# LncRNA BC083743 Silencing Exacerbated Osteoporosis by Regulating the miR-103-3p/SATB2 Axis to Inhibit Osteogenic Differentiation

Authors: Feng Lu; Ling Tang
Journal: Computational Intelligence and Neuroscience
 (2022)

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## Abstract

Objective. The target of the present paper was to reveal the influence of LncRNA BC083743 on osteogenesis in human bone marrow mesenchymal stem cells (hBMSCs). Methods. Serum specimens from osteoporotic patients and normal subjects were collected to isolate hBMSCs from femoral head tissue. The levels of BC083743 and miR-103-3p in serum and hBMSCs were measured by QRT-PCR. Alkaline phosphatase (ALP) activity test and alizarin red dyeing were used to identify ALP activity and mineralization forming ability of hBMSCs after transfection with si-BC083743 (siRNA-targeting BC083743). In addition, QRT-PCR and immunoblotting were conducted to identify the expressing levels of Runt-related transcription factor 2(Runx2), osteoprotegerin (OPG), and bone morphogenetic protein 2 (BMP2) in hBMSCs. Dual-luciferase reporter gene and RNA pull-down assays were employed to substantiate the binding of BC083743 to miR-103-3p and miR-103-3p to SATB2. Results. BC083743 expression was significantly downregulated in sera from patients with osteoporosis, and osteogenic differentiation-related genes and BC083743 expression were obviously upregulated as the time to osteogenic differentiation increased. BC083743 knockdown hindered the osteogenic differentiation of hBMSCs. BC083743 was aimed at miR-103-3p and miR-103-3p inhibitors partially reversed the inhibitory effect of BC083743 downregulation on hBMSCs osteogenesis. BC083743 silencing downregulated SATB2 through uptake of miR-103-3p, thereby inhibiting hBMSCs osteogenesis to exacerbate osteoporosis. Conclusion. BC083743/miR-103-3p/SATB2 axis inhibited osteogenic differentiation and exacerbated osteoporosis, which may offer brand-new molecular aims for the treatment of clinical osteoporosis.


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## Body

## 1. Introduction

Osteoporosis (OP) is a degenerative bone disease that occurs mostly in middle-aged and elderly people. With the ageing of the population in China, its incidence is increasing, and it has become a hot spot for treatment research [1]. It is estimated that the number of people with OP is over 75 million worldwide, with over 1.5 million OP-related fractures each year and hip fractures expected to reach 6.3 million by 2050 [2]. Increased bone loss is thought to be the main cause of the development of osteoporosis [3]. There are many preventive treatments for OP, including preventive measures such as a balanced diet and moderate exercise, as well as medication [4, 5]. Medication can stop and treat OP and is of clinical importance, mainly including drugs that excite bone forming and prevent bone absorption. Although these drugs target systemic osteoporosis, they have significant side effects [6]. At present, the clinical usefulness, safety, and resistance of OP drug treatment still need to be investigated, and the machinery for osteoporosis is not yet entirely expounded. The progress of this objective of the molecular biology of osteoporosis has opened up new ideas for the treatment of OP and has important clinical guidance significance.lncRNAs are structurally diverse and functionally complex and make a momentous part in biological development including genomic imprinting, cell cycle adjustion, cell multipotency, dosage compensation effects, reverse transcription translocon reticence, and chromosome telomere extending [7]. lncRNAs regulate a variety of human physiological and pathological processes by mediating chromatin modifications, regulating transcriptional processes, and mediating posttranscriptional control [8, 9]. This in turn affects the course of osteosarcoma [10], osteoarthritis [11], degenerative disc disease [12], and other bone and joint diseases. Currently, the regulatory role of lncRNAs in OP is also receiving increasing clinical attention [13]. lncRNA BC083743 as a novel lncRNA, Gao et al. demonstrated that BC083743 encourages cell multiplication and axonal reprocess after sciatic nerve crush by miR-103-3p/BDNF axis [14]. It is found that lncRNAs can be directly involved in the osteogenic differentiation of BMSCs and also regulate the osteogenic differentiation process of BMSCs by regulating miRNA expression. Previous studies have confirmed that miR-103-3p prevents bone growth and promotes osteoporosis in vivo [15]. Likewise, miR-103-3p is aimed at SATB2 to inhibit osteogenic differentiation [16]. However, the role of BC083743 in regulating miR-103-3p in OP is unclear.Bone marrow mesenchymal stem cells (BMSCs) originate from mesodermal cells with strong proliferative capacity and multiple differentiation profiles [17]. They can break up into osteoblasts, myoblasts, chondrocytes, stromal cells, and neuronal cells under different induction conditions [18] and have functions such as self-renewal, osteogenic differentiation, and immune regulation. Bone regeneration is a complex and highly regulated process, with two different approaches: intramembranous ossification and endochondral ossification. During intramembranous ossification, osteoblasts derived from the osteogenic differentiation of BMSCs form bone, so bone regeneration can be promoted by regulating the osteo-differentiation of BMSCs [19]. The osteogenic differentiation of BMSCs holds prodigious potentiality in bone tissue engineering for application. Differences in the expression of lncRNAs in osteoblasts and osteoclasts of BMSCs in osteoporosis have been found [20]. Investigating the molecular mechanisms regulating osteogenic differentiation of BMSCs can do a significant part in the therapeutics of OP. Given the above research base, this study explored the serum BC083743 levels in OP patients and, subsequently, assessed whether BC083743 knockdown could make a role in the osteogenic differentiation of hBMSCs. We found that BC083743 silencing downregulated SATB2 through uptake of miR-103-3p, thereby inhibiting osteogenesis of hBMSCs to exacerbate OP. This offers a therapeutic direction and experimental rationale for BC083743 as a potential target for the treatment of osteoporosis.


