Effect of low temperature plasma on the transcriptome of breast cancer cells

Effect of low temperature plasma on the transcriptome of breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of low temperature plasma on the transcriptome of breast cancer cells Lei Liu, Zhuna Yan, Fang Liu, Hongzhi Wang, Wencheng Song This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4316950/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Breast cancer is one of the most commonly diagnosed cancers in women, and due to the limitations of its therapeutic methods, new therapeutic method need to be developed. More and more evidences had shown the potential of low-temperature plasma (LTP) in cancer treatment. In this study, we investigated the inhibitory effect of LTP on the in vitro toxicity of breast cancer cells through the MTT assay, extracellular reactive oxygen species (ROS), and transcriptomics analysis. The results showed that under a certain treatment time, the cell viability was gradually decreased, and the extracellular ROS was gradually increased. In addition, the results of transcriptomics analysis showed that 1272 DEGs, 1573 DEGs, and 1272 DEGs were obtained in the 15s, 30s, and 45s treatment groups, respectively. DEGs were involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, chemical carcinogenesis-reactive oxygen species and mTOR Signaling pathway. In summary, our data suggested that LTP inhibited cell viability while inducing the activation of differentially expressed genes, representing a potential new therapy for breast cancer. LTP Transcriptomics analysis KEGG enrichment Breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights The inhibitory effect of LTP on the in vitro toxicity of breast cancer cells. DEGs were involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway. LTP inhibited cell viability while inducing the activation of differentially expressed genes. Introduction With breast cancer accounting for 11.7% of all cancers, it has already surpassed lung cancer as the main cause of cancer incidence globally in 2020 [1]. It is one of the most frequently diagnosed cancers among women in China, accounting for 19.2% of total cases [2]. Breast cancer is routinely classified based on lymph node status and distant metastasis, and includes expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2(HER2) [3]. Treatments for breast cancer are tumor-metastasized, and specifically include breast-conserving surgery [4], chemoradiotherapy [5], or drug-adjuvant therapy [6]. The current meta-analysis indicated that breast-conserving therapy had an increased risk of local recurrence [7]. As some breast cancers, such as triple-negative breast cancers, are metastatic and resistant to therapy, new treatments need to be developed [8]. However, low temperature plasma in breast cancer treatment has made significant progress [9]. Low temperature plasma (LTP) refers to the fourth state of substances other than solid, gas and liquid. When the applied voltage reaches the breakdown voltage, the gas molecules are ionized to produce a mixture including electrons, ions, atoms and radicals. It can be applied to various aspects in the biomedical field, manifested as bactericidal effect [10], wound healing [11], tumor treatment, dental treatment [12], coagulation [13], etc. Recent studies have shown that low temperature plasma (LTP) is a new alternative cancer therapy, for example, lung cancer [14], melanoma [15], pancreatic cancer [16], colorectal cancer [17], esophageal cancer [18], etc. Cancer cells are mostly killed by low temperature plasma through the production of reactive oxygen and nitrogen species (RONS). The ROS produced by cancer cells has exceeded the cell survival threshold, leading to cell death. However, the ROS produced by normal cells is below the survival threshold [4]. Low temperature plasma was found to reduce cell viability and inhibit migration activity of metastatic breast cancer cells (MDA-MB-231) and also interfere with the life process of non-metastatic breast cancer cells (MCF7) [19]. Some clinical studies have shown that low temperature plasma technology does not have serious adverse reactions and side effects. The AT3 cells were derived from a transgenic MTAG mouse and were the primary breast cancer cells of the mouse. MTAG(MMTV-PyMT/B6) mice were originally developed by specifically targeting the expression of T Ag of breast tissue-specific targeted polyomavirus using the MMTV-LTR promoter [20]. Studies have confirmed that MTAG mice are important clinical models to study the effects of breast cancer development and progression on host immune response [21]. Apoptosis is typically induced by low temperature plasma (LTP). Damage to the mitochondrial membrane potential, an elevated BAX/BCL2 ratio, and a decreased pro-caspase 3 are examples of apoptotic markers. The decrease of cell replication capacity is also indicated by the rise in the proportion of cells in the G2/M phase [22]. Experiments demonstrated that low temperature plasma could cause cancer cells to become genotoxic and cytotoxic, and DNA damage in cells was proved by comet assay [18]. Research on B16F10 melanoma cells has shown that low temperature plasma can activate the caspase 3 pathway by activating the active compounds produced by cell culture medium to achieve the goal of apoptosis [23]. At the same time, apoptosis can also be brought on by low-temperature plasma by boosting Sestrin2 expression and activating the iNOS, Fas, and p38 MAPK signaling pathways [24]. After treatment, the mitochondrial membrane potential of the cells would decrease due to the accumulation of intracellular ROS [25]. Moreover, low-temperature plasma treatment has been shown to result in a decrease of intracellular ATP and downregulation of the PI3K/AKT/mTOR and RAS/MEK pathways [26]. According to the results of RNA sequencing on A875 cells before and after treatment, cell death could be caused by low-temperature plasma through autophagy and necrosis pathways in addition to MAPK and p53 apoptotic signaling pathways [27]. Even though low temperature plasma has been applied to some studies of breast cancer cells [4, 19, 28, 29], the AT-3 cell is not being studied. Therefore, it is necessary to continue researching the impact of low temperature plasma on AT-3. The mechanism of low-temperature plasma on breast cancer cells at the transcriptome level is also unclear. In this study, transcriptome sequencing was used to investigate the specific mechanism by which diverse low-temperature plasma treatment times affect AT-3 cells. Materials and methods Cell culture The mouse breast cancer cell AT-3 was routinely cultured in a 60mm culture dish in DMEM(GIBCO, USA) containing 10% fetal bovine serum (Lonsera, shanghai), 100 U/ml penicillin, and 