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Effects of Simulated Microgravity on the Expression Profiles of RNA Effects of simulated microgravity on the expression profiles of RNA during osteogenic differentiation of human bone marrow mesenchymal stem cells Ping Chen, Emory University L Li, Zhejiang University C Zhang, Zhejiang University F-F Hong, Zhejiang University J-F Wang, Zhejiang University Journal Title: CELL PROLIFERATION Volume: Volume 52, Number 2 Publisher: WILEY | 2019-03-01, Pages e12539-e12539 Type of Work: Article Publisher DOI: 10.1111/cpr.12539 Permanent URL: https://pid.emory.edu/ark:/25593/vk2g5 Final published version: http://dx.doi.org/10.1111/cpr.12539 Accessed September 28, 2021 3:40 PM EDT Received: 5 July 2018 | Revised: 16 August 2018 | Accepted: 4 September 2018 DOI: 10.1111/cpr.12539 ORIGINAL ARTICLE Effects of simulated microgravity on the expression profiles of RNA during osteogenic differentiation of human bone marrow mesenchymal stem cells Liang Li1 | Cui Zhang1 | Jian‐ling Chen1 | Fan‐fan Hong1 | Ping Chen2 | Jin‐fu Wang1 1Institute of Cell and Development Biology, College of Life Sciences, Zhejiang University, Abstract Hangzhou, China Objectives: Exposure to microgravity induces many adaptive and pathological 2 Departments of Cell Biology and changes in human bone marrow mesenchymal stem cells (hBMSCs). However, the Otolaryngology, Emory University School of Medicine, Atlanta, Georgia underlying mechanisms of these changes are poorly understood. We revealed the gene expression patterns of hBMSCs under normal ground (NG) and simulated mi‐ Correspondence Jin‐Fu Wang, Institute of Cell and crogravity (SMG), which showed an interpretation for these changes by gene regula‐ Development Biology, College of Life tion and signal pathways analysis. Sciences, Zhejiang University, Hangzhou, China. Materials and methods: In this study, hBMSCs were osteogenically induced for 0, 2, Email: [email protected] 7 and 14 days under normal ground gravity and simulated microgravity, followed by and Ping Chen, Departments of Cell Biology and analysis of the differences in transcriptome expression during osteogenic differentia‐ Otolaryngology, Emory University School of tion by RNA sequencing and some experimental verification for these results. Medicine, Atlanta, Georgia. Email: [email protected] Results: The results indicated that 837, 399 and 894 differentially expressed genes (DEGs) were identified in 2, 7 and 14 days samples, respectively, out of which 13 Funding information National Basic Research Program of China, genes were selected for qRT-PCR analysis to confirm the RNA-sequencing results. Grant/Award Number: 2014CB541705; After analysis, we found that proliferation was inhibited in the early stage of induc‐ Strategically Guiding Scientific Special Project from Chinese Academy of Sciences, tion. In the middle stage, osteogenic differentiation was inhibited, whereas adipo‐ Grant/Award Number: XDA04020202- genic differentiation benefited from SMG. Moreover, SMG resulted in the 23; National Development Program of Important Scientific Instrument, Grant/ up‐regulation of genes specific for tumorigenesis in the later stage. Award Number: 2013YQ030595; TZ-1 Conclusion: Our data revealed that SMG inhibits the proliferation and inhibits the Application Program, Grant/Award Number: KYTZ01-0901-FB-003; Chinese National differentiation towards osteoblasts but promotes adipogenesis. SMG also selects Science Foundation, Grant/Award Number: highly tumorigenic cells for survival under prolonged SMG. U1738102, 81570932; Chinese Academy of Sciences 1 | INTRODUCTION stem cells (hBMSCs) into osteoblasts detected by a decrease in ALPL, COL1A1, SPARC and RunX2.3 Under microgravity, osteoblasts show Previous studies have shown that microgravity induces significant shorter and wavier microtubules, smaller and fewer focal adhesions, bone loss in weight‐bearing bones at a rate of approximately 1%‐2% and thinner cortical actin and stress fibres. Simultaneously, micro‐ per month.1 Bone loss under microgravity may be due to the inhibi‐ gravity causes an increase in bone resorption by osteoclasts and tion of osteoblast development and the increase in osteoclastic ac‐ a decrease in osteoblast cellular integrity.4 Microgravity also has a tivity.2 Exposure to microgravity for a few days has shown a marked significant effect on cell proliferation. Mouse embryonic stem (ES) suppression of differentiation of human bone marrow mesenchymal cells cultured under the SMG condition had a significantly reduced This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd. Cell Proliferation. 