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Ju et al. Cell Biosci (2021) 11:30 https://doi.org/10.1186/s13578-021-00545-0 Cell & Bioscience RESEARCH Open Access Transcriptomic and metabolomic profling reveal the p53-dependent benzeneacetic acid attenuation of silica‐induced epithelial– mesenchymal transition in human bronchial epithelial cells Zhao Ju1, Jianlin Shao2, Meiling Zhou1, Jing Jin1, Huiji Pan1, Ping Ding1 and Ruixue Huang1* Abstract Background: Silica exposure underlies the development of silicosis, one of the most serious occupational hazards worldwide. We aimed to explore the interaction of the silica-induced epithelial–mesenchymal transition (EMT)-related transcripts with the cellular metabolism regulated by p53. Methods: We knocked out p53 using CRISPR/Cas9 in the human bronchial epithelial (HBE) cell line. The transcrip- tomic and metabolomic analyses and integrative omics were conducted using microarrays, GC–MS, and MetaboAna- lyst, respectively. Results: Fifty-two mRNAs showed signifcantly altered expression in the HBE p53-KO cells post-silica exposure. A total of 42 metabolites were putatively involved in p53-dependent silica-mediated HBE cell dysfunction. Through integrated data analysis, we obtained fve signifcant p53-dependent metabolic pathways including phenylalanine, glyoxylate, dicarboxylate, and linoleic acid metabolism, and the citrate cycle. Through metabolite screening, we fur- ther identifed that benzeneacetic acid, a key regulation metabolite in the phenylalanine metabolic pathway, attenu- ated the silica-induced EMT in HBE cells in a p53-dependent manner. Interestingly, despite the extensive p53-related published literature, the clinical translation of these studies remains unsubstantial. Conclusions: Our study ofers new insights into the molecular mechanisms by which epithelial cells respond to silica exposure and provide fresh perspective and direction for future clinical biomarker research and potential clinically sustainable and translatable role of p53. Keywords: p53, Silica, EMT Introduction Silica, a carcinogenic substance, is most commonly associated with a variety of lung diseases including sili- cosis, lung cancer risk, and the development of autoim- mune alteration [1, 2]. Occupational exposure to silica *Correspondence: [email protected] in the mining industry has been observed for almost 150 1 Department of Occupational and Environmental Health, Xiangya School of Public Health, Central South University, Changsha 410078, Hunan, years [3]. Massive inhalation of silica-based particles China leads to lung fbrosis and lung dysfunction. However, at Full list of author information is available at the end of the article present, the rapid increase in the global population and © The Author(s) 2021. 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 link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ju et al. Cell Biosci (2021) 11:30 Page 2 of 18 the corresponding growth in economic development Although certain studies of p53 involvement in silica- have exponentially increased the number of work envi- induced diseases had valuable discoveries, there remains ronments with silica exposure [4–7]. In the United States much to learn about the roles and regulation of p53 from (1999–2018), the number of silicosis-related deaths other new viewpoints. Recently, owing to the develop- decreased by 40.4 % [8], whereas globally, the numbers ment of transcriptomics and metabolomics technology, increased by 66.0% from 36,000 to 1990 to 60,000 in 2017 an increasing number of new p53 functions have been [9]. China has reported the highest number (640,000) of discovered. Wang et al. indicated that a critical metabo- silicosis cases over the past three decades [10]. Barnes lite, 20(S)-protopanaxatriol, promoted p53 binding with et al. noted that silica-associated silicosis is a persistent DNA to form an efective anticancer network through old-world occupational hazard in modern industries [11]. multi-omics detection and analysis [19]. Another study Although the research in the past 20 years has examined also used integrative omics analysis to obtain a more the role of silica dust in the development of silicosis, lung in-depth understanding of p53-dependent regulation of injury, and autoimmune diseases, our progress has been metabolism, providing a more comprehensive view of limited by the following questions: (1) How does silica its roles in metabolism [20]. Our previous study revealed exposure lead to varied prognoses and clinical charac- that through integrative transcriptomics and metabo- teristics?; (2) What are the potential mechanisms of lung lomics analysis, in response to radiation insult, p53 may injury caused by new sources of silica exposure?; (3) How dysregulate nitrogen, glutathione, and arachidonic acid exactly have studies tried to translate to clinic in silica- metabolism, and glycolysis or gluconeogenesis [21]. Tis induced diseases? Our focus in this feld at present is to indicates the importance of p53-regulated metabolism identify evidence that was either previously ignored or in response to environmental toxins and their insults. missing instead of trying to approach research with mot- Hence, we hypothesized that in the silica insulation, lung tos such as “100 % preventive” or “zero tolerance in the epithelial cells may be regulated through p53-dependent workplace” [12]. Consequently, a better understanding metabolic pathways, which may be afected by p53 and of the pathogenesis of silica-induced diseases would help contribute to the silica-induced lung fbrosis. To verify discover more precise cellular targets and active signaling this, the human bronchial epithelial (HBE) cell line was pathways to decipher the natural silica-associated lung used to knockout p53 using CRISPR/Cas9. Integrative diseases. transcriptomic and metabolomic analyses were per- Te p53 protein was discovered in 1979 [13] in nor- formed. Moreover, since p53 research is a hotspot and mal and cancerous cells through four diferent groups in attracted major attention in scientifc community, to fur- England, the United States and France simultaneously. ther illustrate the p53 research trends globally and pro- Over the past 40 years, p53 has attracted considerable vide more information for translating p53 research into attention and fascination due to its fundamental and clinical application, we also present research and funding important role in gene regulation that safeguards cells trends over the past 10 years. against hazardous insults [14]. According to the search results in PubMed (https ://pubme d.ncbi.nlm.nih.gov/), Methods and materials more than 100,000 hits were found for literature related Tis study has three parts. Te frst part was the tran- to p53 (cumulative of epidemiological and molecular scriptomics and metabolomics analysis, the second part mechanistic research). A typical fnding is that p53 func- was the cell mechanism experiment, and the third was tion is context-dependent, dependent on the stresses, the p53 research trends analysis. activating diferent signaling pathways, and cell states [15]. Indeed, more than 50% of human cancers illus- Cell line, construction of p53‑knockout (p53‑KO) cell line trates p53 mutation, and some of mutant p53 proteins and multi‐omics detection not lose activity but acquire oncogenic function [14]. Te HBE cell line was purchased from ATCC (American p53 is also involved in silica-induced silicosis progres- Type Culture Collection, USA) and stored under the con- sion. Cheng et al. found that circRNA-012091 promotes ditions described in our published literature [21, 22]. Te lung fbroblast proliferation and migration by regulating P53-KO cells were constructed using the CRISPR/Cas9 a major member of the p53 family, PPP1R13B, in L929 technique from Syngentech Ltd. (Beijing, China). Te cells [16]. Another study indicated that silica treatment construction details and constructive efciency were pre- in lung fbroblasts increased p53 expression, enhanc- sented in our previous report. ing cell migration through activation of the p53/PUMA Prior to multi-omics detection, HBE cells were divided signaling pathway [17]. Wang et al. considered that the into two groups: the p53-wild type (p53-wt) and p53-KO abnormal regulation of p53 by silica might contribute groups. Cells in the two groups were harvested 24 h to the development of lung cancer and lung cancer [18]. post 12.5 µg/mL silica treatment. Cells were shipped to Ju et al. Cell Biosci (2021) 11:30 Page 3 of 18 OE Biotech Ltd. (Shanghai, China) for further transcrip- (Bio-Rad Laboratories, Hercules, CA, USA), to validate tomic

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