lncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived from Adipose-Derived Stem Cells for the Treatment of Osteoporosis

Background Osteoporosis is a worldwide medical and socioeconomic burden characterized by systemic impairment of bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been confirmed to play effective roles in the repair of various tissues and organs. This study was aimed at investigating the role of ADSCs-Exos and a novel long noncoding RNA KCNQ1OT1 played in osteoporosis as well as the underlying mechanism. Methods Primary osteoblasts were treated with different doses of tumor necrosis factor-α (TNF-α) (0, 1, 2.5, 5, and 10 ng/ml) and then cocultured with ADSCs-Exos or exosome-derived from lnc-KCNQ1OT1-modified ADSCs (KCNQ1OT1-Exos). The expression of miRNA-141-5p (miR-141-5p) and lnc-KCNQ1OT1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of cleaved-caspase-3, caspase-3, and Bax was determined by Western blot. Cell viability and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis, respectively. The binding sites between KCNQ1OT1 and miR-141-5p were validated by dual-luciferase reporter assay. Results TNF-α dose-dependently increased miR-141-5p expression, inhibited viability, and promoted apoptosis of osteoblasts. However, miR-141-5p silencing or cocultured with ADSCs-Exos attenuated these effects. In addition, KCNQ1OT1-Exos could more significantly attenuate the induced cytotoxicity and apoptosis compared to ADSCs-Exos. Moreover, miR-141-5p was confirmed as the target of KCNQ1OT1 by luciferase reporter assay. Conclusions ADSCs-Exos can attenuate cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos have a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos can be promising agents in osteoporosis treatment.


Introduction
Osteoporosis is a common skeletal disease characterized by structural disorders of bone mass caused by increased osteoclast activity and reduced osteoblast generation [1]. Clinical treatment strategies are often based on the promotion of osteoblast proliferation and osteoclast inhibition [2,3]. In osteoporosis pathogenesis, tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine which has been revealed to contribute to osteoporosis by regulating both osteoblasts and osteoclasts [3,4]. TNF-α can suppress bone formation and inhibit osteoblast differentiation by inducing cytotoxicity and apoptosis [5,6]. Therefore, exploring new pathways to arrest TNF-αinduced cytotoxicity and apoptosis in osteoporosis is necessary.
As a subset of mesenchymal stem cells (MSCs), adiposederived stem cells (ADSCs) can be easily obtained from adipose tissues and possess the potential of multidifferentiation and self-renewal [7,8]. ADSCs are found in abundant quantities and can be easily obtained by minimally invasive procedures [9]. Autologous ADSCs can be used to promote bone regeneration under osteoporotic conditions [10]. Thus, the application of ADSCs might be a promising MSC-based strategy for bone formation and structural remodeling in osteoporosis [11,12].
Exosomes are small, membrane-bound extracellular vesicles that are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates [13]. Exosomes have been proved to play a significant role in the etiology of bone metabolic diseases, especially osteoporosis [14]. Exosomes derived from ADSCs (ADSCs-Exos) contain bioactive substances of ADSCs and might play a similar role to ADSCs [15]. Recent researches have demonstrated that ADSCs-Exos had stimulating effects in the repair of a variety of tissues and organs [10,16,17]. Nevertheless, the potential therapeutic effects of ADSCs-Exos on osteoporosis and the underlying mechanism need further investigations.
Exosomes are important mediators between cells by transferring molecules, such as long noncoding RNAs (lncRNA), microRNAs (miRNAs), and cytokines [18][19][20][21]. Evidence indicates that exosomes derived from mmu_circ_ 0000250-modified ADSCs are able to promote wound healing in diabetic mice [22]. Additionally, exosomes derived from miR-188-3p-modified ADSCs can protect Parkinson's disease [23]. Recently, KCNQ1overlapping transcript 1 (lnc-KCNQ1OT1) was found to be able to positively regulate osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by acting as a ceRNA to regulate BMP2 expression through sponging miR-214 [24]. However, few studies have investigated the potential role of KCNQ1OT1 in the treatment of osteoporosis. This study was aimed at investigating the role of ADSCs-Exos and KCNQ1OT1 played in osteoporosis as well as the underlying mechanism.
2. Methods 2.1. Isolation and Culture of ADSCs. The Institutional Animal Care and Use Committee of Southeast University approved the protocol for the use of animals in this study. Adipose tissues were collected from the inguinal fat pad of 2-3-week-old C57BL/6 mice (SLAC, Shanghai, China) and then rinsed in phosphate-buffered saline (PBS) and cut into 1 × 1 mm pieces. The collected tissues were digested by collagenase type II (Sigma-Aldrich, USA) at 37°C for 1 h. After digestion, tissues were centrifuged at room temperature (1000 rpm, 5 min) and the resultant cell pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) at the cell density of 5 × 10 6 /ml. Cells were then cultured at 37°C under a 5% CO 2 atmosphere. The culture medium was replaced every 2-3 days, and cells after 3 passages were used in the present study.

