Ferroptosis Altered microRNAs Expression in HT-1080 Fibrosarcoma Cells Based on Small RNA Sequencing and Bioinformatics Analysis
<p>The mechanisms of iron and miRNAs regulating ferroptosis. Ferroptosis is an iron-dependent non-apoptotic form of cell death and is regulated by iron metabolism, lipid metabolism, glutathione-GPX4 pathway, glutamate/cystine transport, and other processes. miRNAs influence ferroptosis by regulating the above processes. Cys—cystine; Glu—glutamate; GPX4—glutathione peroxidase 4; GSH—glutathione; GSSG—glutathione disulfide; miRNAs—microRNAs; PUFAs—polyunsaturated fatty acids; ROS—reactive oxygen species; TF—transferrin; and TfR1—transferrin receptor 1. By Figdraw.</p> "> Figure 2
<p>Ferroptosis cell model establishment. (<b>A</b>) Cell viability of HT-1080 cells treated with ferric ammonium citrate (FAC) for 24 h using CCK-8 assay. (<b>B</b>–<b>F</b>) Morphology of HT-1080 cells treated with FAC under different concentrations (0, 2, 4, 6, and 8 mM) for 24 h, scale bar = 100 μm. Data are presented as mean ± SD (<span class="html-italic">n</span> = 6). <sup>a, b, c, d</sup> Values of the bars without a common letter differ significantly at <span class="html-italic">p</span> < 0.05.</p> "> Figure 3
<p>Differentially expressed miRNAs (DE-miRNAs) between the control group (CO) and iron group (IO). (<b>A</b>) Volcano plot. Red color represents significantly up-regulated miRNAs, and green color represents significantly down-regulated miRNAs (<span class="html-italic">p</span> adj < 0.05). Blue color represents miRNAs with no significance. (<b>B</b>) Hierarchical clustering analysis of relatively high expression miRNAs (red) and relatively low expression miRNAs (blue).</p> "> Figure 4
<p>Validation of DE-miRNAs in HT-1080 cells between the control group (CO) and the iron group (IO) using qRT-PCR. (<b>A</b>–<b>D</b>) Up-regulated DE-miRNAs: miR-3529-3p (<b>A</b>), miR-425-5p (<b>B</b>), miR-26b-5p (<b>C</b>), and miR-22-3p (<b>D</b>). (<b>E</b>–<b>H</b>) Down-regulated DE-miRNAs: miR-518c-5p (<b>E</b>), miR-16-2-3p (<b>F</b>), miR-125b-1-3p (<b>G</b>), and miR-744-5p (<b>H</b>). Data are represented as the mean ± SD (<span class="html-italic">n</span> = 3), * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01; ns, not significant.</p> "> Figure 5
<p>Predicted transcription factors (TFs) of DE-miRNAs. EGR1—early growth response 1; FOXO1—forkhead box O1; MEF2A—myocyte enhancer factor 2A; POU2F1—POU class 2 homeobox 1; RREB1—Ras-responsive element binding protein 1, SP1—Sp1 transcription factor; SP4—Sp4 transcription factor; TAL1—basic helix-loop-helix (bHLH) transcription factor 1; TCF3—transcription factor 3; and ZFP161—zinc finger protein 161.</p> "> Figure 6
<p>The Venn diagram of the predicted target genes using miRDB, TargetScan, and miRBase databases.</p> "> Figure 7
<p>Gene ontology (GO) enrichment analysis of target genes. (<b>A</b>) Bar plot. (<b>B</b>) Scatter plot. The diameter of a point represents the number of target genes enriched in a specific item, and the adjusted <span class="html-italic">p</span>-value represents the degree of enrichment.</p> "> Figure 8
<p>Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of target genes. (<b>A</b>) Bar plot. (<b>B</b>) Scatter plot. The diameter of a point represents the number of target genes enriched in a specific item, and the adjusted <span class="html-italic">p</span>-value represents the degree of enrichment.</p> "> Figure 9
<p>PPI network of hub genes. Red indicates a higher degree of nodes in the PPI network.</p> "> Figure 10
