Genomic analyses identify critical significant biological processes in prunetin treated gastric cancer cells

doi:10.21203/rs.3.rs-1537547/v1

Download PDF

Research Article

Genomic analyses identify critical significant biological processes in prunetin treated gastric cancer cells

https://doi.org/10.21203/rs.3.rs-1537547/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Gastric cancer is a malignant tumor in the digestive system and surgery is the only radical treatment. Prunetin is a kind of O-methylated flavonoid that belongs to the group of isoflavone executing beneficial activities, which recently showed treatment effects on a variety of cancers. However, the mechanism of prunetin in gastric cancer is still unclear. Here, we aim to discover the key genes and signaling pathways by analyzing the RNA-seq data. The GSE198930 was created by the Illumina NovaSeq 6000 (Homo sapiens). The KEGG and GO analyses revealed that "Biosynthesis of cofactors" and "Carbon metabolism" were the main changed biological processes. Moreover, we revealed several interactive genes including MYC, SRC, ASNS, SHMT2, PSAT1, CXCL8, EIF4EBP1, PSPH, SLC1A5, and MTHFD2. Our study may provide novel insights into the treatment of gastric cancer.

Gastric cancer is a leading cause of global cancer mortality that accounts for more than 700,000 deaths annually¹. The incidence of gastric cancer is geographically distinct across the world, with a high prevalence in Asia, and Africa². In Western countries, gastric cancer incidence has steadily decreased over the past years³. These geographical differences have been contributed to a variety of factors such as Helicobacter pylori infection, lifestyle factors, and genetic risk factors⁴. Gastric cancer is a kind of heterogeneous disease, with patients stratified by disease stages⁵. Previous genomic studies have uncovered many gastric cancer-related genes⁶. The gastric cancer genes can be divided into two classes: frequent mutation genes such as TP53, ARID1A, ERBB2, FGFR2, and others such as CDH1, RHOA, and GATA⁷.

Prunetin is a kind of O-methylated flavonoid that belongs to the group of isoflavone executing beneficial activities⁸. Prunetin indicates an anti-cancer feature according to previous studies. For example, Prunetin promoted apoptosis and arrested the cell cycle in the G0/G1 phase⁹. Prunetin exposure was associated with increases in CASP3 and TNF-α gene expression^{9, 10}.

In our study, we analyzed the effects of prunetin-treated gastric cancer cells by using the RNA-seq data. We identified a number of DEGs and significant signaling pathways. We then performed the gene enrichment and protein-protein interaction (PPI) network analysis to obtain the interacting map and clusters. These genes and biological processes may help understand the mechanism for prunetin-treated gastric cancer.

Data resources

Gene dataset GSE198930 was obtained from the GEO database. The data was produced by the Illumina NovaSeq 6000 (Homo sapiens) (Gyeongsang National University, Gajwa-dong, Jinju, Singapore, South Korea). The analyzed dataset includes three controls and three flavonoid prunetin-treated groups.

Data acquisition and processing

The data were organized and conducted by the R package as previously described^11–15. We used a classical t-test to identify DEGs with P < 0.05 and fold change ≥ 1.5 as being statistically significant.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO)

KEGG and GO analyses were performed by the R package (ClusterProfiler) and Reactome. P < 0.05 was considered statistically significant.

Protein-protein interaction (PPI) networks

The Molecular Complex Detection (MCODE) was used to create the PPI networks. The significant modules were produced from constructed PPI networks and String networks (https://string-db.org/). The biological processes analyses were performed by using Reactome (https://reactome.org/), and P < 0.05 was considered significant.

Identification of DEGs in gastric cancer cells treated with flavonoid prunetin

To determine the impacts of flavonoid prunetin in gastric cancer cells, we analyzed the RNA-seq data from the GEO database. A total of 603 genes were identified with a threshold of P < 0.001. The top up- and down-regulated genes were shown by the heatmap and volcano plot (Figure 1). The top ten DEGs were listed in Table 1.