## 2. Materials and Methods

### 2.1. Serum Samples

Serum samples were collected from OP patients and normal individuals. This study excluded some patients getting cancer, rheumatoid arthritis, and other metabolic diseases. All serum samples were frozen in liquid nitrogen and retained in a fridge at −80°C. The research was permitted by the local medical ethics committee.


### 2.2. hBMSCs Culture

hBMSCs were purchased from ScienCell (USA). hBMSCs were cultivated in DMEM medium (Hyclone, USA) involving 10% FBS, 100 IU-mL−1 penicillin, and 100 mg-L−1 phytomycin (Solebro Technology, Beijing, China) at 37°C and 5% CO2.


### 2.3. Induction of Osteogenic Differentiation of hBMSCs

When hBMSCs grew to 80%–90% density, the medium was displaced by an osteoblast-specific induction medium (containing 10% FBS, 10 mmol-L−1 sodium β-glycerophosphate, 0.1 μmol-L−1 dexamethasone, and 0.2 mmol-L−1 ascorbic acid phosphate in low sugar DMEM), and the medium was changed every 2 d and incubated for 0, 1, 3, 7, and 14 d under 37°C and 5% CO2.


### 2.4. Cell Transfection

After the process of osteogenic induction, hBMSCs cells were inoculated in 6-well cell culture dishes with 4 × 105 cells/well, and when cell density reached 60%–70%, transfection of miR-103-3p and miR-NC was performed according to Lipofectamine 3000 instructions (Invitrogen, USA), both at a final concentration of 60 nmol-L. Cells were cultivated under 37°C and 5% CO2 for 48 h. For transfection of hBMSCs with lentivirus, the cell culture medium was replaced with lentiviral-pEF-1a/Puro-si-SATB2 (si-SATB2) and lentiviralpGLVU6/Puro-si-NC (the vectors were purchased from GenePharma (China)).


### 2.5. QRT-PCR

The transfected hBMSCs were collected, all RNA was gotten from the samples by the TRIzol method, RNA concentration was determined spectrophotometrically, and cDNA was synthesized by reverse transcription. QRT-PCR was performed according to SYBRPremix ExTaqTM II kit instructions (TaKaTa, Japan). Reaction conditions: 95°C, 0.5 min; 95°C, 5 s, 58°C, 0.5 min, 40 cycles; 95°C, 15 s, 58°C, 0.5 min, 95°C, 15 s. Using U6 and GAPDH as an internal reference, the 2-ΔΔCt way was utilized to get miR-103-3p expression levels and LncRNAs BC083743, SATB2, Runx2, OPG, and BMP2 mRNA expression levels. All primer sequences were synthesized by Guangzhou Ribo Bio (China). All primers were synthesized by Guangzhou Ruibo Co., Ltd.


### 2.6. Western Blot

The transfected hBMSCs cells were gotten, and the protein level was identified by the BCA approach. SDS-PAGE gel electrophoresis separated 30μg of protein, which was transferred to the PVDF membrane. 10% skimmed milk powder was closed at the temperature of room for 3 hours. Runx2 (1:1000), OPG (1:1000), BMP2 (1:1000), and GAPDH (1:2000) were put and cultivated under 4°C all the night. HRP-labelled secondary antibodies (1:2500) were cultivated at the temperature of room for 1 h. The band was analysed by Image J, and the gray level of the targeted protein was expressed as the proportion of the gray value of the targeted protein band to the gray value of the GAPDH band. All antibodies were bought from Abcam (UK).


### 2.7. Alkaline Phosphatase (ALP) Activity Measurement

The instructions of the kit (Biyuntian, China) reported that the cells were stable in 4% PFA for 0.5 h, cleaned 3 times in PBS, and configured into BCIP/NBT staining working solution (Sigma, Germany). Since the last wash was completed, the washing solution was moved away, and a proper number of BCIP/NBT staining working solution was put in to ensure that the sample was covered and cultivated under ambient temperature in the dark and incubated for 5–30 min under ambient temperature in the dark until stained to desired shade. The staining solution was removed, and the colour development reaction was terminated by washing twice with distilled water and photographed under the microscope. The absorbance (A) values of each group of cells were measured at 405 nm on an enzyme marker for quantification of ALP levels.


### 2.8. Alizarin Red S (ARS) Staining

The hBMSCs were discarded from the osteogenic induction medium, washed in PBS buffer, and fixed in 4% paraformaldehyde. After washing the cells in PBS buffer, the cells were incubated in 0.1% ARS staining solution (Sigma, Germany) for 20 min under ambient temperature. The staining solution was removed and cleaned in distilled water. To quantify calcium salt deposition, 10% cetylpyridinium chloride containing 10 mmol-L−1 sodium phosphate was put into the cells, and the A value at 562 nm was checked for each sample using an enzyme marker.


### 2.9. Dual-Luciferase Reporter Gene Assay

StarBase 3.0 predicts target binding sites between BC083743 and miR-103-3p, and TargetScan 7.1 predicts target common sites between miR-103-3p and SATB2. Wild-type (WT) and mutant (MUT) reporter gene plasmids were performed against the BC083743 3’-UTR end and SATB23′-UTR end sequences. BC083743-WT and BC083743-MUT (or SATB2-WT and SATB2-MUT) were cotransfected into HEK-293T cells with NC mimics and miR-103-3p mimics, separately, and luciferase activity was assayed after 48h.