Characterization of Isolated ADSCs.
ADSCs were cultured in a 24-well plate at a density of 4 × 10 4 cells/well with basic culture medium (DMEM-LG) containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. They were subjected to induced differentiation by culturing them in osteogenic (Cyagen Biosciences, Rasmx-90021), adipogenic (Cyagen Biosciences, Rasmx-90031), and chondrogenic (Cyagen Biosciences, Rasmx-90041) medium, respectively. The outcomes were evaluated by Alizarin Red, Oil Red O, and Alcian Blue staining, respectively. In addition, MSC surface markers (CD90 + and CD34-) were detected by flow cytometric analysis. Briefly, adherent cells from passage 4 were resuspended and then incubated in FACS buffer containing 2 μl of Fc receptor-blocking reagent (FcX Blocker, BioLegend, San Diego, CA, USA). Cells were stained with reagent to exclude dead cells (Zombie NIR, Japan). After washing the cells, IC fixation buffer (Affymetrix, Japan) was added. Permeabilization buffer (Affymetrix, Japan) was diluted with purified water for 10 times. After permeabilization, CD90 and CD34 antibodies were used to stain the cells. All antibodies were from BD Biosciences (Tokyo, Japan). Stained cells were sorted by flow cytometry (Accuri C6, BD), and data was analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Isolation and Characterization of Primary Osteoblasts.
The isolation and characterization of primary osteoblasts were conducted as described before [25]. Briefly, calvarium tissues from C57BL/6 mice were collected and digested in 0.25% trypsin (Thermo Fisher, USA) containing 0.02% EDTA for 25 min at 37°C. Then, the tissues were digested in 5 ml Hanks solution containing 0.1% collagenase I (Thermo Fisher, USA) and 0.05% trypsin for 1 h in a shaking incubator at 37°C with a shaking speed of 200 r/min. The released cells were collected by centrifugation for 10 minutes at 1500 r/min. The cells were suspended in 5 ml of a-MEM (containing 1 g/L D-Glucose and L-Glutamine, BI, USA) containing 10% FBS and then transferred to 25 cm 2 plastic culture flasks (Nest, China). The culture flasks were incubated at 37°C in a 5% CO 2 incubator. The images of cell morphology were taken using a microscope attaching camera (OLYMPUS, IX51). The primary osteoblasts of passage 4 were prepared for further experiments and characterized by alkaline phosphatase staining and Alizarin Red S staining.
2.5. Isolation and Characterization of Exosomes. At 48 h posttransfection, a Total Exosome Isolation kit (Invitrogen, USA) was applied to isolate the total exosomes from the supernatant of ADSC culture medium and culture medium transfected with pcDNA-lnc-KCNQ1OT1 or NC (ADSCs-Exos, LV-KCNQ1OT1-Exos, or LV-NC-Exos, respectively) according to the manufacturer's protocol [26]. Bicinchoninic acid (BCA) protein assay kit (MACGENE, China) was used Meanwhile, the expression of lncRNA-KCNQ1OT1 was evaluated by PrimeScript™ Master Mix (Takara, Japan) and GAPDH was employed as the loading control. The data was calculated using the 2 −ΔΔCt method. Primers used in this study are shown in Table 1.
2.12. Statistical Analysis. Statistical analysis was performed with SPSS 20.0 software (IBM, Armonk, NY, USA). All quantitative data were described as mean ± SD. One-way ANOVA was used to analyze the statistical differences among three or more groups while unpaired Student's t -test was applied to analyze the statistical differences
The red fluorescence of PKH26 label was observed in primary osteoblasts (Figure 3(d)), indicating primary osteoblasts could uptake ADSCs-Exos.