100µg/ml streptomycin in a humidified incubator at 37 ℃ and 5% CO 2 . Cells in logarithmic phase with the growth rate up to 80% were used for subsequent experiments. Cells not treated with low-temperature plasma were used as the control group, and cells treated with low-temperature plasma for 15 s, 30 s and 45 s were used as the experimental group. Low temperature plasma treatment The low temperature plasma equipment designed in our laboratory was schematically illustrated in Fig. 1 . Four plasma reactors were placed in a hollow quartz glass cylinder with two orifices in that reaction chamber: one for injecting experimental gases such as helium and one for exhausting gas from the reaction chamber. What's more, a ground electrode and a high-voltage electrode are included in the device, between which a low-temperature plasma with a high energy density is produced. The high-voltage electrodes were also covered with a 1mm thick quartz glass as an insulating dielectric barrier. There are four holding grooves on the base, and a culture dish with a diameter of 60mm can be placed in each groove. The gas used in the laboratory was helium with a purity of 99.999% at a gas flow rate of 1L/min. Before the start of the experiment, helium of 90s was required to exhaust the air in the reaction chamber, and then the low-temperature plasma device was turned on. During the experiment, the medium was placed in a containment vessel of the low-temperature plasma device, and the cells were treated at predetermined intervals. Cell viability experiments AT-3 cells were respectively inoculated in 60mm culture dishes at appropriate concentrations and cultivated to attachment in DMEM medium containing 10% FBS. The cells were exposed to low-temperature plasma for a predetermined period of time after reaching a 70% growth rate. After the treated cells were cultured for 24h, 1 mL of MTT working solution was added into each culture dish. After standing for 4h in the incubator, the remaining MTT was discarded, and then 1 mL of DMSO was added for full dissolution. The absorbance (OD) was then determined at 490 nm by sucking 200µl into a 96-well plate. Extracellular ROS detection The H 2 O 2 detection kit was purchased from Beyotime Biotech. AT-3 cells were inoculated into 5ml culture dishes and treated with low temperature plasma for 0s, 15s, 30s, and 45s after growing to 70%. After LTP treatment, 50µl of medium was immediately added into a 96-well plate based on culture medium, followed by 100µl of H 2 O 2 detection reagent, which was then incubated with shaking at room temperature for 30 min. Finally, the OD value was measured at 560nm to calculate the H 2 O 2 concentration in the medium. RNA extraction AT-3 cells were collected by centrifugation after LTP treatment and stored at -80°C for subsequent experiments. Total RNA was extracted from the cells in triplicate using the TRIZOL kit (Invitrogen, Carlsbad, CA, USA) according to the kit instructions. RNA integrity was assessed using the Agilent 2100 bioanalytical system (Santa Clara, CA, USA). cDNA preparation and sequence After DNase I was used to digest the total RNA, Oligo (dT) magnetic beads were used to enrich mRNA from the total RNA. The mRNA was then broken into short fragments by using a breaking reagent and used as a template to synthesize cDNA. After the preliminary synthesis of cDNA, end repair was performed, followed by the A-tailing and ligation adaptor, and the ligation product was amplified by PCR. The PCR product was cycled to obtain the final library. Single-stranded cyclic DNA molecules were replicated by rolling rings to form DNA nanospheres (DNB) for sequencing on computer. Transcriptome data analysis The data resulting from sequencing are referred to raw reads or raw data, and raw reads are subsequently controlled for suitability for subsequent analysis. After quality control, sequencing data were filtered to remove reads containing linkers, reads with an unknown base N content greater than 5%, and low-quality reads. After filtration, the obtained clean reads were aligned to the reference sequence. The second quality control was conducted to determine whether the comparison result passed the second quality control by counting the comparison rate and the distribution of reads on the reference sequence. The gene expression was examined following the comparison. The DEGs of the control group and the experimental group were screened with the corrected P value (false discovery rate, FDR ≤ 0.05) and the absolute value of the logarithm of the multiple of difference (ัlog2FoldChangeั,ัFCั) ≥ 1) as the threshold. GO enrichment and KEGG pathway enrichment were performed using the Huada multi-omics system. Results and discussion Effect of LTP on cell viability The cell viability of LTP on breast cancer cells was shown in Fig. 2 . Breast cancer cells showed time-dependent cytotoxicity at all treatment times, and the cell viability gradually decreased with the treatment time. At 45 s, cell viability had decreased to 50%. Impact of LTP on the generation of extracellular ROS The effect of LTP on extracellular ROS production in breast cancer cells is shown in Fig. 3 . LTP induced ROS production in the medium, and the extracellular ROS concentration also showed a significant increase with the increasing treatment time. control. Transcriptome comparative analysis of breast cancer cells Compared with the control cells, we found that 812 genes were up-regulated and 460 genes were down-regulated in the 15s-treated group (Fig. 4 A); After 30s treatment, 1156 genes were up-regulated and 417 genes were down-regulated (Fig. 4 B); 878 genes were up-regulated and 787 genes were down-regulated in the 45s-treated group (Fig. 4 C). With the increase of LTP treatment time, the different expressed genes were also gradually increased, indicating that LTP on AT-3 cells showed a dose-dependent damage. GO enrichment analysis of DEGs In order to reflect the impact of various low temperature plasma treatment times on breast cancer cells, GO enrichment primarily examines biological process (BP), cell composition (CC), and molecular function (MF). After treatment for 15s, BP analysis revealed that the up-regulated genes were involved in the positive regulation of protein localization, the positive regulation of cellular catabolic process, and the regulation of protein-containing complex assembly, while the down-regulated genes were involved in organelle fission, nuclear division, and chromosome segregation (Fig. 5 A); According to CC analysis, DEGs that were up-regulated were involved in the apical part of cell, nuclear envelope, and cell leading edge, whereas genes that were down-regulated were involved in the spindle, microtubule, and chromosomal region (Fig. 5 B); MF analysis showed that