### 2.10. RNA Pull-down

Totally, 1 × 107 cells were gotten, subjected to lysis and ultrasonic breaking. The BC083743 probe was cultivated with CMurl-1 magnet beads (Life Technology, California, USA) under 25°C for 2 hours to make beads coated with probes. The cellular lysate involving the BC083743 probe or oligonucleotide probe was cultivated under 4°C for one night. Since a washing buffer was used to wash, RNA mix was escaped with RNasy Minikit (QIAGEN, Germany) for QRT-PCR or timely PCR.


### 2.11. Statistical Research

This study used the SPSS 20.0 statistical software to analyze. Statistical data were shown as average ± SD, one-way ANOVA was utilized to make a comparison between groups, and an LSD experiment was utilized for two-way comparison; test levelα = 0.05.


## 2.1. Serum Samples

Serum samples were collected from OP patients and normal individuals. This study excluded some patients getting cancer, rheumatoid arthritis, and other metabolic diseases. All serum samples were frozen in liquid nitrogen and retained in a fridge at −80°C. The research was permitted by the local medical ethics committee.


## 2.2. hBMSCs Culture

hBMSCs were purchased from ScienCell (USA). hBMSCs were cultivated in DMEM medium (Hyclone, USA) involving 10% FBS, 100 IU-mL−1 penicillin, and 100 mg-L−1 phytomycin (Solebro Technology, Beijing, China) at 37°C and 5% CO2.


## 2.3. Induction of Osteogenic Differentiation of hBMSCs

When hBMSCs grew to 80%–90% density, the medium was displaced by an osteoblast-specific induction medium (containing 10% FBS, 10 mmol-L−1 sodium β-glycerophosphate, 0.1 μmol-L−1 dexamethasone, and 0.2 mmol-L−1 ascorbic acid phosphate in low sugar DMEM), and the medium was changed every 2 d and incubated for 0, 1, 3, 7, and 14 d under 37°C and 5% CO2.


## 2.4. Cell Transfection

After the process of osteogenic induction, hBMSCs cells were inoculated in 6-well cell culture dishes with 4 × 105 cells/well, and when cell density reached 60%–70%, transfection of miR-103-3p and miR-NC was performed according to Lipofectamine 3000 instructions (Invitrogen, USA), both at a final concentration of 60 nmol-L. Cells were cultivated under 37°C and 5% CO2 for 48 h. For transfection of hBMSCs with lentivirus, the cell culture medium was replaced with lentiviral-pEF-1a/Puro-si-SATB2 (si-SATB2) and lentiviralpGLVU6/Puro-si-NC (the vectors were purchased from GenePharma (China)).


## 2.5. QRT-PCR

The transfected hBMSCs were collected, all RNA was gotten from the samples by the TRIzol method, RNA concentration was determined spectrophotometrically, and cDNA was synthesized by reverse transcription. QRT-PCR was performed according to SYBRPremix ExTaqTM II kit instructions (TaKaTa, Japan). Reaction conditions: 95°C, 0.5 min; 95°C, 5 s, 58°C, 0.5 min, 40 cycles; 95°C, 15 s, 58°C, 0.5 min, 95°C, 15 s. Using U6 and GAPDH as an internal reference, the 2-ΔΔCt way was utilized to get miR-103-3p expression levels and LncRNAs BC083743, SATB2, Runx2, OPG, and BMP2 mRNA expression levels. All primer sequences were synthesized by Guangzhou Ribo Bio (China). All primers were synthesized by Guangzhou Ruibo Co., Ltd.


## 2.6. Western Blot

The transfected hBMSCs cells were gotten, and the protein level was identified by the BCA approach. SDS-PAGE gel electrophoresis separated 30μg of protein, which was transferred to the PVDF membrane. 10% skimmed milk powder was closed at the temperature of room for 3 hours. Runx2 (1:1000), OPG (1:1000), BMP2 (1:1000), and GAPDH (1:2000) were put and cultivated under 4°C all the night. HRP-labelled secondary antibodies (1:2500) were cultivated at the temperature of room for 1 h. The band was analysed by Image J, and the gray level of the targeted protein was expressed as the proportion of the gray value of the targeted protein band to the gray value of the GAPDH band. All antibodies were bought from Abcam (UK).


## 2.7. Alkaline Phosphatase (ALP) Activity Measurement

The instructions of the kit (Biyuntian, China) reported that the cells were stable in 4% PFA for 0.5 h, cleaned 3 times in PBS, and configured into BCIP/NBT staining working solution (Sigma, Germany). Since the last wash was completed, the washing solution was moved away, and a proper number of BCIP/NBT staining working solution was put in to ensure that the sample was covered and cultivated under ambient temperature in the dark and incubated for 5–30 min under ambient temperature in the dark until stained to desired shade. The staining solution was removed, and the colour development reaction was terminated by washing twice with distilled water and photographed under the microscope. The absorbance (A) values of each group of cells were measured at 405 nm on an enzyme marker for quantification of ALP levels.


