Effect of miR-105-5p on Cell Proliferation, Colony Formation, and hTERT/P53/P21 Gene Expression in Multiple Cancer Cell Lines

Abstract

Aim: MicroRNAs are small endogenous RNA molecules that do not code for proteins but have the capacity to influence cancer development by promoting or inhibiting it. They play normal biological roles in regulating cell metabolism, development, apoptosis, and metastasis. While numerous studies on the functional mechanisms of miRNAs exist, there is limited research in the literature on miR-105-5p, a member of the miR-105 family. This study investigated the effects of miR-105-5p on cell proliferation, colony formation, and the expression of hTERT, P21, and P53 genes in five cancer cell lines: A549 (lung), Huh7 (liver), HCT116 (colon), MCF-7 (breast), and HeLa (cervix).

Method: The expression of miR-105-5p was both upregulated and downregulated through transfection in the selected cancer cell lines. After transfection, assays were performed to assess cell proliferation, colony-forming ability, and the expression of the hTERT, P21, and P53 genes.

Results: Overexpression of miR-105-5p inhibited proliferation in all cancer cell lines. miR-105-5p suppressed colony formation in A549, MCF-7, HeLa, and Huh7 cells. Differences in the expression levels of hTERT, P53, and P21 genes were observed in different cell lines due to the effect of the miR-105-5p mimic.

Conclusion: Direct or indirect differences in hTERT gene expression levels suggest that miR-105-5p may be useful in targeted therapy.

Keywords

• miR-105-5p
• hTERT
• P53
• P21
• transfection

Supporting Institution

Çukurova Üniversitesi

Project Number

FYL-2021-14344, TYL-2021-13415

Ethical Statement

In this study, we affirm that all regulations outlined in the "Higher Education Institutions Scientific Research and Publication Ethics Directive" have been strictly followed. Furthermore, no actions contrary to the principles described under "Actions Against Scientific Research and Publication Ethics" have been undertaken.

Thanks

We would like to thank the Scientific Research Unit of Çukurova University for their support in the conduct of this study with projects numbered FYL-2021-14344 and BAP: TYL-2021-13415. We would also like to thank the Central Laboratory and Biotechnology Center of Cukurova University for providing space and equipment for conducting of the experimental stages of the project.

miR-105-5p'nin Çeşitli Kanser Hücre Hatlarında Hücre Proliferasyonu, Koloni Oluşumu ve hTERT/P53/P21 Gen Ekspresyonuna Etkisi

Abstract

Amaç: MikroRNA'lar, protein kodlamayan ancak kanser gelişimini teşvik ederek veya engelleyerek etkileme kapasitesine sahip küçük endojen RNA molekülleridir. Hücre metabolizması, gelişim, apoptoz ve metastazın düzenlenmesinde normal biyolojik roller oynarlar. MikroRNA'ların fonksiyonel mekanizmalarına ilişkin çok sayıda çalışma mevcut olmasına rağmen, miR-105 ailesinin bir üyesi olan miR-105-5p hakkında literatürde sınırlı araştırma bulunmaktadır. Bu çalışma, beş kanser hücresi hattında (A549 (akciğer), Huh7 (karaciğer), HCT116 (kolon), MCF-7 (meme) ve HeLa (serviks)) miR-105-5p'nin hücre çoğalması, koloni oluşumu ve hTERT, P21 ve P53 genlerinin ekspresyonu üzerindeki etkilerini araştırmıştır.

Yöntem: Seçilen kanser hücre hatlarında miR-105-5p ekspresyonu, transfeksiyon yoluyla hem yukarı hem de aşağı doğru düzenlenmiştir. Transfeksiyondan sonra, hücre çoğalması, koloni oluşturma yeteneği ve hTERT, P21 ve P53 genlerinin ekspresyonunu değerlendirmek için analizler yapıldı.

Bulgular: miR-105-5p'nin aşırı ekspresyonu tüm kanser hücre hatlarında çoğalmayı inhibe etti. miR-105-5p, A549, MCF-7, HeLa ve Huh7 hücrelerinde koloni oluşumunu baskıladı. miR-105-5p taklidinin etkisi nedeniyle farklı hücre hatlarında hTERT, P53 ve P21 genlerinin ekspresyon seviyelerinde farklılıklar gözlendi.

Sonuç: hTERT gen ekspresyon seviyelerindeki doğrudan veya dolaylı farklılıklar, miR-105-5p'nin hedefli tedavide yararlı olabileceğini düşündürmektedir.

Keywords

• miR-105-5p
• hTERT
• P53
• P21
• transfeksiyon

Supporting Institution

Çukurova Üniversitesi Bilimsel Araştırma Birimi

Project Number

FYL-2021-14344, TYL-2021-13415

Ethical Statement

Bu çalışmada, "Yükseköğretim Kurumları Bilimsel Araştırma ve Yayın Etiği Yönergesi"nde belirtilen tüm düzenlemelere harfiyen uyulduğunu ve "Bilimsel Araştırma ve Yayın Etiğine Aykırı Eylemler" başlığı altında açıklanan ilkelere aykırı herhangi bir eylemde bulunulmadığını beyan ederiz.