the up-regulated and down-regulated genes were mainly involved in the protein serine/threonine kinase activity, GTPase regulator and transcription coregulator (Fig. 5 C). In the 30s treatment group, BP analysis revealed that the up-regulated genes were involved in the regulation of the apoptotic signaling pathway, muscle tissue development, and neuron death, while the down-regulated genes were involved in histone modification. Organelle fission and small GTPase mediated signal transduction (Fig. 6 A); CC analysis showed that the up-regulated genes were involved in nuclear speck, cell leading edge, and RNA polymerase II transcription regulator complex, while the down-regulated genes were involved in cell leading edge, microtubule, and chromosomal region (Fig. 6 B); MF analysis showed that up-regulated genes were mainly involved in ubiquitin-like protein ligase binding, DNA-binding transcription repressor activity, mRNA binding, while down-regulated genes were mainly involved in GTPase regulator activity. transcription coregulator activity, tubulin binding (Fig. 6 C). In the 45s treatment group, BP analysis revealed that the up-regulated genes were primarily connected to the regulation of cell growth, the regulation of apoptotic signaling pathway, and myeloid cell differentiation, while the down-regulated genes were involved in histone modification, Small GTPase mediated signal transduction, and positive regulation of cell projection organization (Fig. 7 A); CC analysis showed that the up-regulated genes were involved in ribosome, protein-DNA complex, ribosomal subunit; while the down-regulated genes were involved in microtubule, cell leading edge, and chromosomal region (Fig. 7 B); MF analysis showed that the up-regulated genes were mainly involved in ubiquitin-like protein transfer activity, while the down-regulated genes were involved in the transcription coregulator activity, GTPase regulator activity, nucleoside-triphosphatase regulator activity (Fig. 7 C). The above results indicated that the down-regulated genes in the three experimental groups all involved the expression of GTPase family. Compared with healthy tissues, the GTPase family showed a significant increase in expression in breast cancer [30]. Studies have shown that GTPase family may be related to cell migration and cell cycle process [31]. These results suggested that LTP may have the ability to inhibit the migration of breast cancer cells and decrease GTPase expression. KEGG pathway enrichment analysis of DEGs Through KEGG pathway enrichment analysis, the most significant signaling pathway for DEGs can be discovered. The pathway is considered to be highly enriched when the p value is less than 0.05. The MAPK signaling pathway, transcriptional misregulation in cancer, and FoxO signaling pathway were found to be highly enriched in the up-regulated genes in the 15s and 30s treatment groups after comparison of all experimental groups with the control group, as shown in Fig. 8 A and Fig. 8 C. In addition, the down-regulated genes in the 15s-treated group were mainly concentrated in cell cycle, Wnt signaling pathway, and Hippo signaling pathway, as shown in Fig. 8 B; In the 30s treatment group, down-regulated genes were mainly enriched in Rap 1 signaling pathway, TGF-beta signaling pathway, and Herpes simplex virus 1 infection, as shown in Fig. 7 D. After treatment for 45s, the main pathways for up-regulated genes were enriched in ribosome, Transcriptional misregulation in cancer and chemical carcinogenesis-reactive oxygen species, but the main pathway for down-regulated genes were enriched in herpes simplex virus 1 infection, and mTOR Signaling pathway, as shown in Fig. 7 E and 7 F. The MAPK signaling pathway is involved in cancer-related cellular activities, including proliferation, differentiation, apoptosis and inflammation [32]. The MAPK signaling pathways were mainly divided into three subfamilies, named p38, extracellular signal-regulated protein kinase (ERK), and the Jun N-terminal kinases (JNK) [33]. Akter et al. demonstrated that when U87 MG cells were treated with LTP, cell proliferation was effectively inhibited and the expressions of apoptotic proteins and MAPK-related proteins (including p38, cleaved caspase-3, and PARP) were significantly increased [34]. This is similar to the results of this study. FoxO signaling pathway is involved in such biological processes as apoptosis, cell cycle arrest and antioxidant stress [35]. Previous studies have reported that up-regulation of FoxO signaling pathway can significantly inhibit cell proliferation, cell invasion, and promote apoptosis [36]. Analysis of 14 published GWAS data sets by Wang et al. suggested that FBXO32 in the FoxO pathway might play a protective role in breast cancer risk [37]. The Wnt signaling pathway is a highly conservative signaling pathway. According to whole genome sequencing and gene expression profile analysis, researchers have found that the Wnt signaling pathway is involved in the proliferation and metastasis of breast cancer [38]. In addition, studies have confirmed that under the interaction of PROX1 and hnRNPK, the Wnt signaling pathway can be activated to promote the invasion and metastasis of breast cancer [39]. Paclitaxel combined with XAV939 can induce apoptosis and inhibit Wnt signaling pathway [40]. In the breast cancer cells treated for 15s, the down-regulated genes were concentrated in the Wnt signaling pathway, indicating that low temperature plasma effectively inhibited the migration of cancer cells. Transforming growth factor β(TGF-β), as a pluripotent cytokine and multifunctional growth factor, plays an important role in the normal breast development and breast cancer [41]. After resveratrol was applied to MDA231 cells, it was found that resveratrol could reverse TGF-β1 and inhibit the migration of MDA231 cells [42]. The down-regulated differential genes in the cells treated for 30s were enriched in TGF-β signaling pathway, indicating that low temperature plasma could reduce the expression of TGF-β signaling pathway to a certain extent. ROS is a group of short-lived, highly reactive, oxygen-containing molecules that can induce DNA damage [43]. In addition, ROS could also trigger oxidative stress to promote cell death [44]. Studies had confirmed that LTP can inhibit multiple myeloma and prolong survival time by inducing ROS production and inhibiting Notch signaling pathway [45]. In this experiment, it was found that the active oxygen content increased with the treatment time, but the cell viability decreased, which was consistent with the previous study. Besides, plasma activation medium (PAM) at a certain ratio can also reduce cell viability and change the mTOR pathway, which is crucial for cancer cell viability [46]. Conclusion In this