Effectiveness of a Novel CTGF LNA GapmeR Sequence in Gastric Cancer Cells

Abstract

Gastric cancer is the third most common cause of malignancy worldwide and the prognosis is poor due to drug resistance and molecular diversity of the disease. Therefore, development of novel therapies is required. Connective Tissue Growth Factor (CTGF) is involved in the extracellular matrix production, cell proliferation and migration which makes it a target for the treatment of disease. Nucleic acid-based therapies are used to reduce the expression of specific mRNA sequences. The purpose of this study was to reduce the migration, and proliferation of gastric cancer cells through the inhibition of CTGF expression. On this purpose, a novel locked nucleic acid GapmeR sequence was identified as an inhibitor of CTGF expression, and the effectiveness of the sequence was shown in the gastric cancer cells. The gastric adenocarcinoma cells were transfected with GapmeR and changes in gene expressions of CTGF and collagen type I (COL1A1) were studied by qRT-PCR. The CTGF protein levels and proliferation were studied by Western Blot analysis and Alamar Blue Assay. The sequence caused significant reductions in CTGF and COL1A1 mRNA levels and proliferation of cells. These results might lead to the development of delivery system towards gastric cancer cells by using this sequence.

Keywords

• Gastric Cancer
• LNA GapmeR
• CTGF

References

1. E. C. Smyth, M. Nilsson, H. I. Grabsch, N. Ct Van Grieken, and F. Lordick, “Gastric cancer Epidemiology and risk factors”, The Lancet, 396, 635–48, 2020.
2. H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, and F. Bray, “Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries”, CA: A Cancer Journal for Clinicians, 71(3), 209–249, 2021.
3. S. S. Joshi and B. D. Badgwell, “Current treatment and recent progress in gastric cancer”, CA: A Cancer Journal for Clinicians, 71(3), 264–279, 2021.
4. S. W. T. Ho and P. Tan, “Dissection of gastric cancer heterogeneity for precision oncology”, Cancer Science, 110, 3405–3414, 2019.
5. X. F. Zhang, C. M. Huang, H. S. Lu, X. Y. Wu, C. Wang, G. X. Guang, J. Z. Zhang, and C. H. Zheng, “Surgical treatment and prognosis of gastric cancer in 2 613 patients”, World Journal of Gastroenterology, 10(23), 3405-3408, 2004.
6. P. Bork, “The modular architecture of a new family of growth regulators related to connective tissue growth factor”, Federation of European Biochemical Societies, 327(2), 125-130, 1993.
7. L. F. Lau and S. C-T Lam, “The CCN Family of Angiogenic Regulators: The Integrin Connection”, Experimental Cell Research, 248, 44–57, 1999.
8. L. Kular, J. Pakradouni, P. Kitabgi, M. Laurent, and C. Martinerie, “The CCN family: A new class of inflammation modulators?”, Biochimie, 93(3), 377–388, 2011.

