Huang et al. Experimental & Molecular Medicine (2021) 53:894–906 https://doi.org/10.1038/s12276-021-00621-y Experimental & Molecular Medicine ARTICLE Open Access PAX8-AS1 knockdown facilitates cell growth and inactivates autophagy in osteoblasts via the miR-1252-5p/GNB1 axis in osteoporosis Caiqiang Huang1,RunguangLi 2, Changsheng Yang1,RuiDing1,QingchuLi1, Denghui Xie3, Rongkai Zhang3 and Yiyan Qiu1 Abstract Osteoporosis (OP) is the most common systematic bone disorder among elderly individuals worldwide. Long noncoding RNAs (lncRNAs) are involved in biological processes in various human diseases. It has been previously revealed that PAX8 antisense RNA 1 (PAX8-AS1) is upregulated in OP. However, its molecular mechanism in OP remains unclear. Therefore, we specifically designed this study to determine the specific role of PAX8-AS1 in OP. We first established a rat model of OP and then detected PAX8-AS1 expression in the rats with RT-qPCR. Next, to explore the biological function of PAX8-AS1 in osteoblasts, in vitro experiments, such as Cell Counting Kit-8 (CCK-8) assays, flow cytometry, western blotting and immunofluorescence (IF) staining, were conducted. Subsequently, we performed bioinformatic analysis and luciferase reporter assays to predict and identify the relationships between microRNA 1252-5p (miR-1252-5p) and both PAX8-AS1 and G protein subunit beta 1 (GNB1). Additionally, rescue assays in osteoblasts clarified the regulatory network of the PAX8-AS1/miR-1252-5p/GNB1 axis. Finally, in vivo loss-of-function studies verified the role of PAX8-AS1 in OP progression. The results illustrated that PAX8-AS1 was upregulated in the proximal tibia of OP 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; rats. PAX8-AS1 silencing promoted the viability and inhibited the apoptosis and autophagy of osteoblasts. PAX8-AS1 interacted with miR-1252-5p. GNB1 was negatively regulated by miR-1252-5p. In addition, the impacts of PAX8-AS1 knockdown on osteoblasts were counteracted by GNB1 overexpression. PAX8-AS1 depletion suppressed OP progression by inhibiting apoptosis and autophagy in osteoblasts. In summary, PAX8-AS1 suppressed the viability and activated the autophagy of osteoblasts via the miR-1252-5p/GNB1 axis in OP. Introduction current aging societies2. OP is a degenerative bone disease Osteoporosis (OP), the most common systematic bone characterized by excessive bone resorption and decreased disorder in elderly individuals1, has a high incidence in bone formation, which lead to deterioration of bone – microstructure3 5. Bone remodeling caused by the action of osteoblasts can repair microfractures and adapt bones – Correspondence: Yiyan Qiu ([email protected]) to mechanical loads and strains6 8. However, deregulation 1 Division of Spine Surgery, Section II, Department of Orthopedics, The Third of bone remodeling can lead to bone diseases, including Affiliated Hospital of Southern Medical University, Guangdong Provincial Key Laboratory of Bone and Joint Degeneration Diseases, Southern Medical OP. Autophagy is defined as a natural process of self University, Academy of Orthopedics of Guangdong Province, Guangzhou, cannibalization that maintains cellular homeostasis by Guangdong, China 2 degrading and recycling abnormal cell organelles or Division of Foot and Ankle Surgery, Department of Orthopedics, The Third 9 Affiliated Hospital of Southern Medical University, Guangdong Provincial Key macromolecules . Autophagy is active during the Laboratory of Bone and Joint Degeneration Diseases, Southern Medical maturation of bone tissue and bone cells and has been University, Academy of Orthopedics of Guangdong Province, Guangzhou, revealed to be associated with OP10. Accumulating evi- Guangdong, China Full list of author information is available at the end of the article dence has demonstrated that autophagy exerts an These authors contributed equally: Caiqiang Huang, Runguang Li © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Korean Society for Biochemistry and Molecular Biology Huang et al. Experimental & Molecular Medicine (2021) 53:894–906 895 – influence on OP11 14. Regulation of autophagy is thus a ovaries. An AAV vector was generated after cloning vital factor for OP. However, this mechanism has been small interfering RNA (siRNA) fragments targeting incompletely explored in OP. PAX8-AS1 into the adenoviral