Thanks

Bu çalışmanın yürütülmesinde FYL-2021-14344 ve BAP: TYL-2021-13415 numaralı projelerle bize destek olan Çukurova Üniversitesi Bilimsel Araştırma Birimi'ne teşekkür ederiz. Ayrıca, projenin deneysel aşamalarının yürütülmesi için mekan ve ekipman sağlayan Çukurova Üniversitesi Merkez Laboratuvarı ve Biyoteknoloji Merkezi'ne de teşekkür ederiz.

References

1. 1. Ratti M, Lampis A, Ghidini M, et al. MicroRNAs (miRNAs) and ong Non-Coding RNAs (lncRNAs) as new tools for cancer therapy: First steps from bench to bedside. Target Oncol. 2020;15(3):261-78. doi: 10.1007/s11523-020-00717-x.
2. 2. Smolarz B, Durczyński A, Romanowicz H, Szyłło K, Hogendorf P. miRNAs in cancer (review of literature). Int J Mol Sci. 2022;23(5). doi: 10.3390/ijms23052805.
3. 3. Otmani K, Lewalle P. Tumor suppressor miRNA in cancer cells and the tumor microenvironment: Mechanism of deregulation and clinical ımplications. Front Oncol. 2021;11:708765. doi: 10.3389/fonc.2021.708765.
4. 4. Slaby O, Bienertova-Vasku J, Svoboda M, Vyzula R. Genetic polymorphisms and microRNAs: New direction in molecular epidemiology of solid cancer. J Cell Mol Med. 2012;16(1):8-21. doi: 10.1111/j.1582-4934.2011.01359.x.
5. 5. Landi D, Gemignani F, Barale R, Landi S. A catalog of polymorphisms falling in microRNA-binding regions of cancer genes. DNA Cell Biol. 2008;27(1):35-43.
6. 6. Seyhan AA. Trials and tribulations of MicroRNA therapeutics. Int J Mol Sci. 2024;25(3).
7. 7. Khan S, Zhang DY, Zhang JY, et al. The key role of microRNAs in initiation and progression of hepatocellular carcinoma. Front Oncol. 2022;12:950374.
8. 8. Li J, Zhang Z, Chen F, et al. The diverse oncogenic and tumor suppressor roles of microRNA-105 in cancer. Front Oncol. 2019;9:518.