Enrichment analysis of DEGs in gastric cancer cells treated with flavonoid prunetin

To further study the mechanisms among the DEGs, we performed the KEGG and GO analyses (Figure 2). We identified the top ten KEGG items including “Biosynthesis of cofactors”, “Carbon metabolism”, “Glycine, serine and threonine metabolism”, “Biosynthesis of amino acids”, “Complement and coagulation cascades”, “Arachidonic acid metabolism”, “One carbon pool by folate”, “Folate biosynthesis”, “Glyoxylate and dicarboxylate metabolism”, and “Thiamine metabolism”. We identified the top ten biological processes of GO including “carboxylic acid biosynthetic process”, “organic acid biosynthetic process”, “vascular process in circulatory system”, “negative regulation of response to wounding”, “L−alpha−amino acid transmembrane transport”, “one−carbon metabolic process”, “serine family amino acid metabolic process”, “serine family amino acid biosynthetic process”, “tetrahydrofolate metabolic process”, and “tetrahydrofolate interconversion”. We figured out the top ten cellular components of GO, including “actin−based cell projection”, “basal part of cell”, “cell cortex”, “myofibril”, “contractile fiber”, “sarcomere”, “microvillus”, “filopodium”, “platelet alpha granule lumen”, and “microvillus membrane”. We identified the top ten molecular functions of GO, including “actin binding”, “actin filament binding”, “modified amino acid binding”, “L−amino acid transmembrane transporter activity”, “amino acid transmembrane transporter activity”, “neutral amino acid transmembrane transporter activity”, “translation repressor activity”, “hydrolase activity, acting on carbon−nitrogen (but not peptide) bonds, in cyclic amidines”, “oxidoreductase activity, acting on the CH−NH group of donors, NAD or NADP as acceptor”, and “aromatic amino acid transmembrane transporter activity”.

PPI network and Reactome analyses

To explore the potential associations among the DEGs, we created the PPI network by using 443 nodes and 780 edges. The combined score > 0.2 was set as a cutoff by using the Cytoscape software. Table 2 indicated the top ten genes with the highest scores. The top two significant modules were presented in Figure 3. We further analyzed the PPI and DEGs with a Reactome map (Figure 4) and identified the top ten biological processes including "Response of EIF2AK1 (HRI) to heme deficiency", "RUNX3 regulates WNT signaling", "Binding of TCF/LEF:CTNNB1 to target gene promoters", "RHO GTPases Activate Rhotekin and Rhophilins", "GRB2:SOS provides linkage to MAPK signaling for Integrins", "Attenuation phase", "p130Cas linkage to MAPK signaling for integrins", "Post-chaperonin tubulin folding pathway", and "Common Pathway of Fibrin Clot Formation" (Supplemental Table S1).

Gastric adenocarcinoma is one of the most common cancers in the world¹⁶. In the past years, surgery is the only chance of curing it¹. Prunetin possesses anti-cancer properties and may be a candidate compound for the prevention of gastric cancer¹⁷. Thus, our study analyzed the potential mechanisms of Prunetin-treated cancer cells.

By performing the KEGG and GO analyses, we found that "Biosynthesis of cofactors" and "Carbon metabolism" are the major signaling pathways after prunetin treatment in gastric cancer cells. Similarly, Krishnan Baskran et al found that there are several cofactors that are associated with cancer¹⁸. Alice C Newman et al summarized that carbon metabolism is the key progress during cancer development¹⁹.