9. Z. Mao, X. Ma, Y. Rong, L. Cui, X. Wang, W. Wu, J. Zhang, and D. Jin, “Connective tissue growth factor enhances the migration of gastric cancer through downregulation of E-cadherin via the NF-κB pathway”, Cancer Science, 102(1), 104–110, 2011.
10. J. Hutchenreuther, K. Vincent, C. Norley, M. Racanelli., S. B. Gruber, T. M. Johnson, D. R. Fullen, L. Raskin, B. Perbal, D. W. Holdsworth, L. M. Postovit, and A. Leask, “Activation of cancer-associated fibroblasts is required for tumor neovascularization in a murine model of melanoma”, Matrix Biology, 74, 52–61, 2018.
11. L. Y. Liu, Y. C. Han, S. H. Wu, and Z. H. Lv, “Expression of connective tissue growth factor in tumor tissues is an independent predictor of poor prognosis in patients with gastric cancer”, World Journal of Gastroenterology, 14(13), 2110–2114, 2008.
12. A. M. Moreira, J. Pereira, S. Melo, M. S. Fernandes, P. Carneiro, R. Seruca, and J. Figueiredo, “The Extracellular Matrix: An Accomplice in Gastric Cancer Development and Progression”, Cells, 9(2), 394-417, 2020.
13. S. Z. Chen, H. Q. Yao, S. Z. Zhu, Q. Y. Li, G. H. Guo, and J. Yu, “Expression levels of matrix metalloproteinase-9 in human gastric carcinoma”, Oncology Letters, 9: 915-919, 2015.
14. Q. N. Zhang, H.L. Zhu, M.T. Xia, J. Liao, X.T. Huang, J.W. Xiao, and C. Yuan, “A panel of collagen genes are associated with prognosis of patients with gastric cancer and regulated by microRNA-29c-3p: An integrated bioinformatics analysis and experimental validation”, Cancer Management and Research.11, 4757–4772, 2019.
15. J. Li, Y. Ding, and A. Li, “Identification of COL1A1 and COL1A2 as candidate prognostic factors in gastric cancer”, World Journal of Surgical Oncology, 14, 297, 2016.
16. Y. Zhao, T. Zhou, A. Li, H. Yao, F. He, L. Wang, and J. Si, “A potential role of collagens expression in distinguishing between premalignant and malignant lesions in stomach”, Anatomical Record, 292(5), 692–700, 2009.
17. Y. Guo, G. Lu, H. Mao, S. Zhou, X. Tong, J. Wu, Q. Sun, H. Xu, and F. Fang, “miR-133b Suppresses Invasion and Migration of Gastric Cancer Cells via the COL1A1/TGF-β Axis”, OncoTargets and Therapy, 13: 7985-7995, 2020.
18. Q. Wang and J. Yu, “MiR-129-5p suppresses gastric cancer cell invasion and proliferation by inhibiting COL1A1” Biochemistry and Cell Biolology, 96: 19-25, 2018.
19. A. Baghbanzadeh, E. Baghbani, K. Hajiasgharzadeh, S. Noorolyai, V. Khaze, B. Mansoori, M. Shirmohamadi, B. Baradaran, and A. Mokhtarzadeh, “microRNA-193a-5p Suppresses the Migratory Ability of Human KATO III Gastric Cancer Cells through Inhibition of Vimentin and MMP-9”, Advanced Pharmaceutical Bulletin, 12(1): 169-175, 2022.
20. C. G. Jiang, L. Lv, F. R. Liu, Z. N. Wang, F. N. Liu, Y. S. Li, C. Y. Wang, H. Y. Zhang, Z. Sun, and H. M. Xu, “Downregulation of connective tissue growth factor inhibits the growth and invasion of gastric cancer cells and attenuates peritoneal dissemination”, Molecular Cancer. 28 (10), 122, 2011.
21. I. Radziejewska, K. Supruniuk, J. Nazaruk, E. Karna, B. Popławska, A. Bielawska, and A. Galicka, “Rosmarinic acid influences collagen, MMPs, TIMPs, glycosylation and MUC1 in CRL-1739 gastric cancer cell line”, Biomedicine & Pharmacotherapy 107: 397–407, 2018.
22. Y. Song, J. S. Kim, E. K. Choi, J. Kim, K. M. Kim, and H. R. Seo, “TGF-β–independent CTGF induction regulates cell adhesion mediated drug resistance by increasing collagen I in HCC”, Oncotarget, 8(13): 21650-21662, 2017.
23. H. Sun, Y. Wang, S. Wang, Y. Xie, K. Sun, S. Li, W. Cui, and K. Wang, “The involvement of collagen family genes in tumor enlargement of gastric cancer”, Scientific Reports, 13(1), 100-111, 2023.
24. S. Karaki, C. Paris, and P. Rocchi, “Antisense Oligonucleotides, A Novel Developing Targeting Therapy”, in Antisense Therapy, Editors, S. Sharad, S. Kapur, doi: 10.5772/intechopen.73736, 2019.
25. C. F. Bennett and E. E. Swayze, “RNA targeting therapeutics: Molecular mechanisms of antisense oligonucleotides as a therapeutic platform”, Annual Review of Pharmacology and Toxicology, 50, 259–293, 2010.
26. T. Tabaglio, D. H. P. Low, W. K. L. Teo, P. A. Goy, P. Cywoniuk, H. Wollmann, J. Ho, D. Tan, J. Aw, A. Pavesi, K. Sobczak, D. K. B. Wee, and E. Guccione, “MBNL1 alternative splicing isoforms play opposing roles in cancer”, Life Science Alliance, 1(5): e201800157, 2018.
27. P. Sengupta, Y. Xu, L. Wang, R. Widom, and B. D. Smith, “Collagen alpha1(I) gene (COL1A1) is repressed by RFX family”, Journal of Biological Chemistry, 280(22): 21004–21014, 2005.
28. M. Hayashi, S. Nomoto, M. Hishida, Y. Inokawa, M. Kanda, and Y. Okamura, “Identification of the collagen type 1 alpha 1 gene (COL1A1) as a candidate survival-related factor associated with hepatocellular carcinoma”, BMC Cancer, 14: 108, 2014.
29. Y. Shi, Z. Duan, X. Zhang, X. Zhang, G. Wang, and F. Li, “Down-regulation of the let-7i facilitates gastric cancer invasion and metastasis by targeting COL1A1” Protein & Cell. Feb;10(2):143-148. 2019.
30. K. I. Hulkower and R. L. Herber, “Cell Migration and Invasion Assays as Tools for Drug Discovery” Pharmaceutics, 3, 107-124, 2011.
31. H. S. Kim, M. H. Kim, M. Jeong, Y. S. Hwang, S. H. Lim, B. A. Shin, B. W. Ahn, and Y. D. Jung, “EGCG blocks tumor promoter-induced MMP-9 expression via suppression of MAPK and AP-1 activation in human gastric AGS cells”, Anticancer Research, 24(2B):747-53, 2004.
32. Y. Z. Deng, P.P. Chen, Y. Wang, D. Yin, H. P. Koeffler, B. Li, X. J. Tong, and D. Xie, “Connective tissue growth factor is overexpressed in esophageal squamous cell carcinoma and promotes tumorigenicity through beta-catenin-T-cell factor/Lef signaling”, Journal of Biological Chemistry, 14;282(50):36571-81, 2007.