<p>Network of interaction between DE-miRNAs and target genes via Cytoscape. Red color represents up-regulated DE-miRNAs, green color represents down-regulated DE-miRNAs, and blue color represents target genes.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Cell Culture
2.2. Construction of Ferroptosis Model in HT-1080 Cells
2.3. Total RNA Extraction
2.4. Small RNA Sequencing
2.5. Differential Expression of miRNAs
2.6. Prediction of Transcription Factors (TFs) and Target Genes
2.7. Functional Annotation and Pathway Enrichment
2.8. Protein–Protein Interaction (PPI) Network Establishment and Analysis of Hub Genes
2.9. Validation
2.10. Statistical Analysis
3. Results
3.1. Ferroptosis Cell Model Establishment
3.2. Overview of Small RNA Sequencing
3.3. miRNA Differential Expression Analysis
3.4. Validation of DE-miRNAs
3.5. Prediction of TFs of DE-miRNAs
3.6. Prediction of Target Genes of DE-miRNAs
3.7. GO and KEGG Enrichment Analyses
3.8. Construction of Target Genes PPI Network and Hub Genes Network
3.9. Construction of the DE-miRNAs-Target Genes Regulatory Network
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
DE-miRNA | differentially expressed miRNA |
FAC | ferric ammonium citrate |
GO | Gene ontology |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
miRNA | microRNA |
PPI | protein–protein interaction |
ROS | reactive oxygen species |
TF | transcription factor |
References
- Bogdan, A.R.; Miyazawa, M.; Hashimoto, K.; Tsuji, Y. Regulators of iron homeostasis: New players in metabolism, cell death, and disease. Trends Biochem. Sci. 2016, 41, 274–286. [Google Scholar] [CrossRef]
- Van Swelm, R.P.L.; Wetzels, J.F.M.; Swinkels, D.W. The multifaceted role of iron in renal health and disease. Nat. Rev. Nephrol. 2020, 16, 77–98. [Google Scholar] [CrossRef]
- Ren, Y.; Li, S.; Song, Z.; Luo, Q.; Zhang, Y.; Wang, H. The regulatory roles of polysaccharides and ferroptosis-related phytochemicals in liver diseases. Nutrients 2022, 14, 2303. [Google Scholar] [CrossRef]
- Fuhrmann, D.C.; Mondorf, A.; Beifuß, J.; Jung, M.; Brüne, B. Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis. Redox Biol. 2020, 36, 101670. [Google Scholar] [CrossRef]
- Dixon, S.J.; Lemberg, K.M.; Lamprecht, M.R.; Skouta, R.; Zaitsev, E.M.; Gleason, C.E.; Patel, D.N.; Bauer, A.J.; Cantley, A.M.; Yang, W.S.; et al. Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell 2012, 149, 1060–1072. [Google Scholar] [CrossRef]
- Wang, Y.; Lv, M.; Zhao, W. Research on ferroptosis as a therapeutic target for the treatment of neurodegenerative diseases. Ageing Res. Rev. 2023, 91, 102035. [Google Scholar] [CrossRef]
- Li, Y.; Ran, Q.; Duan, Q.; Jin, J.; Wang, Y.; Yu, L.; Wang, C.; Zhu, Z.; Chen, X.; Weng, L.; et al. 7-Dehydrocholesterol dictates ferroptosis sensitivity. Nature 2024, 626, 411–418. [Google Scholar] [CrossRef]
- Tong, X.; Tang, R.; Xiao, M.; Xu, J.; Wang, W.; Zhang, B.; Liu, J.; Yu, X.; Shi, S. Targeting cell death pathways for cancer therapy: Recent developments in necroptosis, pyroptosis, ferroptosis, and cuproptosis research. J. Hematol. Oncol. J 2022, 15, 174. [Google Scholar] [CrossRef]
- Chan, J.J.; Tay, Y. Noncoding RNA: RNA regulatory networks in cancer. Int. J. Mol. Sci. 2018, 19, 1310. [Google Scholar] [CrossRef]
- Ha, M.; Kim, V.N. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 2014, 15, 509–524. [Google Scholar] [CrossRef]
- Zhao, Z.; Sun, W.; Guo, Z.; Zhang, J.; Yu, H.; Liu, B. Mechanisms of lncRNA/microRNA interactions in angiogenesis. Life Sci. 2020, 254, 116900. [Google Scholar] [CrossRef]