## 2.8. Alizarin Red S (ARS) Staining

The hBMSCs were discarded from the osteogenic induction medium, washed in PBS buffer, and fixed in 4% paraformaldehyde. After washing the cells in PBS buffer, the cells were incubated in 0.1% ARS staining solution (Sigma, Germany) for 20 min under ambient temperature. The staining solution was removed and cleaned in distilled water. To quantify calcium salt deposition, 10% cetylpyridinium chloride containing 10 mmol-L−1 sodium phosphate was put into the cells, and the A value at 562 nm was checked for each sample using an enzyme marker.


## 2.9. Dual-Luciferase Reporter Gene Assay

StarBase 3.0 predicts target binding sites between BC083743 and miR-103-3p, and TargetScan 7.1 predicts target common sites between miR-103-3p and SATB2. Wild-type (WT) and mutant (MUT) reporter gene plasmids were performed against the BC083743 3’-UTR end and SATB23′-UTR end sequences. BC083743-WT and BC083743-MUT (or SATB2-WT and SATB2-MUT) were cotransfected into HEK-293T cells with NC mimics and miR-103-3p mimics, separately, and luciferase activity was assayed after 48h.


## 2.10. RNA Pull-down

Totally, 1 × 107 cells were gotten, subjected to lysis and ultrasonic breaking. The BC083743 probe was cultivated with CMurl-1 magnet beads (Life Technology, California, USA) under 25°C for 2 hours to make beads coated with probes. The cellular lysate involving the BC083743 probe or oligonucleotide probe was cultivated under 4°C for one night. Since a washing buffer was used to wash, RNA mix was escaped with RNasy Minikit (QIAGEN, Germany) for QRT-PCR or timely PCR.


## 2.11. Statistical Research

This study used the SPSS 20.0 statistical software to analyze. Statistical data were shown as average ± SD, one-way ANOVA was utilized to make a comparison between groups, and an LSD experiment was utilized for two-way comparison; test levelα = 0.05.


## 3. Results

### 3.1. BC083743 Expression Was Upregulated in Osteoporosis Patients and hBMSCs

Serum BC083743 levels were measured in osteoporotic patients and normal controls. The level of BC083743 was prominently descended in osteoporotic patients (P<0.05, Figure 1(a)). Human hBMSCs separated from femoral head tissue were used in this study. The expression level of BC083743 prominently strengthened, with the time of osteogenic differentiation lengthen. Meanwhile, the expression levels of osteogenic genes Runx2, OPG, and BMP2 significantly increased (P<0.05, Figures 1(c)–1(e)). In conclusion, BC083743 may be referred to in the process of OP.Figure 1
BC083743 expression was upregulated in osteoporosis patients and hBMSCs. (a) QRT-PCR was applied to detect serum BC083743 levels in OP patients and normal controls. (b) QRT-PCR detects BC083743 levels in hBMSCs as the time to osteogenic differentiation (0 d, 1 d, 3 d, 7 d, 14 d) is prolonged. (c) The expression levels of osteogenic genes Runx2, (d) OPG, and (e) BMP2 were detected by QRT-PCR as the time of osteogenic differentiation was prolonged.∗∗∗P<0.001, compared with 0 d group.
(a)(b)(c)(d)(e)


### 3.2. Knockout of BC083743 Inhibited Osteogenic Differentiation of hBMSCs

In subsequence, to investigate, hBMSCs were transfected with si-BC083743 in order to assess the effect of BC083743 on osteogenesis in hBMSCs. And BC083743 effectively declined (P<0.05, Figure 2(a)). Knockdown of the BC083743 gene inhibited the expression of osteogenesis-related genes Runx2, OPG, and BMP2 mRNA (P<0.05, Figure 2(b)). Similarly, Western blot showed that silencing of BC083743 decreased Runx2, OPG, and BMP2 protein expression (P<0.05, Figure 2(c)). Likewise, alkaline phosphatase (ALP) activity assay and alizarin red staining showed that knockdown of BC083743 inhibited ALP activity in hBMSCs and attenuated hBMSCs’ mineralization-forming ability (P<0.05, Figure 2(d), 2(e)). Our findings indicate that BC083743 downregulation could restrain hBMSCs osteogenic differentiation.Figure 2
Knockout of BC083743 inhibited osteogenic differentiation of hBMSCs. The si-BC083743 was transfected with hBMSCs, and (a) QRT-PCR was used to detect BC083743 levels. ((b), (c)) QRT-PCR and Western blot were used to detect the expression levels of osteogenic genes Runx2, OPG, and BMP2. (d) Alkaline phosphatase (ALP) activity assay for ALP activity. (e) ARS assay for the mineralization-forming ability of hBMSCs.∗∗∗P<0.001, compared with the si-NC group.
(a)(b)(c)(d)(e)