study, the transcriptomics method was mainly used to investigate the effect of LTP on AT-3 in breast cancer cells. With the increase of treatment time, it was found that the cell viability was gradually decreased, while the extracellular ROS showed an increasing trend. GO enrichment was analyzed from three aspects: biological process (BP), cellular component (CC), and molecular function (MF). In the 15s treatment group, there were 812 up-regulated genes and 460 down-regulated genes; in the 30s treatment group, there were 1156 up-regulated genes and 417 down-regulated genes; and in the 45s treatment group, there were 878 up-regulated genes and 787 down-regulated genes. The KEGG pathway enrichment analysis indicated that DEGs was involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, chemical carcinogenesis-reactive oxygen species and mTOR Signaling pathway. As a result, LTP significantly inhibited the growth of breast cancer, which provided a theoretical basis for future research. Declarations Authorship contributions Lei Liu and Zhuna Yan contributed equally to this paper. Lei Liu: Writing–original draft, Validation, Investigation . Zhuna Yan: Writing–original draft , Validation, Investigation . Fang Liu: Validation, Investigation . Hongzhi Wang: Writing – review & editing, Validation . Wencheng Song: Writing – review & editing, Validation, Supervision, Project administration, Investigation, Funding acquisition. Conflicts of interest All authors declare that they have no actual or potential conflicts. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Financial support This study was supported by the Natural Science Foundation of China (21876179, 22142007 and 22006001), the Anhui Province University Natural Science Research Project (KJ2021A1452, 2023AH04019，2022AH040277, 2022AH052058), the Key projects of Anhui province university outstanding youth talent support program (gxyqZD2021144), and the Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions. Ethical approval and consent to participate Not applicable. 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Molecules (Basel, Switzerland), 27:5832 Yoshikawa N, Liu W, Nakamura K, Yoshida K, Ikeda Y, Tanaka H (2020) Plasma-activated medium promotes autophagic cell death along with alteration of the mTOR pathway. Sci Rep 10:1614 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4316950","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299616773,"identity":"823ed186-de00-4fe5-991c-a9820474f6a2","order_by":0,"name":"Lei Liu","email":"","orcid":"","institution":"Anhui Vocational and Technical College","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Liu","suffix":""},{"id":299616775,"identity":"907ee9f5-6580-4532-9f65-82c732531bf2","order_by":1,"name":"Zhuna Yan","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhuna","middleName":"","lastName":"Yan","suffix":""},{"id":299616777,"identity":"b7effa5e-cf4e-4c80-8f05-7a5b09d42f16","order_by":2,"name":"Fang Liu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Liu","suffix":""},{"id":299616779,"identity":"15a9af61-bd66-4c1b-a553-841a9ef94a3f","order_by":3,"name":"Hongzhi Wang","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hongzhi","middleName":"","lastName":"Wang","suffix":""},{"id":299616781,"identity":"5e23bf49-de03-41e7-8bae-578f5e055f49","order_by":4,"name":"Wencheng Song","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDADftK1SDYAiQMkaTE4QKwWg+O9Bz/+qLhjt/n46bTHHxjs5BnYz+LXaXDmXLI0z5lnydvO5G4H2pRs2MCTl4BXi9mNHDNmxrbDyWY3eLdJHGBgTmCQ4DHAr+X+GzPGn0AtxjPAWuqJ0HKDx4yBt+2wnYEEWMthwlrsz+QYA/1yOEEC5JczBscN23hy8GuRbD9jCAyxw/b87We3PaioqJbnZz+DXwsMJDYwMLABAxBMEgfsGUhQPApGwSgYBSMMAABU1UO6jfgDXwAAAABJRU5ErkJggg==","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Wencheng","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2024-04-24 09:11:51","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4316950/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4316950/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56075364,"identity":"5575b148-fbca-4515-9af8-27007d99e9f3","added_by":"auto","created_at":"2024-05-08 08:24:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":113944,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic diagram of the LTP device. (B) Diagram of the LTP device in the laboratory.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/1e6a017903bd7655e9596c1d.png"},{"id":56075366,"identity":"5f59406f-e860-4626-aa18-bb24c9ef97c9","added_by":"auto","created_at":"2024-05-08 08:24:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65151,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability assay on AT-3 cells. At 24 hours after LTP treatment, cell viability was measured by the MTT assay. Data represent the mean ± SD of three independent experiments. \u003csup\u003e∗\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e∗∗\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e∗∗∗\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 with ANOVA compared with the control.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/93102151e94b938c64c036b6.png"},{"id":56075841,"identity":"8289e74f-ed4d-4303-a684-d64fc789d102","added_by":"auto","created_at":"2024-05-08 08:32:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":70859,"visible":true,"origin":"","legend":"\u003cp\u003eThe concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the cell culture medium after LTP treatment for 0 s, 15 s, 30 s, and 45 s. Data represent the mean ± SD of three independent experiments. \u003csup\u003e∗\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e∗∗\u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, and \u003csup\u003e∗∗∗\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 with ANOVA compared with the\u003c/p\u003e\n\u003cp\u003econtrol.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/7cda2f8c1eb1ad58cc5d727c.png"},{"id":56075363,"identity":"3d93c373-3a01-454e-bb03-d1633bc5e157","added_by":"auto","created_at":"2024-05-08 08:24:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95775,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential expressed genes volcano map. (A) 15s VS 0s; (B) 30s VS 0s; (C) 45s VS 0s. The up-regulated genes are indicated by red dots and down-regulated genes by green dots.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/cf088f287dbe4cfd3d7c3fb5.png"},{"id":56076555,"identity":"bcb6c158-0eca-42cc-84ab-e0374b919a8c","added_by":"auto","created_at":"2024-05-08 08:40:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":424277,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment of 15s treatment group on AT-3 cells. (A) biological process; (B) cell composition; (C) molecular function.