- Bartel, D.P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004, 116, 281–297. [Google Scholar] [CrossRef]
- Jiang, Z.; Zhou, J.; Deng, J.; Li, L.; Wang, R.; Han, Y.; Zhou, J.; Tao, R.; Peng, L.; Wang, D.; et al. Emerging roles of ferroptosis-related miRNAs in tumor metastasis. Cell Death Discov. 2023, 9, 193. [Google Scholar] [CrossRef]
- Jin, S.; Liu, P.S.; Zheng, D.; Xie, X. The interplay of miRNAs and ferroptosis in diseases related to iron overload. Apoptosis 2024, 29, 45–65. [Google Scholar] [CrossRef]
- Davis, M.; Clarke, S. Influence of microRNA on the maintenance of human iron metabolism. Nutrients 2013, 5, 2611–2628. [Google Scholar] [CrossRef]
- Li, Y.; Lin, L.; Li, Z.; Ye, X.; Xiong, K.; Aryal, B.; Xu, Z.; Paroo, Z.; Liu, Q.; He, C.; et al. Iron homeostasis regulates the activity of the microRNA pathway through poly(C)-binding protein 2. Cell Metab. 2012, 15, 895–904. [Google Scholar] [CrossRef]
- Stockwell, B.R.; Angeli, J.P.F.; Bayir, H.; Bush, A.I.; Conrad, M.; Dixon, S.; Fulda, S.; Gascon, S.; Hatzios, S.K.; Kagan, V.; et al. Ferroptosis: A regulated cell death nexus linking metabolism, redox biology, and disease. Cell 2017, 171, 273–285. [Google Scholar] [CrossRef]
- Fang, S.; Yu, X.; Ding, H.; Han, J.; Feng, J. Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors. Biochem. Biophys. Res. Commun. 2018, 503, 297–303. [Google Scholar] [CrossRef]
- Wen, M.; Shen, Y.; Shi, S.; Tang, T. miREvo: An integrative microRNA evolutionary analysis platform for next-generation sequencing experiments. BMC Bioinform. 2012, 13, 140. [Google Scholar] [CrossRef]
- Friedländer, M.R.; Mackowiak, S.D.; Li, N.; Chen, W.; Rajewsky, N. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res. 2012, 40, 37–52. [Google Scholar] [CrossRef]
- Yang, Z.; Wang, L. Regulation of microRNA expression and function by nuclear receptor signaling. Cell Biosci. 2011, 1, 1–9. [Google Scholar] [CrossRef] [PubMed]
- Stefani, G.; Slack, F.J. Small non-coding RNAs in animal development. Nat. Rev. Mol. Cell Biol. 2008, 9, 219–230. [Google Scholar] [CrossRef]
- Zhang, Y.; Liao, J.M.; Zeng, S.X.; Lu, H. P53 downregulates down syndrome-associated DYRK1A through miR-1246. EMBO Rep. 2011, 12, 811–817. [Google Scholar] [CrossRef]
- Miyazawa, M.; Bogdan, A.R.; Hashimoto, K.; Tsuji, Y. Regulation of transferrin receptor-1 mRNA by the interplay between IRE-binding proteins and miR-7/miR-141 in the 3′-IRE stem-loops. RNA 2018, 24, 468–479. [Google Scholar] [CrossRef]
- Tomita, K.; Nagasawa, T.; Kuwahara, Y.; Torii, S.; Igarashi, K.; Roudkenar, M.H.; Roushandeh, A.M.; Kurimasa, A.; Sato, T. MiR-7-5p is involved in ferroptosis signaling and radioresistance thru the generation of ROS in radioresistant HeLa and SAS cell lines. Int. J. Mol. Sci. 2021, 22, 8300. [Google Scholar] [CrossRef] [PubMed]
- Pitchika, A.; Kühn, J.P.; Schipf, S.; Nauck, M.; Dörr, M.; Lerch, M.M.; Kromrey, M.L.; Felix, S.B.; Markus, M.R.P.; Rathmann, W.; et al. Hepatic steatosis and hepatic iron overload modify the association of iron markers with glucose metabolism disorders and metabolic syndrome. Liver Int. 2021, 41, 1841–1852. [Google Scholar] [CrossRef] [PubMed]
- Masi, L.N.; Lotufo, P.A.; Ferreira, F.M.; Rodrigues, A.C.; Serdan, T.D.A.; Souza-Siqueira, T.; Braga, A.A.; Saldarriaga, M.E.G.; Alba-Loureiro, T.C.; Borges, F.T.; et al. Profiling plasma-extracellular vesicle proteins and microRNAs in diabetes onset in middle-aged male participants in the ELSA-Brasil study. Physiol. Rep. 2021, 9, e14731. [Google Scholar] [CrossRef]