### 3.3. BC083743 was Aimed at miR-103-3p

LncRNAs regulate gene expression by interacting with their target microRNAs (ceRNAs) [21]. The miRNA targets of BC083743 were predicted using the Starbase online database. The prediction results indicated that BC083743 was aimed at BC083743 (Figure 3(a)). Thereafter, prior to confirming the combination between both, hBMSCs cells were transfected with miR-103-3p mimics or NC mimics. The levels of miR-103-3p were detected by QRT-PCR (P<0.05, Figure 3(b)). After transfection for 48 h, the relationship between BC083743 and miR-103-3p was verified by dual-luciferase assay. The results showed that the relative luciferase activity was significantly lower in the WT group compared to the control group (P<0.05, Figure 3(c)). However, the luciferase activity was not reduced in the MUT group (Figure 3(c)). In addition, the relationship between BC083743 and miR-103-3p was also verified by RNA pull-down assays. As displayed, miR-103-3p was precipitated by the BC083743 probe (P<0.05, Figure 3(d)). Furthermore, the QRT-PCR outcome demonstrated that the level of miR-103-3p level was strengthened after BC083743 was knocked down (P<0.05, Figure 3(e)). Thereafter, serum miR-103-3p levels were examined in osteoporotic patients and normal controls. Serum miR-103-3p levels were significantly increased in osteoporotic patients (P<0.05, Figure 3(f)). In addition, with the time of osteogenic differentiation lengthen, miR-103-3p level declined dramatically (P<0.05, Figure 3(g)). This suggests that BC083743 can target miR-103-3p and negatively regulate its expression during osteogenic differentiation of bone marrow mesenchymal stem cells.Figure 3
miR-103-3p was a downstream effector of BC083743. (a) Starbase online database predicts miR-103-3p as a target miRNA for BC083743. (b) Transfection of miR-103-3p mimics or NC mimics into hBMSCs cells. The levels of miR-103-3p were detected by QRT-PCR. (c) The relationship between BC083743 and miR-103-3p was verified using a dual-luciferase assay. (d) The RNA pull-down assay verified the binding between BC083743 and miR-103-3p. (e) QRT-PCR detection of miR-103-3p expression after BC083743 overexpression knockdown. (f) QRT-PCR was used to detect serum miR-103-3p levels in OP patients and normal controls. (g) QRT-PCR detection of miR-103-3p levels in hBMSCs as osteogenic differentiation time was prolonged.∗∗P<0.01, ∗∗∗P<0.001, compared with miR-NC/si-NC/pcDNA3.1-NC.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. SATB2 was a Downstream Gene of miR-103-3p

In subsequence, we focused on the hBMSCs osteogenic differentiation regulatory gene SATB2 to assess the impact of miR-103-3p in hBMSCs osteogenic differentiation. The TargetScan website predicted that SATB2 could be a potential target of miR-103-3p (Figure4(a)). Dual-luciferase reporter gene analysis showed that miR-103-3p directly bound to the 3’UTR of SATB2 (P<0.05, Figure 4(b)). miR-103-3p knockdown significantly enhanced SATB2 expression, while miR-103-3p overexpression decreased SATB2 expression (P<0.05, Figure 4(c)). Furthermore, enhanced SATB2 was an important gene for promoting osteogenic differentiation of BMSCs [22]. Consistent with the above studies, the present study found that serum SATB2 levels were significantly reduced in osteoporotic patients (P<0.05, Figure 4(d)). Meanwhile, SATB2 expression levels increased significantly with the prolongation of osteogenic differentiation time (P<0.05, Figure 4(e)). To further confirm the role of miR-103-3p/SATB2 axis in osteoporosis, miR-103-3p inhibitors and si-SATB2 were cotransfected with hBMSCs, effectively downregulating miR-103-3p and SATB2 (P<0.05, Figure 4(f), 4(g)). miR-103-3p knockdown significantly upregulated Runx2, OPG, and BMP2, while silencing of SATB2 reversed the upregulation of Runx2, OPG, and BMP2 (P<0.05, Figure 4(h)). ALP activity assay and ARS showed that knockdown of miR-103-3p promoted ALP activity and enhanced mineralization formation in hBMSCs. In contrast, si-SATB2 partially reversed the role of miR-103-3p inhibitors in promoting osteogenic differentiation (P<0.05, Figure 4(i), 4(j)). The above results suggest that in osteoporosis, SATB2 is a target of miR-103-3p, and knockdown of SATB2 can partially reverse the promotion of osteogenic differentiation of hBMSCs by miR-103-3p inhibitors.Figure 4
SATB2 was a target of miR-103-3p. (a) The TargetScan website predicts that SATB2 may be a potential target for miR-103-3p. (b) Dual-luciferase reporter gene analysis indicates that miR-103-3p binds directly to the 3’UTR of SATB2. (c) Levels of SATB2 were detected by QRT-PCR after miR-103-3p overexpression or knockdown. (d) QRT-PCR was used to detect serum SATB2 levels in OP patients and normal controls. (e) QRT-PCR detects SATB2 levels in hBMSCs as osteogenic differentiation time increases. miR-103-3p inhibitors and si-SATB2 were cotransfected with hBMSCs, and ((f), (g)) QRT-PCR detects miR-103-3p and SATB2 expression. (h) QRT-PCR to detect the expression levels of osteogenic genes Runx2, OPG, and BMP2. (i) Alkaline phosphatase (ALP) activity assay for ALP activity. (j) ARS assay for the mineralization-forming ability of hBMSCs.∗∗P<0.01, ∗∗∗P<0.001, ns P>0.05, compared with NC mimics/NC inhibitors/NC.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