9. 9. Lu G, Fu D, Jia C, et al. Reduced miR-105-1 levels are associated with poor survival of patients with non-small cell lung cancer. Oncol Lett. 2017;14(6):7842-8.
10. 10. Zhou W, Fong MY, Min Y, et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell. 2014;25(4):501-15.
11. 11. Shang JC, Yu GZ, Ji ZW, Wang XQ, Xia L. MiR-105 inhibits gastric cancer cells metastasis, epithelial-mesenchymal transition by targeting SOX9. Eur Rev Med Pharmacol Sci. 2019;23(14):6160-9. doi: 10.26355/eurrev_201907_18429.
12. 12. Lin S, Gregory RI. MicroRNA biogenesis pathways in cancer. Nat Rev Cancer. 2015;15(6):321-33. doi: 10.1038/nrc3932.
13. 13. Gao R, Wang Z, Liu Q, Yang C. MicroRNA-105 plays an independent prognostic role in esophageal cancer and acts as an oncogene. Cancer Biomark. 2020;27(2):173-80.
14. 14. Al Bitar S, Gali-Muhtasib H. The role of the cyclin dependent kinase ınhibitor p21(cip1/waf1) in targeting cancer: Molecular mechanisms and novel therapeutics. Cancers (Basel). 2019;11(10). doi: 10.3390/cancers11101475.
15. 15. Wang H, Guo M, Wei H, Chen Y. Targeting P53 pathways: Mechanisms, structures and advances in therapy. Signal Transduction and Targeted Therapy. 2023;8(1):92.
16. 16. Henderson YC, Breau RL, Liu TJ, Clayman GL. Telomerase activity in head and neck tumors after introduction of wild-type P53, P21, p16, and E2F-1 genes by means of recombinant adenovirus. Head Neck. 2000;22(4):347-54.
17. 17. Miliotis C, Slack FJ. miR-105-5p regulates PD-L1 expression and tumor immunogenicity in gastric cancer. Cancer Lett. 2021;518:115-126. doi: 10.1016/j.canlet.2021.05.037.
18. 18. Davis-Dusenbery BN, Hata A. MicroRNA in cancer: The involvement of aberrant microrna biogenesis regulatory pathways. Genes Cancer. 2010;1(11):1100-14.
19. 19. Menon A, Abd-Aziz N, Khalid K, Poh CL, Naidu R. miRNA: A promising therapeutic target in cancer. Int J Mol Sci. 2022;23(19). doi: 10.3390/ijms231911502.
20. 20. Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol. 2007;302(1):1-12. doi: 10.1016/j.ydbio.2006.08.028.
21. 21. Fasoulakis Z, Daskalakis G, Diakosavvas M, et al. MicroRNAs determining carcinogenesis by regulating oncogenes and tumor suppressor genes during cell cycle. Microrna. 2020;9(2):82-92. doi: 10.2174/2211536608666190919161849.
22. 22. Dong B, Zhang F, Zhang W, Gao Y. IncRNA EPB41L4A-AS1 mitigates the proliferation of non-small-cell lung cancer cells through the mir-105-5p/gımap6 axis. Crit Rev Eukaryot Gene Expr. 2023;33(2):27-40.
23. 23. Pan G, Jiang B, Yi Z, Yin J, Liu Y. Exosomal miR-105-5p derived from bladder cancer stem cells targets for GPR12 to promote the malignancy of bladder cancer. BMC Urol. 2023;23(1):155. doi: 10.1186/s12894-023-01326-2.
24. 24. Chen T, Liu Y, Chen J, et al. Exosomal miR-3180-3p inhibits proliferation and metastasis of non-small cell lung cancer by downregulating FOXP4. Thorac Cancer. 2021;12(3):372-81. doi: 10.1111/1759-7714.13759.
25. 25. Chen W, Lu Q, Li S, Zhang X, Xue X. microRNA-1298 inhibits the malignant behaviors of breast cancer cells via targeting ADAM9. Biosci Rep. 2020;40(12).
26. 26. Cui G, Cui M, Li Y, et al. MiR-186 targets ROCK1 to suppress the growth and metastasis of NSCLC cells. Tumour Biol. 2014;35(9):8933-7. doi: 10.1007/s13277-014-2168-6.
27. 27. Cui J, Pan G, He Q, et al. MicroRNA-545 targets ZEB2 to inhibit the development of non-small cell lung cancer by inactivating Wnt/β-catenin pathway. Oncol Lett. 2019;18(3):2931-8. doi: 10.3892/ol.2019.10619.
28. 28. Wang CJ, Stratmann J, Zhou ZG, Sun XF. Suppression of microRNA-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells. BMC Cancer. 2010;10:616. doi: 10.1186/1471-2407-10-616.
29. 29. Cheng Z, Jiang S, Tao R, Ge H, Qin J. Activating transcription factor 3-activated long noncoding RNA forkhead box P4-antisense RNA 1 aggravates colorectal cancer progression by regulating microRNA-423-5p/nucleus accumbens associated 1 axis. Bioengineered. 2022;13(2):2114-29. doi: 10.1080/21655979.2021.2023798.
30. 30. He L, Wang J, Chang D, et al. Effect of miRNA-200b on the proliferation and apoptosis of cervical cancer cells by targeting RhoA. Open Med (Wars). 2020;15(1):1019-27.
31. 31. Wen Z, Lei Z, Jin-An M, et al. The inhibitory role of miR-214 in cervical cancer cells through directly targeting mitochondrial transcription factor A (TFAM). Eur J Gynaecol Oncol. 2014;35(6):676-82.
32. 32. Ma J, Yan H, Zhang J, Tan Y, Gu W. Long-Chain Non-Coding RNA (lncRNA) MT1JP suppresses biological activities of lung cancer by regulating miRNA-423-3p/Bim Axis. Med Sci Monit. 2019;25:5114-26. doi: 10.12659/msm.914387.
33. 33. Li X, An M, Gao Z. In Hepatocellular carcinoma, mirna-296-3p targets msl2 and suppresses cell proliferation and ınvasion. J Oncol. 2021;2021:7430468.
34. 34. Dogan F, Forsyth NR. Telomerase regulation: A role for epigenetics. Cancers (Basel). 2021;13(6). doi: 10.3390/cancers13061213.
35. 35. Salamati A, Majidinia M, Asemi Z, et al. Modulation of telomerase expression and function by miRNAs: Anti-cancer potential. Life Sci. 2020;259:118387.
36. 36. Marei HE, Althani A, Afifi N, et al. P53 signaling in cancer progression and therapy. Cancer Cell Int. 2021;21(1):703. doi: 10.1186/s12935-021-02396-8.
37. 37. Yao Y, Bellon M, Shelton SN, Nicot C. Tumor suppressors P53, p63TAα, p63TAy, p73α, and p73β use distinct pathways to repress telomerase expression. J Biol Chem. 2012;287(24):20737-47. doi: 10.1074/jbc.M111.319236.
38. 38. Fang SS, Guo JC, Zhang JH, et al. A P53-related microRNA model for predicting the prognosis of hepatocellular carcinoma patients. J Cell Physiol. 2020;235(4):3569-78.
39. 39. Jung SJ, Park JH, Hwang I, Lee JH. Different TERT expression between colorectal adenoma and serrated polyp. Medicina (Kaunas). 2020;56(9).
40. 40. Jones M, Lal A. MicroRNAs, wild-type and mutant P53: More questions than answers. RNA Biol. 2012;9(6):781-91. doi: 10.4161/rna.20146
41. 41. Hengstermann A, Linares LK, Ciechanover A, Whitaker NJ, Scheffner M. Complete switch from Mdm2 to human papillomavirus E6-mediated degradation of P53 in cervical cancer cells. Proc Natl Acad Sci USA. 2001;98(3):1218-23. doi: 10.1073/pnas.98.3.1218.