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/1136f04862b7ab7246a91c7f.png"},{"id":56075370,"identity":"f5a788fa-b65a-47f4-93e6-6d6b8fb5099a","added_by":"auto","created_at":"2024-05-08 08:24:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":333048,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment of 30s treatment group on AT-3 cells. (A) biological process; (B) cell composition; (C) molecular function.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/56f780f3c46b28697410f063.png"},{"id":56075368,"identity":"6f59f301-5306-4125-a407-0cb6b06cb53f","added_by":"auto","created_at":"2024-05-08 08:24:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":356346,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment of 45s treatment group on AT-3 cells. (A) biological process; (B) cell composition; (C) molecular function.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/93e8f0447cd3860731ed4aa5.png"},{"id":56075369,"identity":"38ca1b9a-ba56-4a99-8726-64dc1c1ff41d","added_by":"auto","created_at":"2024-05-08 08:24:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":287322,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment analysis. (A) KEGG enrichment of up-regulated genes in the 15s treatment group; (B) KEGG enrichment of down-regulated genes in the 15s treatment group; (C) KEGG enrichment of up-regulated genes in the 30s treatment group (D) KEGG enrichment of down-regulated genes in the 30s treatment group (E) KEGG enrichment of up-regulated genes in the 45s treatment group (F) KEGG enrichment of down-regulated genes in the 45s treatment group\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/d4e29496ccfb05237c5568a9.png"},{"id":69299448,"identity":"65e9a3b5-5cb6-4c2d-af3d-6be99cd547b3","added_by":"auto","created_at":"2024-11-19 02:23:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2193050,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4316950/v1/2363a822-5838-48a9-b4d1-cd536f3b6155.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of low temperature plasma on the transcriptome of breast cancer cells","fulltext":[{"header":"Highlights","content":"\u003cp\u003eThe inhibitory effect of LTP on the in vitro toxicity of breast cancer cells.\u003c/p\u003e\n\u003cp\u003eDEGs were involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway.\u003c/p\u003e\n\u003cp\u003eLTP inhibited cell viability while inducing the activation of differentially expressed genes.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eWith breast cancer accounting for 11.7% of all cancers, it has already surpassed lung cancer as the main cause of cancer incidence globally in 2020 [1]. It is one of the most frequently diagnosed cancers among women in China, accounting for 19.2% of total cases [2]. Breast cancer is routinely classified based on lymph node status and distant metastasis, and includes expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2(HER2) [3]. Treatments for breast cancer are tumor-metastasized, and specifically include breast-conserving surgery [4], chemoradiotherapy [5], or drug-adjuvant therapy [6]. The current meta-analysis indicated that breast-conserving therapy had an increased risk of local recurrence [7]. As some breast cancers, such as triple-negative breast cancers, are metastatic and resistant to therapy, new treatments need to be developed [8]. However, low temperature plasma in breast cancer treatment has made significant progress [9].\u003c/p\u003e \u003cp\u003eLow temperature plasma (LTP) refers to the fourth state of substances other than solid, gas and liquid. When the applied voltage reaches the breakdown voltage, the gas molecules are ionized to produce a mixture including electrons, ions, atoms and radicals. It can be applied to various aspects in the biomedical field, manifested as bactericidal effect [10], wound healing [11], tumor treatment, dental treatment [12], coagulation [13], etc. Recent studies have shown that low temperature plasma (LTP) is a new alternative cancer therapy, for example, lung cancer [14], melanoma [15], pancreatic cancer [16], colorectal cancer [17], esophageal cancer [18], etc. Cancer cells are mostly killed by low temperature plasma through the production of reactive oxygen and nitrogen species (RONS). The ROS produced by cancer cells has exceeded the cell survival threshold, leading to cell death. However, the ROS produced by normal cells is below the survival threshold [4]. Low temperature plasma was found to reduce cell viability and inhibit migration activity of metastatic breast cancer cells (MDA-MB-231) and also interfere with the life process of non-metastatic breast cancer cells (MCF7) [19]. Some clinical studies have shown that low temperature plasma technology does not have serious adverse reactions and side effects. The AT3 cells were derived from a transgenic MTAG mouse and were the primary breast cancer cells of the mouse. MTAG(MMTV-PyMT/B6) mice were originally developed by specifically targeting the expression of T Ag of breast tissue-specific targeted polyomavirus using the MMTV-LTR promoter [20]. Studies have confirmed that MTAG mice are important clinical models to study the effects of breast cancer development and progression on host immune response [21]. Apoptosis is typically induced by low temperature plasma (LTP). Damage to the mitochondrial membrane potential, an elevated BAX/BCL2 ratio, and a decreased pro-caspase 3 are examples of apoptotic markers. The decrease of cell replication capacity is also indicated by the rise in the proportion of cells in the G2/M phase [22]. Experiments demonstrated that low temperature plasma could cause cancer cells to become genotoxic and cytotoxic, and DNA damage in cells was proved by comet assay [18]. Research on B16F10 melanoma cells has shown that low temperature plasma can activate the caspase 3 pathway by activating the active compounds produced by cell culture medium to achieve the goal of apoptosis [23]. At the same time, apoptosis can also be brought on by low-temperature plasma by boosting Sestrin2 expression and activating the iNOS, Fas, and p38 MAPK signaling pathways [24]. After treatment, the mitochondrial membrane potential of the cells would decrease due to the accumulation of intracellular ROS [25]. Moreover, low-temperature plasma treatment has been shown to result in a decrease of intracellular ATP and downregulation of the PI3K/AKT/mTOR and RAS/MEK pathways [26]. According to the results of RNA sequencing on A875 cells before and after treatment, cell death could be caused by low-temperature plasma through autophagy and necrosis pathways in addition to MAPK and p53 apoptotic signaling pathways [27].