Testin Inhibits the Biological Activity of Lung Adenocarcinoma by Regulating the RhoA Pathway_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

ZHANG Yanmin , ZHU Ning , CHEN Lei , MA Lei ,  LI Haiquan

Department of Respiratory Medicine, Second Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu 221006, P.R. China;

Corresponding author：

LI Haiquan, Email: 8711860@qq.com

Keywords：

Testin; RhoA; lung adenocarcinoma; biological activity

DOI：

10.7507/1671-6205.202503055

Video：

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Objective To explore the mechanism by which the tumor suppressor gene Testin affects the proliferation, migration, and invasive biological activity of lung adenocarcinoma cell lines by regulating the RhoA pathway. Method The cbioportal tumor gene expression was used to screen for genes with high correlation with TES gene expression in lung adenocarcinoma, and the 200 genes with the highest correlation were selected for pathway enrichment analysis. Upload these 200 genes to the David gene annotation tool for GO_Biological Process pathway analysis, GO Molecular Function pathway analysis, KEGG pathway analysis, and Reactome pathway analysis. The lung adenocarcinoma cell line H1299 was cultured, and an overexpression Testin plasmid was constructed and transfected into H1299 cells. The mRNA and protein expression of RhoA, Rac1, and Cdc42 were detected using qRT PCR and western blot. On the basis of downregulating RhoA expression through overexpression of Testin, the overexpression plasmid of RhoA (TES+RhoA) was transfected simultaneously to induce a downregulation of RhoA expression, and the changes in malignant phenotype of lung adenocarcinoma cells were detected. The biological activity changes of adenocarcinoma cell lines after the above intervention were verified through CCK-8 experiment, Transwell experiment, and Matrigel experiment. Results The results of pathway analysis prediction showed that Testin may be involved in regulating the Rho GTPase signaling pathway. Overexpression of Testin did not affect the mRNA levels of RhoA, Rac1, and Cdc42 (all P>0.05), nor did it affect the protein expression levels of Rac1 and Cdc42 (all P>0.05), but it significantly reduced the protein level of RhoA (P<0.05). Knocking down RhoA in lung adenocarcinoma cell H1299 can significantly inhibit cell proliferation, migration, and invasion ability (all P<0.05). Simultaneously transfecting RhoA overexpression plasmid on the basis of overexpression of Testin can downregulate RhoA expression, but does not affect Testin expression. Conclusions RhoA plays a pro-cancer role in lung adenocarcinoma, and Testin can inhibit RhoA expression. Overexpression of RhoA can rescue Testin's effect on lung adenocarcinoma cell proliferation, migration, and invasion. Testin exerts its anti-cancer biological activity by regulating RhoA.

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8.	Stankiewicz TR, Linseman DA. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration. Front Cell Neurosci 2014, 8: 314.
9.	Schmidt SI, Blaabjerg M, Freude K, et al. RhoA Signaling in Neurodegenerative Diseases. Cells, 2022, 11(9): 1520.
10.	Vega FM, Ridley AJ. Rho GTPases in cancer cell biology. FEBS Lett, 2008, 582(14): 2093-2101.
11.	Ridley AJ. Life at the leading edge. Cell, 2011, 145(7): 1012-1022.
12.	Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nu cleotide-exchange factors. Nat Rev Mol Cell Biol, 2005, 6(2): 167-180.
13.	Schmidt SI, Blaabjerg M, Freude K, Meyer M: RhoA Signaling in Neurodegenerative Diseases. Cells, 2022, 11(9).
14.	Li B, Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer, 2002, 2(2): 133-142 .
15.	Hodge RG, Ridley AJ. Regulating Rho GTPases and their regulators. Nat Rev Mol Cell Biol, 2016, 17(8): 496-510.
16.	Liu M, Bi F, Zhou X, et al. Rho GTPase regulation by miRNAs and covalent modifications. Trends Cell Biol, 2012, 22(7): 365-373.
17.	Croft DR, Olson MF. Transcriptional regulation of Rho GTPase signaling. Transcription, 2011, 2(5): 211-215.
18.	Berthold J, Schenkova K, Rivero F. Rho GTPases of the RhoBTB subfamily and tumorigenesis. Acta Pharmacol Sin, 2008, 29(3): 285-295.
19.	Prendergast GC, Khosravi-Far R, Solski PA, et al. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene, 1995, 10(12): 2289-2296.
20.	Lin R, Bagrodia S, Cerione R, et al. A novel Cdc42Hs mutant induces cellular transformation. Curr Biol, 1997, 7(10): 794-797.
21.	Porter AP, Papaioannou A, Malliri A. Deregulation of Rho GTPases in cancer. Small GTPases, 2016, 7(3): 123-138.
22.	Cai J, Niu X, Chen Y, et al. Emodin-induced generation of reactive oxygen species inhibits RhoA activation to sensitize gastric carcinoma cells to anoikis. Neoplasia 2008, 10(1): 41-51.

1. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024, 74(3): 229–63.
2. Chen YY, Lin KH, Kuo YS. et al. Therapeutic impact of epidermal growth factor receptor tyrosine kinase inhibitor with various treatment combinations for advanced lung adenocarcinoma. World J Surg Onc. 2023, 21(1)): 326.
3. Ge Y, Zhan Y, He J. et al. PD-(L)1 inhibitors plus bevacizumab and chemotherapy as first-line therapy in PD-L1-negative metastatic lung adenocarcinoma: a real-world data. J Cancer Res Clin Oncol ournal, 2024, 150(3): 135.
4. Kapoor S. Testin and its emerging modulatory role in systemic carcinogenesis. Spermatogenesis 2012, 2(4): 304.
5. Wang M, Wang Q, Peng WJ, et al. Testin is a tumor suppressor in non-small cell lung cancer. Oncol Rep 2017, 37(2): 1027-1035.
6. Etienne-Manneville S, Hall A: Rho GTPases in cell biology. Nature, 2002, 420(6916): 629-635.
7. Haga RB, Ridley AJ: Rho GTPases: Regulation and roles in cancer cell biology. Small GTPases 2016, 7(4): 207-221.
8. Stankiewicz TR, Linseman DA. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration. Front Cell Neurosci 2014, 8: 314.
9. Schmidt SI, Blaabjerg M, Freude K, et al. RhoA Signaling in Neurodegenerative Diseases. Cells, 2022, 11(9): 1520.
10. Vega FM, Ridley AJ. Rho GTPases in cancer cell biology. FEBS Lett, 2008, 582(14): 2093-2101.
11. Ridley AJ. Life at the leading edge. Cell, 2011, 145(7): 1012-1022.
12. Rossman KL, Der CJ, Sondek J. GEF means go: turning on RHO GTPases with guanine nu cleotide-exchange factors. Nat Rev Mol Cell Biol, 2005, 6(2): 167-180.
13. Schmidt SI, Blaabjerg M, Freude K, Meyer M: RhoA Signaling in Neurodegenerative Diseases. Cells, 2022, 11(9).
14. Li B, Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer, 2002, 2(2): 133-142 .
15. Hodge RG, Ridley AJ. Regulating Rho GTPases and their regulators. Nat Rev Mol Cell Biol, 2016, 17(8): 496-510.
16. Liu M, Bi F, Zhou X, et al. Rho GTPase regulation by miRNAs and covalent modifications. Trends Cell Biol, 2012, 22(7): 365-373.
17. Croft DR, Olson MF. Transcriptional regulation of Rho GTPase signaling. Transcription, 2011, 2(5): 211-215.
18. Berthold J, Schenkova K, Rivero F. Rho GTPases of the RhoBTB subfamily and tumorigenesis. Acta Pharmacol Sin, 2008, 29(3): 285-295.
19. Prendergast GC, Khosravi-Far R, Solski PA, et al. Critical role of Rho in cell transformation by oncogenic Ras. Oncogene, 1995, 10(12): 2289-2296.
20. Lin R, Bagrodia S, Cerione R, et al. A novel Cdc42Hs mutant induces cellular transformation. Curr Biol, 1997, 7(10): 794-797.
21. Porter AP, Papaioannou A, Malliri A. Deregulation of Rho GTPases in cancer. Small GTPases, 2016, 7(3): 123-138.
22. Cai J, Niu X, Chen Y, et al. Emodin-induced generation of reactive oxygen species inhibits RhoA activation to sensitize gastric carcinoma cells to anoikis. Neoplasia 2008, 10(1): 41-51.

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