Additionally, we figured out a couple of association genes by analyzing the PPI network. Michael J Duffy et al proved that MYC acts as a target for cancer treatment²⁰. Circadian clocks regulate a large number of downstream genes to control biological activities such as immunity, metabolism, and aging^{13, 15, 21–39}. As a key downstream target, MYC is controlled by Bmal1, Cry1, and Cry2 to further affect the oncogene and tumorigenesis⁴⁰. Ayse Caner et al found that SRC signaling is a key for regulating the tumor microenvironment⁴¹. GPCRs and their regulators are involved in the different progression of diseases such as cancer, arthritis, and diabetes^42–57. Interestingly, Natalia Pakharukova et al found that GPCR-beta-arrestin can activate the proto-oncogene kinase Src⁵⁸. Haoxin Li et al identified that ASNS is a key gene for cancer metabolism⁵⁹. Sirt5 can regulate SHMT2 to drive cancer cell proliferation⁶⁰. Song Gao et al found that ATF4 can mediate PSAT1 to enhance cell proliferation through the GSK3β/β-catenin signaling pathway⁶¹. Helen Ha et al identified the CXCL8-CXCR1 axis as an important complex in cancer and inflammatory diseases⁶². Pei Wan et al found that BRDT is a critical regulator of eIF4EBP1 in renal cancer⁶³. The study by Li Liao et al found that PSPH regulated the metastasis and proliferation of non-small-cell lung cancer⁶⁴. Hee Chan Yoo et al found that SLC1A5 is a mitochondrial glutamine transporter for metabolic reprogramming in cancer cells⁶⁵. Jun Huang et al found that MTHFD2 promotes cancer cell proliferation via the AKT signaling pathway⁶⁶.

In summary, this study figured out the significant genes and biological processes after prunetin treatment in gastric cancer cells. The "Biosynthesis of cofactors" and "Carbon metabolism" are the top affected signaling pathways after prunetin treatment. Our study may provide novel insights into the treatment of gastric cancer.

Author Contributions

Mengshan Wang: Methodology and Writing. Tingxiang Chang: Conceptualization, Writing- Reviewing and Editing.

Funding

This work was not supported by any funding.

Declarations of interest

There is no conflict of interest to declare.

