Previous studies only showed its impact on osteosarcoma, in which PCAT1 was up-regulated and promoted osteosarcoma tumourigenicity. 12,13 Here, we found that PCAT1 positively regulated the osteogenic differentiation of hADSCs, suggesting that PCAT1 might be an important target lncRNA for ASC-based therapies related to osteogenic differentiation.
TLR4 was also identified to be associated with the osteogenic differentiation of hADSCs in this study. Similar to PACT1, TLR4 promoted the osteogenic differentiation of hADSCs. Raicevic et al demonstrated a similar effect for TLR4 in adipose tissue, as osteogenesis was increased by triggering TLR3 and TLR4 in BMSCs and adipose tissues. 25 Although studies on the role of TLR4 in ASC osteogenic differentiation are limited, some evidence implies that TLR4 is involved in several biological processes in the skeletal system. TLR4 was reported to participate in the transformation of adventitial fibroblasts to myofibroblasts regulated by osteocalcin. 26 In germ-positive bacterial bone infections, TLR2 and TLR4 were related to the increased production of human beta-defensin-3 in osteoblasts. 27 TLR4 expression was up-regulated by the overexpression of HSP60, one of the heat shock proteins that played a critical role in promoting bone loss, and silencing of TLR4 silencing almost eliminated the HSP60 mediated promotion of BMSC apoptosis. 28 Our findings revealed that TLR4 greatly facilitated the osteogenic differentiation of hADSCs, providing a novel insight into TLR4's biological function. Meanwhile, the TLR pathway was also activated, which was consistent with the up-regulation of TLR4.
A connection between lncRNA PCAT1 and TLR4 was established by miR-145-5p, which was proven to be able to regulate the osteoblastic differentiation. MiR-145-5p expression was decreased during osteogenic differentiation, and transfection of miR-145 mimics in pluripotent mesenchymal precursor cells reduced the expression of osteogenic differentiation markers. 16 The undifferentiated interaction networks of mesenchymal stem cells exhibited an increased miR-145-3p identification. 29 In human osteoblast-like MG-63 cells, suppression of miR-145 could stimulate the expression of osteoprotegerin, a soluble glycoprotein that reduced bone resorption and enhances bone formation. 30 MiR-145 also negatively affected the osteoblastic differentiation by targeting Cbfb, the transcription factors that are essential for bone formation. 15 These previous studies revealed the suppressive role of miR-145 in osteogenic differentiation, which was in line with our study as we found miR-145-5p suppressed the osteogenic differentiation of hADSCs.
Furthermore, miR-145-5p was the common target miRNA for lncRNA PCAT1 and TLR4, suggesting that there is a PCAT1/miR-145-5p/TLR4 axis in the regulation of ASC osteogenic differentiation.
LncRNA PCAT1 served as a ceRNA to sponge miR-145-5p and up-regulate TLR4. Similar regulatory mechanisms have been F I G U R E 8 Western blot analysis showing the effect of PCAT1, miR-145-5p and TLR4 on Toll-like receptor signalling pathway. (A) Western blot analysis of phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated JNK (p-JNK), and JNK protein levels in different groups after osteogenic differentiation for 14 days (noninduced, si-PCAT1, miR-145-5p inhibitor, si-TLR4, mix1, and mix2). (B) The expression levels of the Toll-like receptor signalling pathway downstream proteins, p-ERK1/2/ERK1/2 and p-JNK/JNK value were down-regulated by PCAT1 or TLR4 knockdown in hADSCs and up-regulated by miR-145-5p knockdown. *P < 0.05, compared with NC group F I G U R E 9 A proposed schematic diagram of LncRNA PCAT1/ miR-145-5p/TLR4 mRNA axis during the osteogenic differentiation process. (A) We speculated that LncRNA-PCAT1 sponged miR-145-5p to promote TLRA associated osteogenic differentiation of hADSCs via the activation of Toll-like receptor signalling pathway F I G U R E 7 ALP and ARS staining assay results because of the impact of lncRNA PCAT1, miR-145-5p and TLR4 on osteogenic differentiation. (A and B) TLR4 mRNA and protein expression were increased after miR-145-5p knockdown but reduced in the si-PCAT1 group and si-TLR4 group. Mix1 (si-PCAT1+ miR-145-5p inhibitor) and mix2 (miR-145-5p inhibitor+si-TLR4) showed no significant change (*P < 0.05 compared with NC group). (C-E) ALP staining results demonstrated that knockdown of miR-145-5p significantly strengthened osteogenic differentiation while the transfection of si-PCAT1 or si-TLR4 reduced osteogenic differentiation. The simultaneous transfection of si-PCAT1+ miR-145-5p inhibitor and miR-145-5p inhibitor+si-TLR4 showed hardly any significant difference from the NC group alone (*P < 0.05 compared with the NC group). (D-F) ARS staining results showed similar results. MiR-145-5p knockdown significantly strengthened osteogenic differentiation while transfection of si-PCAT1 or si-TLR4 reduced osteogenic differentiation. The simultaneous transfection of si-PCAT1+ miR-145-5p inhibitor and miR-145-5p inhibitor+si-TLR4 showed no significant differences compared with the NC group (*P < 0.05 compared with the NC group). (G and H) OCN, OPN, and RUNX2 expression was increased after miR-145-5p knockdown but was reduced in the si-PCAT1 group and si-TLR4 groups. Mix1 (si-PCAT1+ miR-145-5p inhibitor) and mix2 (miR-145-5p inhibitor+si-TLR4) showed no significant changes (*P < 0.05 compared with NC group) identified for other lncRNAs in osteogenic differentiation. For example, lncRNA MALA1 promoted the osteogenic differentiation of aortic valve interstitial cells by sponging miR-204, and miR-204's target gene, Smad4, was up-regulated. 31 LncRNA H19 promoted BMSC osteoblast differentiation by targeting miR-675, and the down-regulation of miR-675 subsequently elevated TGF-β1 mRNA and protein expressions. 32 LncRNA H19 also functioned as ceRNA of miR-141 and miR-22, both of which were negative regulators of osteogenesis. 33 Through bioinformatics analysis, Gu et al identified 147 lncRNAs totally that were predicted to interact with miRNAs and compete for miRNA binding sites with mRNAs in osteogenic differentiation of periodontal ligament stem cells, revealing the potential lncRNA ceRNA networks. 34 In hADSCs osteogenic differentiation, the differentially expressed lncRNAs were also identified, and lncRNA H19 was found to significantly influence the expression of osteogenesis-related genes ALPL and Runx2. 4 Our results further confirmed that lncRNAs have a great impact on osteogenic differentiation as their sponging effects on miRNAs could mediate the expressions of osteogenesis-related mRNAs.
Although the functions of lncRNA PCAT1 in ASC osteogenic differentiation have been clearly investigated in this study, GSEA also identified other differentially expressed lncRNAs. The effects of these lncRNAs on osteogenic differentiation and their interactions with miRNAs and mRNAs remained unclear and require further studies in the future. Additionally, this study lacks of in vivo experiments, which ought to be supplemented to confirm the conclusions.
In summary, lncRNA PCAT1 and TLR4 were up-regulated while miR-145-5p was down-regulated in ASC osteogenic differentiation.