\u003c/p\u003e \u003cp\u003eEven though low temperature plasma has been applied to some studies of breast cancer cells [4, 19, 28, 29], the AT-3 cell is not being studied. Therefore, it is necessary to continue researching the impact of low temperature plasma on AT-3. The mechanism of low-temperature plasma on breast cancer cells at the transcriptome level is also unclear. In this study, transcriptome sequencing was used to investigate the specific mechanism by which diverse low-temperature plasma treatment times affect AT-3 cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe mouse breast cancer cell AT-3 was routinely cultured in a 60mm culture dish in DMEM(GIBCO, USA) containing 10% fetal bovine serum (Lonsera, shanghai), 100 U/ml penicillin, and 100\u0026micro;g/ml streptomycin in a humidified incubator at 37 ℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells in logarithmic phase with the growth rate up to 80% were used for subsequent experiments. Cells not treated with low-temperature plasma were used as the control group, and cells treated with low-temperature plasma for 15 s, 30 s and 45 s were used as the experimental group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eLow temperature plasma treatment\u003c/h2\u003e \u003cp\u003eThe low temperature plasma equipment designed in our laboratory was schematically illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Four plasma reactors were placed in a hollow quartz glass cylinder with two orifices in that reaction chamber: one for injecting experimental gases such as helium and one for exhausting gas from the reaction chamber. What's more, a ground electrode and a high-voltage electrode are included in the device, between which a low-temperature plasma with a high energy density is produced. The high-voltage electrodes were also covered with a 1mm thick quartz glass as an insulating dielectric barrier. There are four holding grooves on the base, and a culture dish with a diameter of 60mm can be placed in each groove. The gas used in the laboratory was helium with a purity of 99.999% at a gas flow rate of 1L/min. Before the start of the experiment, helium of 90s was required to exhaust the air in the reaction chamber, and then the low-temperature plasma device was turned on. During the experiment, the medium was placed in a containment vessel of the low-temperature plasma device, and the cells were treated at predetermined intervals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability experiments\u003c/h2\u003e \u003cp\u003eAT-3 cells were respectively inoculated in 60mm culture dishes at appropriate concentrations and cultivated to attachment in DMEM medium containing 10% FBS. The cells were exposed to low-temperature plasma for a predetermined period of time after reaching a 70% growth rate. After the treated cells were cultured for 24h, 1 mL of MTT working solution was added into each culture dish. After standing for 4h in the incubator, the remaining MTT was discarded, and then 1 mL of DMSO was added for full dissolution. The absorbance (OD) was then determined at 490 nm by sucking 200\u0026micro;l into a 96-well plate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExtracellular ROS detection\u003c/h2\u003e \u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detection kit was purchased from Beyotime Biotech. AT-3 cells were inoculated into 5ml culture dishes and treated with low temperature plasma for 0s, 15s, 30s, and 45s after growing to 70%. After LTP treatment, 50\u0026micro;l of medium was immediately added into a 96-well plate based on culture medium, followed by 100\u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detection reagent, which was then incubated with shaking at room temperature for 30 min. Finally, the OD value was measured at 560nm to calculate the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration in the medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction\u003c/h2\u003e \u003cp\u003eAT-3 cells were collected by centrifugation after LTP treatment and stored at -80\u0026deg;C for subsequent experiments. Total RNA was extracted from the cells in triplicate using the TRIZOL kit (Invitrogen, Carlsbad, CA, USA) according to the kit instructions. RNA integrity was assessed using the Agilent 2100 bioanalytical system (Santa Clara, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ecDNA preparation and sequence\u003c/h2\u003e \u003cp\u003eAfter DNase I was used to digest the total RNA, Oligo (dT) magnetic beads were used to enrich mRNA from the total RNA. The mRNA was then broken into short fragments by using a breaking reagent and used as a template to synthesize cDNA. After the preliminary synthesis of cDNA, end repair was performed, followed by the A-tailing and ligation adaptor, and the ligation product was amplified by PCR. The PCR product was cycled to obtain the final library. Single-stranded cyclic DNA molecules were replicated by rolling rings to form DNA nanospheres (DNB) for sequencing on computer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome data analysis\u003c/h2\u003e \u003cp\u003eThe data resulting from sequencing are referred to raw reads or raw data, and raw reads are subsequently controlled for suitability for subsequent analysis. After quality control, sequencing data were filtered to remove reads containing linkers, reads with an unknown base N content greater than 5%, and low-quality reads. After filtration, the obtained clean reads were aligned to the reference sequence. The second quality control was conducted to determine whether the comparison result passed the second quality control by counting the comparison rate and the distribution of reads on the reference sequence.\u003c/p\u003e \u003cp\u003eThe gene expression was examined following the comparison. The DEGs of the control group and the experimental group were screened with the corrected P value (false discovery rate, FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05) and the absolute value of the logarithm of the multiple of difference (ัlog2FoldChangeั,ัFCั)\u0026thinsp;\u0026ge;\u0026thinsp;1) as the threshold. GO enrichment and KEGG pathway enrichment were performed using the Huada multi-omics system.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffect of LTP on cell viability\u003c/h2\u003e \u003cp\u003eThe cell viability of LTP on breast cancer cells was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Breast cancer cells showed time-dependent cytotoxicity at all treatment times, and the cell viability gradually decreased with the treatment time. At 45 s, cell viability had decreased to 50%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImpact of LTP on the generation of extracellular ROS\u003c/h2\u003e \u003cp\u003eThe effect of LTP on extracellular ROS production in breast cancer cells is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. LTP induced ROS production in the medium, and the extracellular ROS concentration also showed a significant increase with the increasing treatment time.