Browse > Article
http://dx.doi.org/10.4062/biomolther.2021.075

Discovery of Cellular RhoA Functions by the Integrated Application of Gene Set Enrichment Analysis  

Chun, Kwang-Hoon (Gachon Institute of Pharmaceutical Sciences, College of Pharmacy, Gachon University)
Publication Information
Biomolecules & Therapeutics / v.30, no.1, 2022 , pp. 98-116 More about this Journal
Abstract
The small GTPase RhoA has been studied extensively for its role in actin dynamics. In this study, multiple bioinformatics tools were applied cooperatively to the microarray dataset GSE64714 to explore previously unidentified functions of RhoA. Comparative gene expression analysis revealed 545 differentially expressed genes in RhoA-null cells versus controls. Gene set enrichment analysis (GSEA) was conducted with three gene set collections: (1) the hallmark, (2) the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and (3) the Gene Ontology Biological Process. GSEA results showed that RhoA is related strongly to diverse pathways: cell cycle/growth, DNA repair, metabolism, keratinization, response to fungus, and vesicular transport. These functions were verified by heatmap analysis, KEGG pathway diagramming, and direct acyclic graphing. The use of multiple gene set collections restricted the leakage of information extracted. However, gene sets from individual collections are heterogenous in gene element composition, number, and the contextual meaning embraced in names. Indeed, there was a limit to deriving functions with high accuracy and reliability simply from gene set names. The comparison of multiple gene set collections showed that although the gene sets had similar names, the gene elements were extremely heterogeneous. Thus, the type of collection chosen and the analytical context influence the interpretation of GSEA results. Nonetheless, the analyses of multiple collections made it possible to derive robust and consistent function identifications. This study confirmed several well-described roles of RhoA and revealed less explored functions, suggesting future research directions.
Keywords
RhoA; Gene Set Enrichment Analysis (GSEA); GSE64714; Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway; Hallmark pathway; Gene ontology;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Watanabe, N., Kato, T., Fujita, A., Ishizaki, T. and Narumiya, S. (1999) Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat. Cell Biol. 1, 136-143.   DOI
2 Zhao, R., Liu, K., Huang, Z., Wang, J., Pan, Y., Huang, Y., Deng, X., Liu, J., Qin, C., Cheng, G., Hua, L., Li, J. and Yin, C. (2015) Genetic variants in Caveolin-1 and RhoA/ROCK1 are associated with clear cell renal cell carcinoma risk in a chinese population. PLoS ONE 10, e0128771.   DOI
3 Shimokawa, H., Sunamura, S. and Satoh, K. (2016) RhoA/Rho-kinase in the cardiovascular system. Circ. Res. 118, 352-366.   DOI
4 Smyth, G. K., Michaud, J. and Scott, H. S. (2005) Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21, 2067-2075.   DOI
5 Suwa, H., Ohshio, G., Imamura, T., Watanabe, G., Arii, S., Imamura, M., Narumiya, S., Hiai, H. and Fukumoto, M. (1998) Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br. J. Cancer 77, 147-152.   DOI
6 Garcia-Mariscal, A., Peyrollier, K., Basse, A., Pedersen, E., Ruhl, R., van Hengel, J. and Brakebusch, C. (2018) RhoA controls retinoid signaling by ROCK dependent regulation of retinol metabolism. Small GTPases 9, 433-444.   DOI
7 Adnane, J., Muro-Cacho, C., Mathews, L., Sebti, S. M. and Munoz-Antonia, T. (2002) Suppression of rho B expression in invasive carcinoma from head and neck cancer patients. Clin. Cancer Res. 8, 2225-2232.
8 Chen, Z., Sun, J., Pradines, A., Favre, G., Adnane, J. and Sebti, S. M. (2000) Both farnesylated and geranylgeranylated RhoB inhibit malignant transformation and suppress human tumor growth in nude mice. J. Biol. Chem. 275, 17974-17978.   DOI
9 Du, W. and Prendergast, G. C. (1999) Geranylgeranylated RhoB mediates suppression of human tumor cell growth by farnesyltransferase inhibitors. Cancer Res. 59, 5492-5496.
10 Ashburner, M., Ball, C. A., Blake, J. A., Botstein, D., Butler, H., Cherry, J. M., Davis, A. P., Dolinski, K., Dwight, S. S., Eppig, J. T., Harris, M. A., Hill, D. P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J. C., Richardson, J. E., Ringwald, M., Rubin, G. M. and Sherlock, G. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25-29.   DOI
11 Carlson, M. (2016) mouse4302.db: Affymetrix Mouse Genome 430 2.0 Array annotation data (chip mouse4302). R package version 3.2.3.