vector GV478 (Genechem Long noncoding RNAs (lncRNAs) are a group of Co., Ltd., Shanghai, China) as previously described25. noncoding RNAs with a length of 200 or more nucleo- Rats were injected intraarticularly with this solution with tides15. It has been revealed that lncRNAs play crucial 33-gauge needles (Hamilton Company, Bonaduz, Swit- roles in bone disease16.Forinstance,thelncRNANKILA zerland) and 25-µl CASTIGHT syringes (Hamilton regulates osteogenesis by modulating the RXFP1/AKT Company). All procedures were conducted following the signaling pathway17. Another study found that the recommendations in the Guide for the Care and Use of lncRNA HOTAIRM1 targets the JNK/AP-1 signaling Laboratory Animals (8th edition, 2011, National pathwaytoregulateosteogenicdifferentiation18.More- Research Council). After being fed for another 6 weeks, over, the lncRNA HIF1A-AS2 promotes osteogenic dif- the rats were sacrificed by cervical vertebra dislocation, ferentiation of ADSCs via PI3K/Akt signaling19.RNA and the proximal tibia and skull were excised and sequencing results have demonstrated that the lncRNA separated. All experimental procedures in the experi- PAX8 antisense RNA 1 (PAX8-AS1) is upregulated in ments were approved by the Animal Experimental Ethics peripheral whole blood of patients with OP20. However, Committee of The Third Affiliated Hospital of Southern the molecular mechanism of PAX8-AS1 in OP remains Medical University. to be fully explored. Numerous studies have elucidated that lncRNAs bind Cell culture and conditioning to microRNAs (miRNAs) as competing endogenous The skulls of sham and OVX rats were separated under RNAs (ceRNAs), thereby affecting and regulating the aseptic conditions and were then covered and digested expression of mRNAs21. These lncRNA-mediated ceRNA several times with type I collagenase, and the super- networks have been revealed to exert profound influ- natant was removed. The collected cells were washed ences on certain diseases. For example, the lncRNA twice using Hank’s solution and were then suspended in ANRIL promotes lymphangiogenesis during the diabetic Dulbecco’smodified Eagle’smedium(DMEM,GIBCO, wound healing process by binding with miR-181a to Grand Island, NY, USA) containing 10% fetal bovine upregulate Prox122.Anotherstudyrevealedthatthe serum (FBS, Sigma–Aldrich, Shanghai, China) and 1% lncRNA GAS5 suppresses adipogenic differentiation of penicillin–streptomycin (Sigma–Aldrich) at 37 °C in 5% 23 MSCs via the miR-18a/CTGF axis . Additionally, CO2. When cell-cell contact was observed, the cells were lncRNA-1604 regulates neural differentiation by spong- passaged to remove fibroblasts. The cell suspension was ing miR-200c and targeting ZEB24. These reports transferred to a glass culture bottle, incubated for prompted us to investigate whether the lncRNA PAX8- 10 min, and then transferred to another culture bottle. AS1 regulates the development of OP as a ceRNA; thus, This procedure was repeated two or three times. Finally, we designed this study. the osteoblasts were suspended in culture medium. In the current study, we assumed that PAX8-AS1 may function as a ceRNA and further explored the role and Cell transfection molecular mechanism of PAX8-AS1 in OP. Our report The PAX8-AS1 knockdown vector (si-PAX8-AS1) or its may provide a novel therapeutic strategy for OP. negative control vector (si-NC), the G protein subunit beta 1 (GNB1) overexpression vector (pcDNA3.1-GNB1) Materials and methods or its negative control vector (pcDNA3.1-vector), and the Animal grouping and study design microRNA 1252-5p (miR-1252-5p) mimic vector (miR- Twenty four 12 week old female Sprague–Dawley rats 1252-5p) or its negative control vector (NC mimics) were (105–145 g) were purchased from Cavens Lab Animal synthesized by GenePharma (Shanghai, China) and were Ltd. (Changzhou, China). Rats were housed in individual then transfected into primary osteoblasts. All transfec- cages at 26 °C on a 12-h light/dark cycle (relative tions were performed using Lipofectamine 2000 (Invi- humidity 50–65%). During the experiments, the trogen, Carlsbad, CA, USA) for 48 h according to the “reduction, replacement, and refinement” animal welfare manufacturer’s instructions. principle was obeyed. Rats were randomly divided into the sham group (sham, n = 12) and ovariectomized Real time quantitative polymerase