Rawla P, Barsouk A: Epidemiology of gastric cancer: global trends, risk factors and prevention. Prz Gastroenterol 2019, 14:26–38.
Rahman R, Asombang AW, Ibdah JA: Characteristics of gastric cancer in Asia. World J Gastroenterol 2014, 20:4483–90.
Collaborators GBDSC: The global, regional, and national burden of stomach cancer in 195 countries, 1990–2017: a systematic analysis for the Global Burden of Disease study 2017. Lancet Gastroenterol Hepatol 2020, 5:42–54.
Bravo D, Hoare A, Soto C, Valenzuela MA, Quest AF: Helicobacter pylori in human health and disease: Mechanisms for local gastric and systemic effects. World J Gastroenterol 2018, 24:3071–89.
Lin X, Zhao Y, Song WM, Zhang B: Molecular classification and prediction in gastric cancer. Comput Struct Biotechnol J 2015, 13:448–58.
Katona BW, Rustgi AK: Gastric Cancer Genomics: Advances and Future Directions. Cell Mol Gastroenterol Hepatol 2017, 3:211–7.
Lim B, Kim JH, Kim M, Kim SY: Genomic and epigenomic heterogeneity in molecular subtypes of gastric cancer. World J Gastroenterol 2016, 22:1190–201.
Van De Wier B, Koek GH, Bast A, Haenen GR: The potential of flavonoids in the treatment of non-alcoholic fatty liver disease. Crit Rev Food Sci Nutr 2017, 57:834–55.
Chen J: The Cell-Cycle Arrest and Apoptotic Functions of p53 in Tumor Initiation and Progression. Cold Spring Harb Perspect Med 2016, 6:a026104.
Auyeung KK, Han QB, Ko JK: Astragalus membranaceus: A Review of its Protection Against Inflammation and Gastrointestinal Cancers. Am J Chin Med 2016, 44:1–22.
Yu G, Wang LG, Han Y, He QY: clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 2012, 16:284–7.
Gu H: Identification of biomarkers and pathways of mouse embryonic fibroblasts with the dysfunction of mitochondrial DNA. bioRxiv 2021:2021.04.05.438453.
Gu H, Yuan G: Identification of potential biomarkers and inhibitors for SARS-CoV-2 infection. medRxiv 2020:2020.09.15.20195487.
Jing L, Letian W, Hanming G: Identification of driver genes and biological signaling for alcoholic myopathy. Research Square 2021.
Gu H, Wang W, Yuan G: Identification of biomarkers and candidate inhibitors for multiple myeloma. bioRxiv 2021:2021.02.25.432847.
Ang TL, Fock KM: Clinical epidemiology of gastric cancer. Singapore Med J 2014, 55:621–8.
Vetrivel P, Kim SM, Ha SE, Kim HH, Bhosale PB, Senthil K, Kim GS: Compound Prunetin Induces Cell Death in Gastric Cancer Cell with Potent Anti-Proliferative Properties: In Vitro Assay, Molecular Docking, Dynamics, and ADMET Studies. Biomolecules 2020, 10.
Baskran K, Kumar PK, Santha K, Sivakamasundari, II: Cofactors and Their Association with Cancer of the Uterine Cervix in Women Infected with High-Risk Human Papillomavirus in South India. Asian Pac J Cancer Prev 2019, 20:3415–9.
Newman AC, Maddocks ODK: One-carbon metabolism in cancer. Br J Cancer 2017, 116:1499–504.
Duffy MJ, O'Grady S, Tang M, Crown J: MYC as a target for cancer treatment. Cancer Treat Rev 2021, 94:102154.
Reppert SM, Weaver DR: Coordination of circadian timing in mammals. Nature 2002, 418:935–41.
Yuan G, Hua B, Yang Y, Xu L, Cai T, Sun N, Yan Z, Lu C, Qian R: The Circadian Gene Clock Regulates Bone Formation Via PDIA3. J Bone Miner Res 2017, 32:861–71.
Zhu Z, Hua B, Shang Z, Yuan G, Xu L, Li E, Li X, Sun N, Yan Z, Qian R, Lu C: Altered Clock and Lipid Metabolism-Related Genes in Atherosclerotic Mice Kept with Abnormal Lighting Condition. Biomed Res Int 2016, 2016:5438589.
Xu L, Cheng Q, Hua B, Cai T, Lin J, Yuan G, Yan Z, Li X, Sun N, Lu C, Qian R: Circadian gene Clock regulates mitochondrial morphology and functions by posttranscriptional way. bioRxiv 2018:365452.