12 Hodge, R. G. and Ridley, A. J. (2016) Regulating Rho GTPases and their regulators. Nat. Rev. Mol. Cell Biol. 17, 496-510.   DOI
13 Clark, E. A., Golub, T. R., Lander, E. S. and Hynes, R. O. (2000) Genomic analysis of metastasis reveals an essential role for RhoC. Nature 406, 532-535.   DOI
14 Duong, K. H. M. and Chun, K. H. (2019) Regulation of glucose transport by RhoA in 3T3-L1 adipocytes and L6 myoblasts. Biochem. Biophys. Res. Commun. 519, 880-886.   DOI
15 Gautier, L., Cope, L., Bolstad, B. M. and Irizarry, R. A. (2004) Affy- -analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20, 307-315.   DOI
16 Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U. and Speed, T. P. (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249-264.   DOI
17 Kanehisa, M., Furumichi, M., Tanabe, M., Sato, Y. and Morishima, K. (2017) KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res. 45, D353-D361.   DOI
18 Kim, J. G., Islam, R., Cho, J. Y., Jeong, H., Cap, K. C., Park, Y., Hossain, A. J. and Park, J. B. (2018) Regulation of RhoA GTPase and various transcription factors in the RhoA pathway. J. Cell. Physiol. 233, 6381-6392.   DOI
19 Wang, J., Wu, Q., Zhang, L. H., Zhao, Y. X. and Wu, X. (2016) The role of RhoA in vulvar squamous cell carcinoma: a carcinogenesis, progression, and target therapy marker. Tumour Biol. 37, 2879-2890.   DOI
20 Taiyun, W. and Viliam, S. (2017) R package "corrplot": Visualization of a Correlation Matrix (Version 0.84). Available from: https://github.com/taiyun/corrplot/.
21 Wickham, H. (2016) ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York.
22 Yoo, H. Y., Sung, M. K., Lee, S. H., Kim, S., Lee, H., Park, S., Kim, S. C., Lee, B., Rho, K., Lee, J. E., Cho, K. H., Kim, W., Ju, H., Kim, J., Kim, S. J., Kim, W. S., Lee, S. and Ko, Y. H. (2014) A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T cell lymphoma. Nat. Genet. 46, 371-375.   DOI
23 Simpson, K. J., Dugan, A. S. and Mercurio, A. M. (2004) Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma. Cancer Res. 64, 8694-8701.   DOI
24 Kakiuchi, M., Nishizawa, T., Ueda, H., Gotoh, K., Tanaka, A., Hayashi, A., Yamamoto, S., Tatsuno, K., Katoh, H., Watanabe, Y., Ichimura, T., Ushiku, T., Funahashi, S., Tateishi, K., Wada, I., Shimizu, N., Nomura, S., Koike, K., Seto, Y., Fukayama, M., Aburatani, H. and Ishikawa, S. (2014) Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma. Nat. Genet. 46, 583-587.   DOI
25 Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A. and Kaibuchi, K. (1996) Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273, 245-248.   DOI
26 Shoop, E., Casaes, P., Onsongo, G., Lesnett, L., Petursdottir, E. O., Donkor, E. K., Tkach, D. and Cosimini, M. (2004) Data exploration tools for the Gene Ontology database. Bioinformatics 20, 3442-3454.   DOI
27 Leung, T., Manser, E., Tan, L. and Lim, L. (1995) A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J. Biol. Chem. 270, 29051-29054.   DOI
28 Zhou, J., Hayakawa, Y., Wang, T. C. and Bass, A. J. (2014) RhoA mutations identified in diffuse gastric cancer. Cancer Cell 26, 9-11.   DOI
29 Zhu, J., Zhao, Q., Katsevich, E. and Sabatti, C. (2019) Exploratory gene ontology analysis with interactive visualization. Sci. Rep. 9, 7793.   DOI
30 Haga, R. B. and Ridley, A. J. (2016) Rho GTPases: regulation and roles in cancer cell biology. Small GTPases 7, 207-221.   DOI
31 Liberzon, A., Birger, C., Thorvaldsdottir, H., Ghandi, M., Mesirov, J. P. and Tamayo, P. (2015) The Molecular Signatures Database (MSigDB) hallmark gene set collection. Cell Syst. 1, 417-425.   DOI
32 Maekawa, M., Ishizaki, T., Boku, S., Watanabe, N., Fujita, A., Iwamatsu, A., Obinata, T., Ohashi, K., Mizuno, K. and Narumiya, S. (1999) Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 285, 895-898.   DOI
33 Gentleman, R., Carey, V., Huber, W. and Hahne, F. (2018) genefilter: methods for filtering genes from high-throughput experiments. R package version 1.64.0.
