Materials Express 2158-5849/2020/10/1836/010 Copyright © 2020 by American Scientific Publishers All rights reserved. doi:10.1166/mex.2020.1822 Printed in the United States of America www.aspbs.com/mex Upregulation of signal transducer and activator of transcription 4 promotes osteoblast activity by activating AMP-activated protein kinase based on cationic liposome transfection Tao Jiang1,4,†, Qingzhen Chen1,2,†,MinShao2,∗, Zhen Shen3, Gang Wang3, Qinsheng Wang2, and Zhenming Zeng2 1The Third Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, PR China 2Department of Orthopedics, The Third Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510240, Guangdong, PR China 3The First Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, PR China 4 Department of Orthopedics, GuangdongIP: 192.168.39.151 Second Traditional On: Thu, Chinese 30 Sep Medicine 2021 19:20:15 Hospital, Guangzhou 510095, Guangdong, PR China Copyright: American Scientific Publishers Delivered by Ingenta Article ABSTRACT Activation of Protein Kinase AMP-Activated Catalytic Subunit Alpha (AMPK) is an important regulatory path- way for osteogenic differentiation. STAT4 acts as a transcriptional activity factor to regulate the transcription of many genes and is potentially a regulatory factor for AMPK transcription activity. To confirm the regulatory effect of STAT4 on AMPK and the effect of STAT4 on osteogenic differentiation, the promoter sequence of AMPK was analyzed via bioinformatics, the STAT4 overexpression vector was constructed and transfected into human osteoblast-like cells MG-63 by cationic liposome, fluorescence quantitative PCR (RT-qPCR) and western blotting technologies were used to detect the effect of STAT4 on the expression of AMPK.MTT and ALP activity assays were also used to verify the effect of STAT4 on the proliferation and maturation of osteoblasts by regulating AMPK expression. Our results showed that STAT4 was a co-transcriptional regu- lator of AMPK1 and AMPK2, which combined the enrichment region of CpG on the promoter sequence of AMPK1/2. Overexpression of STAT4 significantly increased the expression of AMPK1 and AMPK2, which promoted the proliferation and maturation of osteoblasts. We concluded that STAT4 was a transcriptional activa- tor of AMPK and promoting STAT4 expression enhances the proliferation and differentiation activity of AMPK in osteoblasts. Keywords: STAT4, AMPK, Bioinformatics Analysis, Osteogenic Differentiation. 1. INTRODUCTION cause of osteoporosis. Bone formation concerns the growth The discrepancy in bone formation and bone resorption and differentiation of osteoblasts [1–3]. Many studies have processes, caused by metabolic disorders, is an important found that the activation of AMP-activated protein kinase ∗Author to whom correspondence should be addressed. (AMPK) plays a key role in osteogenic differentiation, †These two authors contributed equally to this work. which is due to the regulation of energy metabolism 1836 Mater. Express, Vol. 10, No. 11, 2020 Upregulation of STAT4 promotes osteoblast activity by activating AMPK Materials Express Jiang et al. by AMPK, which affects bone metabolism processes 2. MATERIALS AND METHODS and thus influences osteogenic differentiation [4–9]. For 2.1. Bioinformatic Analysis of Promoter Sequences example, Ginsenoside Rd increases the phosphorylation Common databases such as NCBI and Ensembl were of AMPK to induce differentiation and calcification of used to find gene information of AMPKá1/2. The pro- osteoblasts, inhibition of AMPK expression, and activation moters of common genes were about 1,000 bp before and which significantly affects the differentiation of MC3T3- 1,000 bp after the transcriptional initiation sites. Using E1 cells [8]. AMPK is the core regulatory pathway in the websites BDGP and FPROM, the target gene pro- the process of osteogenic differentiation. It is impor- moters were predicted from the promoter region found tant to explore the mechanism of AMPK in osteogenic using Ensembl. The methylation sites in the promoter differentiation to contribute to the prevention and region were predicted by the websites Ensembl, CpG treatment of osteoporosis. Island, CpG Finder, EMBOSS, and GPMiner to pre- AMPK displays a significant difference in transcription dict the DNA methylation regulatory region. GPMiner level in patients with osteoporosis, which may be related and ROM were used to predict transcription binding to signal transducer and activator of transcription (STAT) factors in this promoter region as well as target gene family proteins. Generally speaking, STAT proteins com- transcription activating factors and their action sites bine with CpG islands, reduce levels of gene methyla- (Table I). tion, and