LncRNA-KCNQ1OT1: A Potential Target in Exosomes Derived From ADSCs for The Treatment of Osteoporosis

Osteoporosis is a worldwide medical and socioeconomic threat characterized by systemic impairment to bone strength and microstructure. Exosomes derived from adipose-derived stem cells (ADSCs-Exos) have been conrmed to play effective roles in the repair of various tissues and organs. This study aimed to investigate the role of ADSCs-Exos and a noval long none coding RNAKCNQ1OT1 (lnc-KCNQ1OT1) played in osteoporosis as well as the underlying mechanism. cytotoxicity and apoptosis of TNF-α-induced MC3T3-E1 cells. KCNQ1OT1-Exos had a more signicant inhibitory effect compared to ADSCs-Exos by the function of sponging miR-141-5p, suggesting that KCNQ1OT1-Exos could be promising agents in osteoporosis treatment.

Isolation and characterization of exosomes A Total Exosome Isolation kit (Invitrogen, 4478359, USA) was applied to isolate the total exosomes from the supernatant of ADSCs culture medium according to the manufacturer's protocol. The experimental procedures were conducted as described before [24]. Bicinchoninic acid assay (BCA) protein assay kit was used to measure the concentration of isolated exosomes. The protein levels of CD9, CD63, CD81 and Alix (representative markers of exosomes) were then detected.
Exosomes were resuspended in the complete culture medium and the PKH26-labeled ADSCs-Exos solution was added into MC3T3-E1 cells for incubation. After 24-48h of culture, MC3T3-E1 cells were xed with 4% formaldehyde for 10 min. DAPI was used to stain the nuclei. Cells were nally observed under a confocal microscope.
Cell apoptosis assay MC3T3-E1 cell apoptosis was evaluated by Annexin V-FITC/PI Apoptosis Assay Kit (Keygen Biotech, China). Brie y, MC3T3-E1 cells were collected and washed with PBS. A total of 500 μl binding buffer was added to suspend cells. Firstly, 5 μl annexin V-FITC was added, and then 5 μl propidium iodide was added for incubation for 5-15min in the dark at room temperature. Cell apoptosis was analyzed by ow cytometry (Becton-Dickinson, FACS Calibur, USA).

Western blot analysis
The total protein of MC3T3-E1 cell was extracted using RIPA lysis buffer (Beyotime, China) and quanti ed by BCA assay (Beyotime, China). Equal amounts of proteins (100μg) were separated via BeyoGel™ Plus PAGE (Beyotime, China) and then transferred to a PVDF membrane (Millipore, USA). After transferring, the membranes were blocked with 5% fat-free milk for 1h. The membranes were incubated with primary antibodies (Bax, ab32503, Caspase-3, ab32351, cleaved-Caspase-3, ab32042), which were purchased from Abcam (USA) and GAPDH, 10494-I-AP which was obtained from Proteintech (China) at 4°C overnight. The membranes were incubated with the second antibodies (Goat anti rabbit IgG HRP SE134, Goat anti mouse IgG HRP SE131, Solarbio, China) at 37°C for 1h. The ECL system (CLINX, China) was used for exposing protein bands. The intensity of the bands was analyzed using Image lab (version 3.0, Bio-Rad, USA).
Gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. For detecting miR-141-5p expression, MicroRNA cDNA Synthesis Kit (Vazyme, China) was applied for reverse transcription of cDNA, followed by qRT-PCR analysis. U6 was employed as the loading control. The primers of miR-141-5p and U6 were purchased from GeneCopoeia (Guangzhou, China). Meanwhile, the expression of lncRNA-KCNQ1OT1 was evaluated by Prime Script TM Master Mix (Takara, Japan) and GAPDH was employed as the loading control. The data was calculated using the Dual-luciferase reporter assay The wild-type (WT) sequence of lncRNA-KCNQ1OT1 containing the miR-141-5p binding sites (KCNQ1OT1-WT) and the mutant sequence (KCNQ1OT1-MUT) were cloned into pMIR vectors (Promega, USA) respectively. MC3T3-E1 and HEK293 cells were co-transfected with miR-141-5p or miR-NC and KCNQ1OT1-WT or KCNQ1OT1-MUT and kept for 24 h. The luciferase activity was detected using Dual-Luciferase Reporter Assay System (Promega, USA) in the dark.

Statistical Analysis
Statistical analysis was performed with SPSS 20.0 software (IBM, Armonk, NY, U.S.A.). 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 between two groups. P< 0.05 was considered statistically signi cant.