### 3.5. BC083743 Promoted Osteogenic Differentiation of hBMSCs via the miR-103-3p/SATB2 Axis

Since BC083743 inhibited the expression of miR-103-3p in hBMSCs and miR-103-3p directly targets SATB2, we further explored whether BC083743 upregulates SATB2 expression by adsorbing miR-103-3p. si-BC083743 and miR-103-3p inhibitors Cotransfection of hBMSCs and downregulation of BC083743 significantly reduced the expression level of SATB2 in hBMSCs, while transfection of miR-103-3p inhibitor partially reversed the decrease in SATB2 expression (P<0.05, Figure 5(a)). Knockdown of BC083743 significantly downregulated Runx2, OPG, and BMP2, while miR-103-3p inhibitors reversed the downregulation of Runx2, OPG, and BMP2 (P<0.05, Figure 5(b)). ALP activity assay and ARS showed that knockdown of BC083743 inhibited the ALP activity of hBMSCs and attenuated the mineralization-forming ability of hBMSCs. In contrast, miR-103-3p inhibitors partially reversed the inhibitory effect of BC083743 knockdown on osteogenic differentiation (P<0.05, Figure 5(c), 5(d)). This implies that BC083743 acts as a ceRNA to promote osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating the miR-103-3p/SATB2 axis.Figure 5
BC083743 promoted osteogenic differentiation of hBMSCs via the miR-103-3p/SATB2 axis. si-BC083743 and miR-103-3p inhibitors were cotransfected with hBMSCs. (a) QRT-PCR was performed to detect SATB2 levels in hBMSCs. (b) QRT-PCR to detect the expression levels of osteogenic genes Runx2, OPG, and BMP2. (c) Alkaline phosphatase (ALP) activity assay for ALP activity. (d) ARS assay for the mineralization-forming ability of hBMSCs.∗∗∗P<0.001, compared with NC.
(a)(b)(c)(d)


## 3.1. BC083743 Expression Was Upregulated in Osteoporosis Patients and hBMSCs

Serum BC083743 levels were measured in osteoporotic patients and normal controls. The level of BC083743 was prominently descended in osteoporotic patients (P<0.05, Figure 1(a)). Human hBMSCs separated from femoral head tissue were used in this study. The expression level of BC083743 prominently strengthened, with the time of osteogenic differentiation lengthen. Meanwhile, the expression levels of osteogenic genes Runx2, OPG, and BMP2 significantly increased (P<0.05, Figures 1(c)–1(e)). In conclusion, BC083743 may be referred to in the process of OP.Figure 1
BC083743 expression was upregulated in osteoporosis patients and hBMSCs. (a) QRT-PCR was applied to detect serum BC083743 levels in OP patients and normal controls. (b) QRT-PCR detects BC083743 levels in hBMSCs as the time to osteogenic differentiation (0 d, 1 d, 3 d, 7 d, 14 d) is prolonged. (c) The expression levels of osteogenic genes Runx2, (d) OPG, and (e) BMP2 were detected by QRT-PCR as the time of osteogenic differentiation was prolonged.∗∗∗P<0.001, compared with 0 d group.
(a)(b)(c)(d)(e)


## 3.2. Knockout of BC083743 Inhibited Osteogenic Differentiation of hBMSCs

In subsequence, to investigate, hBMSCs were transfected with si-BC083743 in order to assess the effect of BC083743 on osteogenesis in hBMSCs. And BC083743 effectively declined (P<0.05, Figure 2(a)). Knockdown of the BC083743 gene inhibited the expression of osteogenesis-related genes Runx2, OPG, and BMP2 mRNA (P<0.05, Figure 2(b)). Similarly, Western blot showed that silencing of BC083743 decreased Runx2, OPG, and BMP2 protein expression (P<0.05, Figure 2(c)). Likewise, alkaline phosphatase (ALP) activity assay and alizarin red staining showed that knockdown of BC083743 inhibited ALP activity in hBMSCs and attenuated hBMSCs’ mineralization-forming ability (P<0.05, Figure 2(d), 2(e)). Our findings indicate that BC083743 downregulation could restrain hBMSCs osteogenic differentiation.Figure 2
Knockout of BC083743 inhibited osteogenic differentiation of hBMSCs. The si-BC083743 was transfected with hBMSCs, and (a) QRT-PCR was used to detect BC083743 levels. ((b), (c)) QRT-PCR and Western blot were used to detect the expression levels of osteogenic genes Runx2, OPG, and BMP2. (d) Alkaline phosphatase (ALP) activity assay for ALP activity. (e) ARS assay for the mineralization-forming ability of hBMSCs.∗∗∗P<0.001, compared with the si-NC group.
(a)(b)(c)(d)(e)


## 3.3. BC083743 was Aimed at miR-103-3p

LncRNAs regulate gene expression by interacting with their target microRNAs (ceRNAs) [21]. The miRNA targets of BC083743 were predicted using the Starbase online database. The prediction results indicated that BC083743 was aimed at BC083743 (Figure 3(a)). Thereafter, prior to confirming the combination between both, hBMSCs cells were transfected with miR-103-3p mimics or NC mimics. The levels of miR-103-3p were detected by QRT-PCR (P<0.05, Figure 3(b)). After transfection for 48 h, the relationship between BC083743 and miR-103-3p was verified by dual-luciferase assay. The results showed that the relative luciferase activity was significantly lower in the WT group compared to the control group (P<0.05, Figure 3(c)). However, the luciferase activity was not reduced in the MUT group (Figure 3(c)). In addition, the relationship between BC083743 and miR-103-3p was also verified by RNA pull-down assays. As displayed, miR-103-3p was precipitated by the BC083743 probe (P<0.05, Figure 3(d)). Furthermore, the QRT-PCR outcome demonstrated that the level of miR-103-3p level was strengthened after BC083743 was knocked down (P<0.05, Figure 3(e)). Thereafter, serum miR-103-3p levels were examined in osteoporotic patients and normal controls. Serum miR-103-3p levels were significantly increased in osteoporotic patients (P<0.05, Figure 3(f)). In addition, with the time of osteogenic differentiation lengthen, miR-103-3p level declined dramatically (P<0.05, Figure 3(g)). This suggests that BC083743 can target miR-103-3p and negatively regulate its expression during osteogenic differentiation of bone marrow mesenchymal stem cells.Figure 3
miR-103-3p was a downstream effector of BC083743. (a) Starbase online database predicts miR-103-3p as a target miRNA for BC083743. (b) Transfection of miR-103-3p mimics or NC mimics into hBMSCs cells. The levels of miR-103-3p were detected by QRT-PCR. (c) The relationship between BC083743 and miR-103-3p was verified using a dual-luciferase assay. (d) The RNA pull-down assay verified the binding between BC083743 and miR-103-3p. (e) QRT-PCR detection of miR-103-3p expression after BC083743 overexpression knockdown. (f) QRT-PCR was used to detect serum miR-103-3p levels in OP patients and normal controls. (g) QRT-PCR detection of miR-103-3p levels in hBMSCs as osteogenic differentiation time was prolonged.∗∗P<0.01, ∗∗∗P<0.001, compared with miR-NC/si-NC/pcDNA3.1-NC.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. SATB2 was a Downstream Gene of miR-103-3p