regulate gene transcription levels; In addition, the STAT family translocates into the nucleus after dimer 2.2. Construction of STAT4 Overexpression Plasmid formation or other activation forms, directly targets the Vector pcDNA3.1(+)-STAT4 target gene transcription initiation region, and activates Full length CDS sequences of STAT4 and the transcription activity. However, there are few studies con- pcDNA3.1+ plasmid vector (Invitrogen, Carlsbad, CA, cerning the transcriptional activators regulating AMPK. In USA) labeled with enhanced green fluorescent protein this study, through bioinformatic analysis of the AMPK (EGFP) gene were digested by HindIII and BamHI promoter sequence, we screened the transcriptional activa- (TAKARA, Tokyo, Japan) at 37 C for 1 hour. T4 Article tors regulating the transcription level of AMPK and veri- DNA ligase (NEB, Hitchin, Herts, UK) was used to fied them at the cell level. In addition,IP: 192.168.39.151 the STAT signaling On: Thu,react 30 Sep at 252021C 19:20:15 for 10 minutes to connect the target pathway is a transcription activator, whichCopyright: correlates American with Scientificgene and Publishers pcDNA3.1(+) plasmid vector. The recombinant the occurrence and development of osteoporosis.Delivered When bypcDNA3.1( Ingenta +)-STAT4 plasmid vector was transfected into the STAT signaling pathway is inhibited, the expression of DH5á competent cells (TAKARA, Tokyo, Japan). Posi- BMP9 and ALP increased, which promotes bone forma- tive clones were selected for amplification. The plasmid- tion [10–14]. STAT4 is predicted to be a potential tran- carrier was extracted via plasmid extraction kit (Thermo scriptional activator of AMPK, but its effect on AMPK Fisher Scientific, Waltham, MA, USA). Subsequently, dou- and osteogenic differentiation needs further clarification. ble digestion and DNA sequencing were performed to Cationic liposomes [15] are effective and important identify the plasmid vectors. materials for the analysis of gene function and they have been widely used in to study osteogenic ability in 2.3. Cell Culture and Plasmid Vector Transfection vitro. Therefore, in this study, cationic liposomes were MG-63 cells were cultured with minimum essential used to transfer STAT4 recombinant gene into MG-63 medium (MEM) including 10% Fetal Bovine Serum and cells, interfere with the expression of STAT4, detect the 1% Penicillin-Streptomycin solution, at 37 C and 5% effect of STAT4 differential expression on AMPK expres- CO2. After the cell fusion rate was above 80%, adher- sion, and ultimately explore its effect on osteogenesis ent cells were made into a single cell suspension via in vitro. hatching with trypsin for 3 minutes, and then subcultured Table I. Websites of various online websites. Website Link address Ensembl http://asia.ensembl.org/Homo_sapiens/Gene/Summary?db=core;g=ENSG00000132356;r=5:40759379-40798374 FPROM http://www.softberry.com/cgi-bin/programs/promoter/fprom.pl BDGP http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl CpG Island https://sites.ualberta.ca/∼stothard/javascript/cpg_islands.html CpG Finder http://www.softberry.com/cgi-bin/programs/promoter/cpgfinder.pl EMBOSS https://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=emboss_cpgplot-I20190829-034717-0929-71406869-p2m GPMiner http://gpminer.mbc.nctu.edu.tw/show_prediction/show.php?OS=human&ID=20190829_112548&scale=3&GC_window=15& TFBS_core=1.00&TFBS_matrix=0.95&OR_zscore=5&OR_number=20&OR_occur=2&stability_window=15& miRNA_MFE=&miRNA_score= Mater. Express, Vol. 10, 2020 1837 Materials Express Upregulation of STAT4 promotes osteoblast activity by activating AMPK Jiang et al. every three days. The plasmid vectors were transfected Electrophoresis was performed at 100 V constant pres- into MG-63 cells by cationic liposomes (Life Technolo- sure to separate the protein and ran for 2 h. Following an gies Corporation, Carlsbad, CA, USA), according to the ice bath and 300 mA of constant current, the protein was instructions [16]. After 24 hours, the biological functions shifted from the SDS-PAGE gel to the nitrocellulose (NC) of the cells and the differences in target gene expression membrane. 5% skimmed milk powder (prepared with Tris- were detected. buffered saline, 0.1% Tween 20 (TBST)) was incubated at 25 C for 1.5 hours, and 1× TBST was washed three times 2.4. Transmission Electron Microscope (TEM) for 5 minutes each time. Specific first antibodies (1:1,000) 200 ng/mL of cationic liposome solution were prepared, were added to incubate overnight at 4 C. Goat anti-rabbit of which 1 L was inserted into a TEM. Under the work- secondary antibody (1:10,000) (Boster Biological Technol- ing condition of electron microscope, the system pressure ogy Co. Ltd., Wuhan,