- Wang, Y.; Lin, C. Exosomes miR-22-3p derived from mesenchymal stem cells suppress colorectal cancer cell proliferation and invasion by regulating RAP2B and PI3K/AKT pathway. J. Oncol. 2021, 2021, e3874478. [Google Scholar] [CrossRef]
- Yoshioka, Y.; Kosaka, N.; Ochiya, T.; Kato, T. Micromanaging iron homeostasis: Hypoxia-inducible micro-RNA-210 suppresses iron homeostasis-related proteins. J. Biol. Chem. 2012, 287, 34110–34119. [Google Scholar] [CrossRef]
- Soubeyrand, S.; Lau, P.; Beehler, K.; McShane, K.; McPherson, R. miR1908-5p regulates energy homeostasis in hepatocyte models. Sci. Rep. 2021, 11, 23748. [Google Scholar] [CrossRef]
- Wan, X.; Yao, B.; Ma, Y.; Liu, Y.; Tang, Y.; Hu, J.; Li, M.; Fu, S.; Zheng, X.; Yin, D. MicroRNA-128-1-5p attenuates myocardial ischemia/reperfusion injury by suppressing Gadd45g-mediated apoptotic signaling. Biochem. Biophys. Res. Commun. 2020, 530, 314–321. [Google Scholar] [CrossRef] [PubMed]
- Ali Syeda, Z.; Langden, S.S.S.; Munkhzul, C.; Lee, M.; Song, S.J. Regulatory mechanism of microRNA expression in cancer. Int. J. Mol. Sci. 2020, 21, 1723. [Google Scholar] [CrossRef] [PubMed]
- Cui, Q.; Yu, Z.; Pan, Y.; Purisima, E.O.; Wang, E. MicroRNAs preferentially target the genes with high transcriptional regulation complexity. Biochem. Biophys. Res. Commun. 2007, 352, 733–738. [Google Scholar] [CrossRef] [PubMed]
- Wang, J.; Lu, M.; Qiu, C.; Cui, Q. TransmiR: A transcription factor-microRNA regulation database. Nucleic Acids Res. 2010, 38, D119–D122. [Google Scholar] [CrossRef] [PubMed]
- Tsang, J.; Zhu, J.; van Oudenaarden, A. MicroRNA-mediated feedback and feedforward loops are recurrent network motifs in mammals. Mol. Cell 2007, 26, 753–767. [Google Scholar] [CrossRef] [PubMed]
- Xia, L.; Gong, N. Identification and verification of ferroptosis-related genes in the synovial tissue of osteoarthritis using bioinformatics analysis. Front. Mol. Biosci. 2022, 9, 956071. [Google Scholar] [CrossRef] [PubMed]
- Ye, G.; Feng, S.; Yang, Y.; Cao, Z.; Zhang, B.; Wang, F. Establishment and comprehensive analysis of underlying microRNA-mRNA interactive networks in ovarian cancer. J. Oncol. 2022, 2022, e5120342. [Google Scholar] [CrossRef]
- McKinsey, T.A.; Zhang, C.L.; Olson, E.N. MEF2: A calcium-dependent regulator of cell division, differentiation and death. Trends Biochem. Sci. 2002, 27, 40–47. [Google Scholar] [CrossRef]
- Kim, J.M.; Hwang, I.H.; Jang, I.S.; Kim, M.; Bang, I.S.; Park, S.J.; Chung, Y.J.; Joo, J.C.; Lee, M.G. Houttuynia cordata Thunb promotes activation of HIF-1A-FOXO3 and MEF2A pathways to induce apoptosis in human HepG2 hepatocellular carcinoma cells. Integr. Cancer Ther. 2017, 16, 360–372. [Google Scholar] [CrossRef]
- Farhan, M.; Wang, H.; Gaur, U.; Little, P.J.; Xu, J.; Zheng, W. FOXO signaling pathways as therapeutic targets in cancer. Int. J. Biol. Sci. 2017, 13, 815–827. [Google Scholar] [CrossRef]
- Lee, H.; Zandkarimi, F.; Zhang, Y.; Meena, J.K.; Kim, J.; Zhuang, L.; Tyagi, S.; Ma, L.; Westbrook, T.F.; Steinberg, G.R.; et al. Energy stress-mediated AMPK activation inhibits ferroptosis. Nat. Cell Biol. 2020, 22, 225–234. [Google Scholar] [CrossRef] [PubMed]
- Li, S.W.; Liu, C.M.; Guo, J.; Marcondes, A.; Deeg, J.; Li, X.; Guan, F. Iron overload induced by ferric ammonium citrate triggers reactive oxygen species-mediated apoptosis via both extrinsic and intrinsic pathways in human hepatic cells. Hum. Exp. Toxicol. 2016, 35, 598–607. [Google Scholar] [CrossRef]