Results
TNF-α increased miR-141-5p expression, suppressed the viability and promoted the apoptosis of MC3T3-
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 was elevated, but the expression of miR-141-5p was down-regulated compared with that in LV-NC treated group ( Fig. 5a and b).

Discussion
With the deepening understanding of osteobiology, skeletal stem cells and osteoblasts are identi ed as signi cant target in the treatment of OP by promoting bone formation and remodeling [25]. Previous studies have proven that decreasing miR-10a-3p and raising CXCL12 can induce the osteogenic differentiation of bone marrow-derived MSCs (BMSCs) [26]. miR-26b could promote BMSCs osteogenesis through directly regulating GSK3β and activating Wnt pathway [27]. MSCs-derived exosomes are involved in multiple physiology and pathology processes, including osteogenesis, bone regeneration and osteoarthritis [28,29]. BMSC-derived exosomes are able to alleviate radiation-induced bone loss via Wnt/ β-catenin signaling pathway. In addition, BMSCs-derived exosomal MALAT1 can promote the progression of osteoblasts [30].
Similar to BMSCs, ADSCs also have the potential of multi-lineage differentiation. In 2001, Zuk et al [31] rstly isolated ADSCs from adipose tissue and found they could differentiate into adipocytes, osteoblasts and chondrocytes under different induction conditions. In addition to the commonness with BMSCs, ADSCs can be easily obtained with abundant sources. Depending on this, ADSCs might be a better choice in the treatment of osteoporosis compared to BMSCs. However, few studies have investigated the roles of ADSCs-Exos, especially in the treatment of osteoporosis. Thus, whether ADSCs-Exos could effectively protect MC3T3-E1cells from the TNF-α-induced cytotoxicity and apoptosis was studied in the present study.
Our results indicated that TNF-α increased miR-141-5p expression, inhibited cell viability and promoted cell apoptosis in MC3T3-E1 cells. 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. However, with the in-depth investigations, the application of ADSCs has the potential risk of iatrogenic infection, malignant transformation, immune rejection safety issues [32][33][34]. Compared to ADSCs, ADSCs-Exos have no risk of malignant transformation and can hardly cause the immune rejections [35,36]. Therefore, ADSCs-Exos can be promising regenerative agents for osteoporosis. To make clear the effects of ADSCs-Exos in osteoporosis, we attempted to culture TNF-α-treated MC3T3-E1 cells with ADSCs-Exos. Interestingly, ADSCs-Exos promoted cell viability and impeded apoptosis, suggesting that ADSCs-Exos can be promising candidates in the treatment of OP.
Although we've found ADSCs-Exos can be bene cial in the treatment of OP, 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 [37,38]. In osteoarthritis, exosomal KLF3-AS1 originated from hMSCs boosted cartilage repair and chondrocyte proliferation [39]. 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 [23]. Evidence has showed that KCNQ1OT1 can promote cell proliferation and migration [40]. KCNQ1OT1 was found to enhance glioma cell proliferation and invasion via regulating the miR-375/YAP pathway and thereby accelerated the progression of glioma [41]. Meanwhile, in colorectal cancer, KCNQ1OT1 can induce cell apoptosis by sponging miR-329-3p [42]. Herein, KCNQ1OT1 was targeted for further investigation in this study. We con rmed the presence of KCNQ1OT1 in ADSCs-Exos, and further investigated the role of KCNQ1OT1 in the treatment of osteoporosis. KCNQ1OT1-Exos had a more signi cant inhibitory effect on TNF-α-induced cytotoxicity and apoptosis compared to ADSCs-Exos. Therefore, KCNQ1OT1-Exos are expected to be superior candidates in osteoporosis treatment.

Conclusions
In conclusion, we demonstrated that ADSCs-Exos attenuated the effect of TNF-α on MC3T3-E1 cells. KCNQ1OT1-Exos had a more signi cant inhibitory effect on TNF-α-induced cytotoxicity and apoptosis compared to ADSCs-Exos. KCNQ1OT1 exerted its role by sponging miR-141-5p, suggesting that KCNQ1OT1-Exos might provide potential precise target for patients with osteoporosis. 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 e cacy based on ADSCs-Exos.

Declarations
Ethics approval The Institutional Animal Care and Use Committee of Southeast University approved the protocol for the use of animals in this study.

Consent for publication
Not applicable.

Availability of data and material
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Authors' contributions SZW performed the experiments, collected data, and drafted the manuscript. JJ particpated in the experimental design and experimentation, and helped collect the data. CHC took part in the study design and revised the manuscript. All authors read and approved the nal manuscript.