34 Mootha, V. K., Lindgren, C. M., Eriksson, K. F., Subramanian, A., Sihag, S., Lehar, J., Puigserver, P., Carlsson, E., Ridderstrale, M., Laurila, E., Houstis, N., Daly, M. J., Patterson, N., Mesirov, J. P., Golub, T. R., Tamayo, P., Spiegelman, B., Lander, E. S., Hirschhorn, J. N., Altshuler, D. and Groop, L. C. (2003) PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately down-regulated in human diabetes. Nat. Genet. 34, 267-273.   DOI
35 Cheng, C., Seen, D., Zheng, C., Zeng, R. and Li, E. (2021) Role of small GTPase RhoA in DNA damage response. Biomolecules 11, 212.   DOI
36 Faried, A., Faried, L. S., Kimura, H., Nakajima, M., Sohda, M., Miyazaki, T., Kato, H., Usman, N. and Kuwano, H. (2006) RhoA and RhoC proteins promote both cell proliferation and cell invasion of human oesophageal squamous cell carcinoma cell lines in vitro and in vivo. Eur. J. Cancer 42, 1455-1465.   DOI
37 Ridley, A. J. (2006) Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol. 16, 522-529.   DOI
38 Kolde, R. (2019) pheatmap: Pretty Heatmaps. Available from: https://CRAN.R-project.org/package=pheatmap/.
39 Liberzon, A., Subramanian, A., Pinchback, R., Thorvaldsdottir, H., Tamayo, P. and Mesirov, J. P. (2011) Molecular signatures database (MSigDB) 3.0. Bioinformatics 27, 1739-1740.   DOI
40 Ridley, A. J. and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389-399.   DOI
41 Satoh, K., Fukumoto, Y. and Shimokawa, H. (2011) Rho-kinase: important new therapeutic target in cardiovascular diseases. Am. J. Physiol. Heart Circ. Physiol. 301, H287-H296.   DOI
42 Porter, A. P., Papaioannou, A. and Malliri, A. (2016) Deregulation of Rho GTPases in cancer. Small GTPases 7, 123-138.   DOI
43 Sato, N., Fukui, T., Taniguchi, T., Yokoyama, T., Kondo, M., Nagasaka, T., Goto, Y., Gao, W., Ueda, Y., Yokoi, K., Minna, J. D., Osada, H., Kondo, Y. and Sekido, Y. (2007) RhoB is frequently downregulated in non-small-cell lung cancer and resides in the 2p24 homozygous deletion region of a lung cancer cell line. Int. J. Cancer 120, 543-551.   DOI
44 Wang, K., Yuen, S. T., Xu, J., Lee, S. P., Yan, H. H., Shi, S. T., Siu, H. C., Deng, S., Chu, K. M., Law, S., Chan, K. H., Chan, A. S., Tsui, W. Y., Ho, S. L., Chan, A. K., Man, J. L., Foglizzo, V., Ng, M. K., Chan, A. S., Ching, Y. P., Cheng, G. H., Xie, T., Fernandez, J., Li, V. S., Clevers, H., Rejto, P. A., Mao, M. and Leung, S. Y. (2014) Wholegenome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer. Nat. Genet. 46, 573-582.   DOI
45 Sakata-Yanagimoto, M., Enami, T., Yoshida, K., Shiraishi, Y., Ishii, R., Miyake, Y., Muto, H., Tsuyama, N., Sato-Otsubo, A., Okuno, Y., Sakata, S., Kamada, Y., Nakamoto-Matsubara, R., Tran, N. B., Izutsu, K., Sato, Y., Ohta, Y., Furuta, J., Shimizu, S., Komeno, T., Sato, Y., Ito, T., Noguchi, M., Noguchi, E., Sanada, M., Chiba, K., Tanaka, H., Suzukawa, K., Nanmoku, T., Hasegawa, Y., Nureki, O., Miyano, S., Nakamura, N., Takeuchi, K., Ogawa, S. and Chiba, S. (2014) Somatic RHOA mutation in angioimmunoblastic T cell lymphoma. Nat. Genet. 46, 171-175.   DOI
46 Sergushichev, A. A. (2016) An algorithm for fast preranked gene set enrichment analysis using cumulative statistic calculation. bioRxiv doi: 10.1101/060012.   DOI
47 Subramanian, A., Tamayo, P., Mootha, V. K., Mukherjee, S., Ebert, B. L., Gillette, M. A., Paulovich, A., Pomeroy, S. L., Golub, T. R., Lander, E. S. and Mesirov, J. P. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U.S.A. 102, 15545-15550.   DOI
48 Wang, D., Dou, K., Xiang, H., Song, Z., Zhao, Q., Chen, Y. and Li, Y. (2007) Involvement of RhoA in progression of human hepatocellular carcinoma. J. Gastroenterol. Hepatol. 22, 1916-1920.   DOI
49 Mazieres, J., Antonia, T., Daste, G., Muro-Cacho, C., Berchery, D., Tillement, V., Pradines, A., Sebti, S. and Favre, G. (2004) Loss of RhoB expression in human lung cancer progression. Clin. Cancer Res. 10, 2742-2750.   DOI
50 Narumiya, S. and Thumkeo, D. (2018) Rho signaling research: history, current status and future directions. FEBS Lett. 592, 1763-1776.   DOI
51 R Core Team (2018) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available from: https://www.R-project.org/.
52 Sherr, C. J. and McCormick, F. (2002) The RB and p53 pathways in cancer. Cancer Cell 2, 103-112.   DOI