Yuan G, Hua B, Cai T, Xu L, Li E, Huang Y, Sun N, Yan Z, Lu C, Qian R: Clock mediates liver senescence by controlling ER stress. Aging 2017, 9:2647–65.
Hanming G, Wei W, Gongsheng Y: Identification of potential biomarkers and inhibitors in SARS-CoV-2 infected macaques. Research Square 2020.
Mao SZ, Fan XF, Xue F, Chen R, Chen XY, Yuan GS, Hu LG, Liu SF, Gong YS: Intermedin modulates hypoxic pulmonary vascular remodeling by inhibiting pulmonary artery smooth muscle cell proliferation. Pulm Pharmacol Ther 2014, 27:1–9.
Zhu Z, Hua B, Xu L, Yuan G, Li E, Li X, Sun N, Yan Z, Lu C, Qian R: CLOCK promotes 3T3-L1 cell proliferation via Wnt signaling. IUBMB Life 2016, 68:557–68.
Gu H, Yuan G: Identification of key genes in SARS-CoV-2 patients on bioinformatics analysis. bioRxiv 2020:2020.08.09.243444.
Hanming G, Gongsheng Y: Identification of key genes and pathways in the hPSC-derived lungs infected by the SARS-CoV-2. Research Square 2021.
Yuan G: Identification of biomarkers and pathways of mitochondria in sepsis patients. bioRxiv 2021:2021.03.29.437586.
Zhu Z, Xu L, Cai T, Yuan G, Sun N, Lu C, Qian R: Clock represses preadipocytes adipogenesis via GILZ. J Cell Physiol 2018, 233:6028–40.
Fan XF, Wang XR, Yuan GS, Wu DH, Hu LG, Xue F, Gong YS: [Effect of safflower injection on endoplasmic reticulum stress-induced apoptosts in rats with hypoxic pulmonary hypertension]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2012, 28:561–7.
Yuan G, Xu L, Cai T, Hua B, Sun N, Yan Z, Lu C, Qian R: Clock mutant promotes osteoarthritis by inhibiting the acetylation of NFkappaB. Osteoarthritis Cartilage 2019, 27:922–31.
Gu H, Yuan G: Identification of potential key genes for SARS-CoV-2 infected human bronchial organoids based on bioinformatics analysis. bioRxiv 2020:2020.08.18.256735.
Fu L, Pelicano H, Liu J, Huang P, Lee C: The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 2002, 111:41–50.
Cai T, Hua B, Luo D, Xu L, Cheng Q, Yuan G, Yan Z, Sun N, Hua L, Lu C: The circadian protein CLOCK regulates cell metabolism via the mitochondrial carrier SLC25A10. Biochim Biophys Acta Mol Cell Res 2019, 1866:1310–21.
Gu H, Yuan G: Identification of specific biomarkers and pathways in the synovial tissues of patients with osteoarthritis in comparison to rheumatoid arthritis. bioRxiv 2020:2020.10.22.340232.
Doi M, Hirayama J, Sassone-Corsi P: Circadian regulator CLOCK is a histone acetyltransferase. Cell 2006, 125:497–508.
Liu Z, Selby CP, Yang Y, Lindsey-Boltz LA, Cao X, Eynullazada K, Sancar A: Circadian regulation of c-MYC in mice. Proc Natl Acad Sci U S A 2020, 117:21609–17.
Caner A, Asik E, Ozpolat B: SRC Signaling in Cancer and Tumor Microenvironment. Adv Exp Med Biol 2021, 1270:57–71.
Rosenbaum DM, Rasmussen SG, Kobilka BK: The structure and function of G-protein-coupled receptors. Nature 2009, 459:356–63.
Yuan G, Yang S, Yang S: Macrophage RGS12 contributes to osteoarthritis pathogenesis through enhancing the ubiquitination. Genes & Diseases 2021.
Yuan G, Huang Y, Yang ST, Ng A, Yang S: RGS12 inhibits the progression and metastasis of multiple myeloma by driving M1 macrophage polarization and activation in the bone marrow microenvironment. Cancer Commun (Lond) 2022, 42:60–4.
Hanming G: nuotrophils arthritis. Research Square 2021.
Fu C, Yuan G, Yang ST, Zhang D, Yang S: RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN. J Dent Res 2020:22034520972095.
Yuan G, Yang S, Yang S, Ng A, Oursler MJ: RGS12 is a critical proinflammatory factor in the pathogenesis of inflammatory arthritis via acting in Cox2-RGS12-NF kappa B pathway activation loop. J Bone Miner Res: WILEY 111 RIVER ST, HOBOKEN 07030 – 5774, NJ USA, 2019. pp. 147-.