KCNQ1OT1-Exos Inhibit TNF-α-Induced Cytotoxicity and Apoptosis of Primary Osteoblasts.
ADSCs were transfected with LV-KCNQ1OT1 or LV-NC for the determination of whether ADSCs transfected LV-KCNQ1OT1 into secreted exosomes. At 24 h post-LV-KCNQ1OT1 transfection, the expression of KCNQ1OT1 in ADSCs or exosomes derived from the ADSCs after different transfections was elevated, but the expression of miR-141-5p was downregulated compared with that in LV-NC-treated group (Figures 5(a) and 5(b)). In order to confirm whether ADSCs-Exos carrying LV-KCNQ1OT1 could deliver KCNQ1OT1 into primary osteoblasts, primary osteoblasts were cocultured with LV-NC-Exos or LV-KCNQ1OT1-Exos. KCNQ1OT1 expression was upregulated in primary osteoblasts treated with LV-KCNQ1OT1-Exos ( Figure 5(c)). Next, to explore whether KCNQ1OT1-Exos could influence TNF-α-induced cytotoxicity and apoptosis, primary osteoblasts were treated with TNF-α and then cocultured with medium, ADSCs-Exos, LV-NC-Exos, or LV-KCNQ1OT1-Exos. The results of CCK-8 showed the coculture of primary osteoblasts with LV-KCNQ1OT1-Exos mitigated the negative effect of TNF-α on cell viability; ADSCs-Exos exerted a weaker stimulative effect on cell viability compared to LV-KCNQ1OT1-Exos ( Figure 5(d)). Flow cytometry analysis indicated that TNF-α-induced cell apoptosis was reversed when primary osteoblasts were cocultured with LV-KCNQ1OT1-Exos; ADSCs-Exos exerted a weaker inhibitory effect on cell apoptosis compared to LV-KCNQ1OT1-Exos ( Figure 5(e)). Consistent with that, the expression of Bax and cleaved caspase-3 in primary osteoblasts was blocked after coculture of LV-KCNQ1OT1-Exos ( Figure 5(f)).

Discussion
With the deepening understanding of osteobiology, skeletal stem cells and osteoblasts are identified as significant targets in the treatment of osteoporosis by promoting bone formation and remodeling [27]. MSC transplantation provides evidences of enhancing osteogenic differentiation, increasing bone mineral density, and halting the deterioration of osteoporosis [28]. In 2001, Zuk et al. [29] isolated ADSCs from adipose tissues and found they are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. As significant bioactive substances released from ADSCs, ADSCs-Exos have exhibited regenerative potential in many diseases [30][31][32]. However, few studies have investigated the role of ADSCs-Exos in the treatment of osteoporosis. Thus, whether ADSCs-Exos can effectively protect primary osteoblasts from the TNF-α-induced cytotoxicity and apoptosis is primarily studied in the present study.
Our results indicate that TNF-α can increase miR-141-5p expression and inhibit the cell proliferation in primary osteoblasts. In addition, TNF-α can increase cell apoptosis. Consistent with this, the expression of cleaved caspase-3 and Bax was also elevated. Currently, ADSCs have been widely used in tissue regeneration and bioengineering [7,33]. However, with the in-depth investigations, the application of ADSCs has the potential risk of iatrogenic infection, malignant transformation, immune rejection, and safety issues [34][35][36][37]. Compared to ADSCs, ADSCs-Exos can hardly cause the immune rejection and malignant transformation [38,39]. To make clear the effects of ADSCs-Exos in osteoporosis, we attempt to culture TNF-α-treated primary osteoblasts with ADSCs-Exos. Interestingly, ADSCs-Exos promote cell viability and decrease cell apoptosis, suggesting that ADSCs-Exos can be promising candidates in the treatment of osteoporosis.
Although we have found ADSCs-Exos can be beneficial in the treatment of osteoporosis, the underlying mechanism has not been revealed. Exosomes derived from MSCs contain multiple lncRNAs, which can be transported and transferred to other cells to regulate biological functions through targeting downstream genes [40,41]. KCNQ1OT1, a lncRNA which is closely related to cell proliferation, migration, and apoptosis, has been reported to be an oncogene in a variety of tumors [42]. Evidence has showed that KCNQ1OT1 can promote cell proliferation and migration [43]. Therefore, we are curious whether KCNQ1OT1 can play a positive role in the treatment of osteoporosis. In the present study, KCNQ1OT1-Exos were confirmed to exert a more significant inhibitory effect on TNF-α-induced cytotoxicity and apoptosis compared to ADSCs-Exos. Thus, KCNQ1OT1-Exos are expected to be promising candidates in osteoporosis treatment.

Conclusions
In the present study, we demonstrate that ADSCs-Exos can attenuate cytotoxicity and apoptosis of TNF-α-induced primary osteoblasts. KCNQ1OT1-Exos have a more significant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos can be promising agents in osteoporosis treatment. Further explorations of the pleiotropic effect of KCNQ1OT1 and the crosstalk between KCNQ1OT1 and miR-141-5p will provide new insights for developing new treatments to improve the therapeutic efficacy based on ADSCs-Exos.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon request.

Disclosure
A preprint related to this study has previously been published and is available at Research Square [49].