In subsequence, we focused on the hBMSCs osteogenic differentiation regulatory gene SATB2 to assess the impact of miR-103-3p in hBMSCs osteogenic differentiation. The TargetScan website predicted that SATB2 could be a potential target of miR-103-3p (Figure4(a)). Dual-luciferase reporter gene analysis showed that miR-103-3p directly bound to the 3’UTR of SATB2 (P<0.05, Figure 4(b)). miR-103-3p knockdown significantly enhanced SATB2 expression, while miR-103-3p overexpression decreased SATB2 expression (P<0.05, Figure 4(c)). Furthermore, enhanced SATB2 was an important gene for promoting osteogenic differentiation of BMSCs [22]. Consistent with the above studies, the present study found that serum SATB2 levels were significantly reduced in osteoporotic patients (P<0.05, Figure 4(d)). Meanwhile, SATB2 expression levels increased significantly with the prolongation of osteogenic differentiation time (P<0.05, Figure 4(e)). To further confirm the role of miR-103-3p/SATB2 axis in osteoporosis, miR-103-3p inhibitors and si-SATB2 were cotransfected with hBMSCs, effectively downregulating miR-103-3p and SATB2 (P<0.05, Figure 4(f), 4(g)). miR-103-3p knockdown significantly upregulated Runx2, OPG, and BMP2, while silencing of SATB2 reversed the upregulation of Runx2, OPG, and BMP2 (P<0.05, Figure 4(h)). ALP activity assay and ARS showed that knockdown of miR-103-3p promoted ALP activity and enhanced mineralization formation in hBMSCs. In contrast, si-SATB2 partially reversed the role of miR-103-3p inhibitors in promoting osteogenic differentiation (P<0.05, Figure 4(i), 4(j)). The above results suggest that in osteoporosis, SATB2 is a target of miR-103-3p, and knockdown of SATB2 can partially reverse the promotion of osteogenic differentiation of hBMSCs by miR-103-3p inhibitors.Figure 4
SATB2 was a target of miR-103-3p. (a) The TargetScan website predicts that SATB2 may be a potential target for miR-103-3p. (b) Dual-luciferase reporter gene analysis indicates that miR-103-3p binds directly to the 3’UTR of SATB2. (c) Levels of SATB2 were detected by QRT-PCR after miR-103-3p overexpression or knockdown. (d) QRT-PCR was used to detect serum SATB2 levels in OP patients and normal controls. (e) QRT-PCR detects SATB2 levels in hBMSCs as osteogenic differentiation time increases. miR-103-3p inhibitors and si-SATB2 were cotransfected with hBMSCs, and ((f), (g)) QRT-PCR detects miR-103-3p and SATB2 expression. (h) QRT-PCR to detect the expression levels of osteogenic genes Runx2, OPG, and BMP2. (i) Alkaline phosphatase (ALP) activity assay for ALP activity. (j) ARS assay for the mineralization-forming ability of hBMSCs.∗∗P<0.01, ∗∗∗P<0.001, ns P>0.05, compared with NC mimics/NC inhibitors/NC.
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)


## 3.5. BC083743 Promoted Osteogenic Differentiation of hBMSCs via the miR-103-3p/SATB2 Axis

Since BC083743 inhibited the expression of miR-103-3p in hBMSCs and miR-103-3p directly targets SATB2, we further explored whether BC083743 upregulates SATB2 expression by adsorbing miR-103-3p. si-BC083743 and miR-103-3p inhibitors Cotransfection of hBMSCs and downregulation of BC083743 significantly reduced the expression level of SATB2 in hBMSCs, while transfection of miR-103-3p inhibitor partially reversed the decrease in SATB2 expression (P<0.05, Figure 5(a)). Knockdown of BC083743 significantly downregulated Runx2, OPG, and BMP2, while miR-103-3p inhibitors reversed the downregulation of Runx2, OPG, and BMP2 (P<0.05, Figure 5(b)). ALP activity assay and ARS showed that knockdown of BC083743 inhibited the ALP activity of hBMSCs and attenuated the mineralization-forming ability of hBMSCs. In contrast, miR-103-3p inhibitors partially reversed the inhibitory effect of BC083743 knockdown on osteogenic differentiation (P<0.05, Figure 5(c), 5(d)). This implies that BC083743 acts as a ceRNA to promote osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating the miR-103-3p/SATB2 axis.Figure 5
BC083743 promoted osteogenic differentiation of hBMSCs via the miR-103-3p/SATB2 axis. si-BC083743 and miR-103-3p inhibitors were cotransfected with hBMSCs. (a) QRT-PCR was performed to detect SATB2 levels in hBMSCs. (b) QRT-PCR to detect the expression levels of osteogenic genes Runx2, OPG, and BMP2. (c) Alkaline phosphatase (ALP) activity assay for ALP activity. (d) ARS assay for the mineralization-forming ability of hBMSCs.∗∗∗P<0.001, compared with NC.
(a)(b)(c)(d)