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003econtrol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome comparative analysis of breast cancer cells\u003c/h2\u003e \u003cp\u003eCompared with the control cells, we found that 812 genes were up-regulated and 460 genes were down-regulated in the 15s-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA); After 30s treatment, 1156 genes were up-regulated and 417 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB); 878 genes were up-regulated and 787 genes were down-regulated in the 45s-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). With the increase of LTP treatment time, the different expressed genes were also gradually increased, indicating that LTP on AT-3 cells showed a dose-dependent damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGO enrichment analysis of DEGs\u003c/h2\u003e \u003cp\u003eIn order to reflect the impact of various low temperature plasma treatment times on breast cancer cells, GO enrichment primarily examines biological process (BP), cell composition (CC), and molecular function (MF). After treatment for 15s, BP analysis revealed that the up-regulated genes were involved in the positive regulation of protein localization, the positive regulation of cellular catabolic process, and the regulation of protein-containing complex assembly, while the down-regulated genes were involved in organelle fission, nuclear division, and chromosome segregation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA); According to CC analysis, DEGs that were up-regulated were involved in the apical part of cell, nuclear envelope, and cell leading edge, whereas genes that were down-regulated were involved in the spindle, microtubule, and chromosomal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB); MF analysis showed that the up-regulated and down-regulated genes were mainly involved in the protein serine/threonine kinase activity, GTPase regulator and transcription coregulator (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the 30s treatment group, BP analysis revealed that the up-regulated genes were involved in the regulation of the apoptotic signaling pathway, muscle tissue development, and neuron death, while the down-regulated genes were involved in histone modification. Organelle fission and small GTPase mediated signal transduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA); CC analysis showed that the up-regulated genes were involved in nuclear speck, cell leading edge, and RNA polymerase II transcription regulator complex, while the down-regulated genes were involved in cell leading edge, microtubule, and chromosomal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB); MF analysis showed that up-regulated genes were mainly involved in ubiquitin-like protein ligase binding, DNA-binding transcription repressor activity, mRNA binding, while down-regulated genes were mainly involved in GTPase regulator activity. transcription coregulator activity, tubulin binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the 45s treatment group, BP analysis revealed that the up-regulated genes were primarily connected to the regulation of cell growth, the regulation of apoptotic signaling pathway, and myeloid cell differentiation, while the down-regulated genes were involved in histone modification, Small GTPase mediated signal transduction, and positive regulation of cell projection organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA); CC analysis showed that the up-regulated genes were involved in ribosome, protein-DNA complex, ribosomal subunit; while the down-regulated genes were involved in microtubule, cell leading edge, and chromosomal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB); MF analysis showed that the up-regulated genes were mainly involved in ubiquitin-like protein transfer activity, while the down-regulated genes were involved in the transcription coregulator activity, GTPase regulator activity, nucleoside-triphosphatase regulator activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The above results indicated that the down-regulated genes in the three experimental groups all involved the expression of GTPase family. Compared with healthy tissues, the GTPase family showed a significant increase in expression in breast cancer [30]. Studies have shown that GTPase family may be related to cell migration and cell cycle process [31]. These results suggested that LTP may have the ability to inhibit the migration of breast cancer cells and decrease GTPase expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eKEGG pathway enrichment analysis of DEGs\u003c/h2\u003e \u003cp\u003eThrough KEGG pathway enrichment analysis, the most significant signaling pathway for DEGs can be discovered. The pathway is considered to be highly enriched when the p value is less than 0.05. The MAPK signaling pathway, transcriptional misregulation in cancer, and FoxO signaling pathway were found to be highly enriched in the up-regulated genes in the 15s and 30s treatment groups after comparison of all experimental groups with the control group, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC. In addition, the down-regulated genes in the 15s-treated group were mainly concentrated in cell cycle, Wnt signaling pathway, and Hippo signaling pathway, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB; In the 30s treatment group, down-regulated genes were mainly enriched in Rap 1 signaling pathway, TGF-beta signaling pathway, and Herpes simplex virus 1 infection, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD. After treatment for 45s, the main pathways for up-regulated genes were enriched in ribosome, Transcriptional misregulation in cancer and chemical carcinogenesis-reactive oxygen species, but the main pathway for down-regulated genes were enriched in herpes simplex virus 1 infection, and mTOR Signaling pathway, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF.\u003c/p\u003e \u003cp\u003eThe MAPK signaling pathway is involved in cancer-related cellular activities, including proliferation, differentiation, apoptosis and inflammation [32]. The MAPK signaling pathways were mainly divided into three subfamilies, named p38, extracellular signal-regulated protein kinase (ERK), and the Jun N-terminal kinases (JNK) [33]. Akter et al. demonstrated that when U87 MG cells were treated with LTP, cell proliferation was effectively inhibited and the expressions of apoptotic proteins and MAPK-related proteins (including p38, cleaved caspase-3, and PARP) were significantly increased [34]. This is similar to the results of this study. FoxO signaling pathway is involved in such biological processes as apoptosis, cell cycle arrest and antioxidant stress [35]. Previous studies have reported that up-regulation of FoxO signaling pathway can significantly inhibit cell proliferation, cell invasion, and promote apoptosis [36]. Analysis of 14 published GWAS data sets by Wang et al. suggested that FBXO32 in the FoxO pathway might play a protective role in breast cancer risk [37]. The Wnt signaling pathway is a highly conservative signaling pathway. According to whole genome sequencing and gene expression profile analysis, researchers have found that the Wnt signaling pathway is involved in the proliferation and metastasis of breast cancer [38]. In addition, studies have confirmed that under the interaction of PROX1 and hnRNPK, the Wnt signaling pathway can be activated to promote the invasion and metastasis of breast cancer [39]. Paclitaxel combined with XAV939 can induce apoptosis and inhibit Wnt signaling pathway [40]. In the breast cancer cells treated for 15s, the down-regulated genes were concentrated in the Wnt signaling pathway, indicating that low temperature plasma effectively inhibited the migration of cancer cells. Transforming growth factor β(TGF-β), as a pluripotent cytokine and multifunctional growth factor, plays an important role in the normal breast development and breast cancer [41]. After resveratrol was applied to MDA231 cells, it was found that resveratrol could reverse TGF-β1 and inhibit the migration of MDA231 cells [42]. The down-regulated differential genes in the cells treated for 30s were enriched in TGF-β signaling pathway, indicating that low temperature plasma could reduce the expression of TGF-β signaling pathway to a certain extent. ROS is a group of short-lived, highly reactive, oxygen-containing molecules that can induce DNA damage [43]. In addition, ROS could also trigger oxidative stress to promote cell death [44]. Studies had confirmed that LTP can inhibit multiple myeloma and prolong survival time by inducing ROS production and inhibiting Notch signaling pathway [45]. In this experiment, it was found that the active oxygen content increased with the treatment time, but the cell viability decreased, which was consistent with the previous study. Besides, plasma activation medium (PAM) at a certain ratio can also reduce cell viability and change the mTOR pathway, which is crucial for cancer cell viability [46].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, the transcriptomics method was mainly used to investigate the effect of LTP on AT-3 in breast cancer cells. With the increase of treatment time, it was found that the cell viability was gradually decreased, while the extracellular ROS showed an increasing trend. GO enrichment was analyzed from three aspects: biological process (BP), cellular component (CC), and molecular function (MF). In the 15s treatment group, there were 812 up-regulated genes and 460 down-regulated genes; in the 30s treatment group, there were 1156 up-regulated genes and 417 down-regulated genes; and in the 45s treatment group, there were 878 up-regulated genes and 787 down-regulated genes. The KEGG pathway enrichment analysis indicated that DEGs was involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, chemical carcinogenesis-reactive oxygen species and mTOR Signaling pathway. As a result, LTP significantly inhibited the growth of breast cancer, which provided a theoretical basis for future research.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eAuthorship contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLei Liu\u003c/strong\u003e and \u003cstrong\u003eZhuna Yan\u003c/strong\u003e contributed equally to this paper.\u003cstrong\u003e\u0026nbsp;Lei Liu:\u0026nbsp;\u003c/strong\u003eWriting\u0026ndash;original draft, Validation, Investigation\u003cstrong\u003e. \u0026nbsp;Zhuna Yan:\u0026nbsp;\u003c/strong\u003eWriting\u0026ndash;original draft\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003eValidation, Investigation\u003cstrong\u003e. Fang Liu:\u0026nbsp;\u003c/strong\u003eValidation, Investigation\u003cstrong\u003e. \u0026nbsp;Hongzhi Wang:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing, Validation\u003cstrong\u003e. Wencheng Song:\u0026nbsp;\u003c/strong\u003eWriting\u0026nbsp;\u0026ndash;\u0026nbsp;review \u0026amp; editing, Validation, Supervision, Project administration, Investigation, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no actual or potential conflicts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of China (21876179, 22142007 and 22006001), the Anhui Province University Natural Science Research Project (KJ2021A1452, 2023AH04019，2022AH040277, 2022AH052058), the Key projects of Anhui province university outstanding youth talent support program (gxyqZD2021144), and the Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Sci Rep 10:1614 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"LTP, Transcriptomics analysis, KEGG enrichment, Breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-4316950/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4316950/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancer is one of the most commonly diagnosed cancers in women, and due to the limitations of its therapeutic methods, new therapeutic method need to be developed. More and more evidences had shown the potential of low-temperature plasma (LTP) in cancer treatment. In this study, we investigated the inhibitory effect of LTP on the in vitro toxicity of breast cancer cells through the MTT assay, extracellular reactive oxygen species (ROS), and transcriptomics analysis. The results showed that under a certain treatment time, the cell viability was gradually decreased, and the extracellular ROS was gradually increased. In addition, the results of transcriptomics analysis showed that 1272 DEGs, 1573 DEGs, and 1272 DEGs were obtained in the 15s, 30s, and 45s treatment groups, respectively. DEGs were involved in MAPK signaling pathway, Fox O signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, chemical carcinogenesis-reactive oxygen species and mTOR Signaling pathway. In summary, our data suggested that LTP inhibited cell viability while inducing the activation of differentially expressed genes, representing a potential new therapy for breast cancer.\u003c/p\u003e","manuscriptTitle":"Effect of low temperature plasma on the transcriptome of breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 08:24:44","doi":"10.21203/rs.3.rs-4316950/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a640909d-9a6a-4229-b8ea-952116e85af1","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-19T02:23:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-08 08:24:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4316950","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4316950","identity":"rs-4316950","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}