- Mayer, I.A.; Arteaga, C.L. The PI3K/AKT pathway as a target for cancer treatment. Annu. Rev. Med. 2016, 67, 11–28. [Google Scholar] [CrossRef] [PubMed]
- Lei, G.; Zhuang, L.; Gan, B. Targeting ferroptosis as a vulnerability in cancer. Nat. Rev. Cancer 2022, 22, 381–396. [Google Scholar] [CrossRef] [PubMed]
- Yu, H.; Guo, P.; Xie, X.; Wang, Y.; Chen, G. Ferroptosis, a new form of cell death, and its relationships with tumourous diseases. J. Cell. Mol. Med. 2017, 21, 648–657. [Google Scholar] [CrossRef] [PubMed]
- Zhou, L.; Guan, Q.; Zhou, W.; Kan, J.; Dong, Y. Ambient synthesis of an iminium-linked covalent organic framework for synergetic RNA interference and metabolic therapy of fibrosarcoma. Chem. Sci. 2022, 13, 7846–7854. [Google Scholar] [CrossRef] [PubMed]
- Zhou, N.; Yuan, X.; Du, Q.; Zhang, Z.; Shi, X.; Bao, J.; Ning, Y.; Peng, L. FerrDb V2: Update of the manually curated database of ferroptosis regulators and ferroptosis-disease associations. Nucleic Acids Res. 2023, 51, D571–D582. [Google Scholar] [CrossRef]
- Poursaitidis, I.; Wang, X.; Crighton, T.; Labuschagne, C.; Mason, D.; Cramer, S.L.; Triplett, K.; Roy, R.; Pardo, O.E.; Seckl, M.J.; et al. Oncogene-selective sensitivity to synchronous cell death following modulation of the amino acid nutrient cystine. Cell Rep. 2017, 18, 2547–2556. [Google Scholar] [CrossRef]
- He, Y.; Sun, M.; Zhang, G.; Yang, J.; Chen, K.; Xu, W.; Li, B. Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduct. Target. Ther. 2021, 6, 425. [Google Scholar] [CrossRef]
- Lee, H.C.; Lin, Y.Z.; Lai, Y.T.; Huang, W.J.; Hu, J.R.; Tsai, J.N.; Tsai, H.J. Glycogen synthase kinase 3 beta in somites plays a role during the angiogenesis of zebrafish embryos. FEBS J. 2014, 281, 4367–4383. [Google Scholar] [CrossRef]
- Gupte, R.; Liu, Z.; Kraus, W.L. PARPs and ADP-ribosylation: Recent advances linking molecular functions to biological outcomes. Genes Dev. 2017, 31, 101–126. [Google Scholar] [CrossRef] [PubMed]
- Zhou, B.; Liu, J.; Kang, R.; Klionsky, D.J.; Kroemer, G.; Tang, D. Ferroptosis is a type of autophagy-dependent cell death. Semin. Cancer Biol. 2020, 66, 89–100. [Google Scholar] [CrossRef] [PubMed]
Sample | Raw Reads | Clean Reads | Total Reads | Mapped Reads | Matured miRNA | |
---|---|---|---|---|---|---|
Known miRNA | Novel miRNA | |||||
CO1 | 14,708,693 | 14,490,295 | 12,890,534 | 11,542,419 | 1012 | 53 |
CO2 | 13,213,177 | 12,863,314 | 10,856,522 | 9,830,156 | 996 | 53 |
CO3 | 12,471,423 | 12,123,043 | 10,138,113 | 9,126,551 | 998 | 53 |
IO1 | 11,236,010 | 11,066,190 | 9,047,944 | 7,702,918 | 958 | 50 |
IO2 | 13,314,751 | 13,114,956 | 10,692,894 | 9,134,142 | 966 | 52 |
IO3 | 12,246,380 | 11,905,219 | 10,680,970 | 8,885,369 | 956 | 48 |
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Zhang, Q.; Wang, Q.; Ding, H.; Hu, C.; Feng, J. Ferroptosis Altered microRNAs Expression in HT-1080 Fibrosarcoma Cells Based on Small RNA Sequencing and Bioinformatics Analysis. Nutrients 2024, 16, 873. https://doi.org/10.3390/nu16060873
Zhang Q, Wang Q, Ding H, Hu C, Feng J. Ferroptosis Altered microRNAs Expression in HT-1080 Fibrosarcoma Cells Based on Small RNA Sequencing and Bioinformatics Analysis. Nutrients. 2024; 16(6):873. https://doi.org/10.3390/nu16060873
Chicago/Turabian StyleZhang, Qian, Qiwen Wang, Haoxuan Ding, Caihong Hu, and Jie Feng. 2024. "Ferroptosis Altered microRNAs Expression in HT-1080 Fibrosarcoma Cells Based on Small RNA Sequencing and Bioinformatics Analysis" Nutrients 16, no. 6: 873. https://doi.org/10.3390/nu16060873