## 4. Discussion

OP is a kind of metabolic illness in bone characterised accompanied by reduced bone mass and altered bone ultrastructure. Patients present with increased bone fragility, which increases their risk of fracture. Osteoporotic fractures are common in the hip, spine, and wrist joints and have a high incidence, seriously affecting the quality of life of patients and increasing the socio-economic burden [23]. A variety of preventive and therapeutic measures, both Chinese and Western, can help maintain bone mass, but fracture osteoporosis is still a major challenge for clinical patients [24]. With advances in the molecular biology of osteoporosis, such as multiple susceptibilities and predictive screening gene indicators, and bone loss-related gene therapy, research has provided a solid foundation for applied basic research and drug development for the treatment of osteoporosis [25]. Lately, in the study of OP, lncRNA has been put more interest [26]. In the current study, a novel lncRNA, BC083743, was found to be involved in the progression of OP.Regulating osteogenic differentiation of BMSCs in OP progression, lncRNA has been put into more attention. Sun et al. confirmed during osteogenic differentiation of BMSCs, the expression of lncRNA Bmcob and selenoprotein P was consistent, and lncRNA Bmcob knockdown resulted in the downregulation of selenoprotein P and the inhibition of BMSCs osteogenic differentiation [27]. What is more, upregulation of the BDNF-AS inversely adjusted brain-derived neurotrophic factor, osteopontin, and Runt-related transcription factor 2 osteogenic signaling pathways, promoting self-proliferation and inhibiting BMSCs osteogenic differentiation [28]. At present, the impact of BC083743 has been assessed in BMSCs osteogenic differentiation. Importantly, we authenticateed BC083743 level that was markedly declined in serum of OP patients, and BC083743 level increased, with the time to osteogenic differentiation. Moreover, BC083743 inhibition could restrain hBMSCs osteogenic differentiation. This suggests that BC083743 knockdown can exacerbate osteoporosis by inhibiting osteogenic differentiation of hBMSCs.BMSCs are pluripotent stem cells, which can differentiate into osteoblasts. BMSCs also can be separated into other cell types (e.g., adipocytes, myoblasts, and chondrocytes), with multiple cytokines and pathways involved in their osteogenic differentiation process [17]. Runx2 expression is an early marker of the stereotypic differentiation of BMSCs into the osteoblast lineage and restrains the directional differentiation of chondrocytes and adipocytes [29]. Runx2 promotes the expression of alkaline phosphatase (ALP) and osteoprotegerin (OPG), the latter two being markers of osteoblast activation and bone formation [29]. Bone morphogenetic protein II (BMP2) has an important role in bone development and reconstruction. The current study found that osteogenic differentiation-related genes (Runx2, OPG, BMP2) were significantly upregulated, with the time to osteogenic differentiation lengthen. And BC083743 deficiency declined the levels of osteogenic differentiation-related genes. This suggests that BC083743 knockdown inhibited osteogenic differentiation.The role of miRNAs in osteoblast growth and differentiation has been extensively studied [30]. Among them, osteoblast differentiation is an important process in bone homeostasis, and miRNAs can regulate the biological process of osteoblast differentiation [31]. It has been shown that miR-375 inhibits osteoblast differentiation by targeting RUNX2 while reducing the activity of several key osteoblast markers [32]. miR-96 promotes osteoblast differentiation by inhibiting HB-EGF/EGF receptor signaling in osteoblasts [33]. Previous studies suggest that miR-103-3p inhibits bone formation and promotes osteoporosis in vivo [15]. In line with the above studies, miR-103-3p expression was significantly upregulated in the serum of OP patients and downregulated as the time of osteogenic differentiation increased. In recent years, the mechanism of osteogenic differentiation in BMSCs has become a hot area of clinical and basic research, and certain progress has been made, including the study of regulatory signals and the discovery of some important osteogenic transcription factors, such as SATB2 [34]. Previous studies suggest that SATB2 has been shown to promote osteogenic differentiation of BMSCs [35]. miR-103-3p targets SATB2 to inhibit osteogenic differentiation [16]. Consistent with the above studies, SATB2 expression was significantly downregulated in the serum of patients with OP, and SATB2 expression was significantly upregulated as the time to osteogenic differentiation increased. LncRNA regulates osteogenic differentiation of BMSCs by targeting miRNAs to regulate OP progression [20]. Therefore, we wondered whether BC083743 could influence OP occurrence by regulating the miR-103-3p/SATB2 axis as a ceRNA. Dual-luciferase assays and RNA pull-down assays confirmed that miR-103-3p binds to BC083743 and SATB2, and functional experiments confirmed that knockdown of BC083743 inhibited ALP activity and attenuated the mineralization-forming ability of hBMSCs. In contrast, miR-103-3p inhibitors partially reversed the inhibitory effect of BC083743 knockdown on osteogenic differentiation. Reference [36].



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*Source: 7066759-2022-06-20.xml*