2019;52:e12539. wileyonlinelibrary.com/journal/cpr | 1 of 11 https://doi.org/10.1111/cpr.12539 2 of 11 | LI ET AL. total cell number compared to cells cultured under normal ground 2.3 | RNA extraction, sequencing and analysis gravity (NG).5 Another study found that the proliferation of murine bone marrow stromal cells was also inhibited in space flight.6 It has RNA from cells was extracted using Trizol reagent (Sigma). All been reported that cells under SMG are more flattened and reached RNA samples were detected and analysed in Vazyme Biotech Co. confluence at a lower cell density.7 (Nanjing, China). RNA integrity and concentration were detected Random positioning machine (RPM) is a kind of 3D clinostat that by 1.2% agarose gel electrophoresis and Agilent 2100 Bioanalyzer. provides continuous random changes in orientation relative to the cDNA libraries were constructed with rRNA elimination and se‐ gravity vector, which can be used to simulate microgravity.8 In this quenced by the Illumina Hiseq X Ten platform as 150-bp pair- study, we used an RPM to simulate microgravity and evaluated the ended reads. Reads were aligned using Hisat2 (v2.0.5, Center for effects of SMG on whole gene expression during osteogenesis of Computational Biology, Johns Hopkins University, Baltimore, MD, hBMSCs. We performed an RNA-seq analysis of hBMSCs induced USA). FPKM estimation was performed with Cuffdiff (v1.3.0, Cole osteogenically under SMG and NG, and clustered differentially ex‐ Trapnell’s lab at the University of Washington, Seattle, WA, USA), pressed genes (DEGs) into several functional groups to analyse the aligned reads were counted with HTSeq and differential expres‐ impacts of SMG on the physiology of hBMSCs. sion analysis was performed with Cuffdiff (v2.2.1). Differentially expressed genes were selected using a cut‐off at a P value of less than 0.05 (FDR adjusted for multiple testing) and the absolute 2 | MATERIALS AND METHODS value of log2 (fold change) greater than or equal to 1. The data discussed in this publication have been deposited in NCBI’s Gene 2.1 | Preparation of hBMSCs Expression Omnibus13,14 and are accessible through GEO Series This experiment was performed in accordance with standard ethical accession number GSE114117 (https://www.ncbi.nlm.nih.gov/geo/ guidelines and approved by the Institutional Review Board and the query/acc.cgi). Ethical Committee of the First Affiliate Hospital, Zhejiang University, China. Informed consent was obtained from all the healthy donors: 2.4 | Validation of gene expression by qRT‐PCR one male donor aged 23 and two female donors aged 19 and 34. Whole bone marrow samples were collected at the First Affiliate Reverse transcription of 1 μg of total RNA was performed using Hospital, and hBMSCs were isolated, cultured and passaged as previ‐ the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, 9 ously described. Cells of 3rd passage were used for the experiments, Shanghai, China). Primers (Table S1) were designed using Primer and the cells examined for the expression profile of special surface an‐ Premier 6.0 Demo (PREMIER Biosoft, Palo Alto, CA, USA) and − − − − − − + tigens expressed CD19 , CD34 , CD14 , CD45 , HLA , DR , CD105 , Oligo 7.36 Demo software (Molecular Biology Insights, Inc., + + + CD90 , CD73 and CD29 (Figure S1). In addition, these cells had os‐ Colorado Springs, CO, USA). Quantitative real-time PCR reactions teogenic and adipogenic potentials indicating that the cells prepared using the LightCycler® 480 SYBR Green qPCR Supermix (Roche, 10,11 from human bone marrow can be accepted as hBMSCs (Figure S2). Shanghai, China) were performed in the CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Berkeley, CA) with 18S rRNA as an internal control. The thermal cycler protocol in‐ 2.2 | Microgravity simulation and induction with cluded denaturation at 95°C for 1 minute followed by 40 cycles of random position machine 95°C for 10 second, 56°C for 30 second and 72°C for 20 second. The SM-31 random position machine (RPM) (Chinese Academy of Quantification of selected gene expression was performed using Science, Beijing, China) was used to simulate microgravity condi‐ the relative quantitation (2−ΔΔCT) method. All experiments were tion. It was operated in random modes of speed (0.1‐10 rpm) inside performed in triplicate, and three independent experiments were a CO2 incubator (5% CO2, 37°C and 100% humidity). After the cells performed. of 3rd passage reached 60%-70% confluency, the medium was re‐ placed with osteogenic medium12 (L-Dulbecco’s modified Eagle’s 2.5 | Cell proliferation assay and cell cycle analysis medium (DMEM; Life Technologies, Shanghai, China) supple‐ by PI staining mented with 10% FBS, 50 μg/mL L‐ascorbic acid (Sigma, Shanghai, China), 10 mmol/L β‐glycerophosphate (Sigma), 0.1
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