Li J, Wang W, Gu H: Identification of biological processes and signaling pathways for the knockout of REV-ERB in mouse brain. bioRxiv 2021:2021.11.22.469579.
Yuan G, Yang S, Ng A, Fu C, Oursler MJ, Xing L, Yang S: RGS12 Is a Novel Critical NF-kappaB Activator in Inflammatory Arthritis. iScience 2020, 23:101172.
Xie K, Martemyanov KA: Control of striatal signaling by g protein regulators. Front Neuroanat 2011, 5:49.
Yuan G, Fu C, Yang ST, Yuh DY, Hajishengallis G, Yang S: RGS12 Drives Macrophage Activation and Osteoclastogenesis in Periodontitis. J Dent Res 2022, 101:448–57.
Zhang M, Wang J, Gu H: Identification of biological processes and signaling pathways in the stretched nucleus pulposus cells. bioRxiv 2021:2021.11.23.469730.
Stewart A, Fisher RA: Introduction: G Protein-coupled Receptors and RGS Proteins. Prog Mol Biol Transl Sci 2015, 133:1–11.
Yuan G, Yang S, Gautam M, Luo W, Yang S: Macrophage regulator of G-protein signaling 12 contributes to inflammatory pain hypersensitivity. Ann Transl Med 2021, 9:448.
Jing W, Hanming G: Identification of biological processes and potential inhibitors for aging skin. Research Square 2021.
Yuan G, Yang S, Liu M, Yang S: RGS12 is required for the maintenance of mitochondrial function during skeletal development. Cell Discov 2020, 6:59.
Tamirisa P, Blumer KJ, Muslin AJ: RGS4 inhibits G-protein signaling in cardiomyocytes. Circulation 1999, 99:441–7.
Pakharukova N, Masoudi A, Pani B, Staus DP, Lefkowitz RJ: Allosteric activation of proto-oncogene kinase Src by GPCR-beta-arrestin complexes. J Biol Chem 2020, 295:16773–84.
Li H, Ning S, Ghandi M, Kryukov GV, Gopal S, Deik A, Souza A, Pierce K, Keskula P, Hernandez D, Ann J, Shkoza D, Apfel V, Zou Y, Vazquez F, Barretina J, Pagliarini RA, Galli GG, Root DE, Hahn WC, Tsherniak A, Giannakis M, Schreiber SL, Clish CB, Garraway LA, Sellers WR: The landscape of cancer cell line metabolism. Nat Med 2019, 25:850–60.
Yang X, Wang Z, Li X, Liu B, Liu M, Liu L, Chen S, Ren M, Wang Y, Yu M, Wang B, Zou J, Zhu WG, Yin Y, Gu W, Luo J: SHMT2 Desuccinylation by SIRT5 Drives Cancer Cell Proliferation. Cancer Res 2018, 78:372–86.
Gao S, Ge A, Xu S, You Z, Ning S, Zhao Y, Pang D: PSAT1 is regulated by ATF4 and enhances cell proliferation via the GSK3beta/beta-catenin/cyclin D1 signaling pathway in ER-negative breast cancer. J Exp Clin Cancer Res 2017, 36:179.
Ha H, Debnath B, Neamati N: Role of the CXCL8-CXCR1/2 Axis in Cancer and Inflammatory Diseases. Theranostics 2017, 7:1543–88.
Wan P, Chen Z, Zhong W, Jiang H, Huang Z, Peng D, He Q, Chen N: BRDT is a novel regulator of eIF4EBP1 in renal cell carcinoma. Oncol Rep 2020, 44:2475–86.
Liao L, Ge M, Zhan Q, Huang R, Ji X, Liang X, Zhou X: PSPH Mediates the Metastasis and Proliferation of Non-small Cell Lung Cancer through MAPK Signaling Pathways. Int J Biol Sci 2019, 15:183–94.
Yoo HC, Park SJ, Nam M, Kang J, Kim K, Yeo JH, Kim JK, Heo Y, Lee HS, Lee MY, Lee CW, Kang JS, Kim YH, Lee J, Choi J, Hwang GS, Bang S, Han JM: A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells. Cell Metab 2020, 31:267 – 83 e12.
Huang J, Qin Y, Lin C, Huang X, Zhang F: MTHFD2 facilitates breast cancer cell proliferation via the AKT signaling pathway. Exp Ther Med 2021, 22:703.

Tables 1-2 are in the supplementary files section.

Download PDF

Version 1

posted

You are reading this latest preprint version

Genomic analyses identify critical significant biological processes in prunetin treated gastric cancer cells

Status:

Version 1

Abstract

Figures

Introduction

Methods

Results

Discussion

Declarations

References

Tables

Supplementary Files

Status:

Version 1