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. 4A); After 30s treatment, 1156 genes were up-regulated and 417 genes were down-regulated (Fig. 4B); 878 genes were up-regulated and 787 genes were down-regulated in the 45s-treated group (Fig. 4C). 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. 5A); 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. 5B); 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. 5C).
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. 6A); 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. 6B); 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. 6C).
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. 7A); 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. 7B); 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. 7C). 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. 8A and Fig. 8C. 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. 8B; 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. 7D. 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. 7E and 7F.
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].