Objective: To investigate the effect of MicroRNA-199a-3p (miR-199a-3p) on proliferation of uveal melanoma cells. Methods: In this experimental research, real-time RT-PCR was performed to determine the level of miR-199a-3p in a human melanocyte cell line (UM96) and uveal melanoma cell line (OCM290).
Lipofectamine was used to transfect miR-199a-3p into uveal melanoma cells to upregulate the expression level of miR-199a-3p. A scrambled oligonucleotide was transfected into uveal melanoma cells as the negative control (NC). The proliferation of uveal melanoma cells was examined by cell proliferation assay (MTS) and clone formation experiments. Flow cytometry was performed to detect the cell cycle of uveal melanoma cells. In addition, Western blot was used to identify the protein level of cell cycle-related proteins. Data analysis was performed by independent t-tests. Results: The level of miR-199a-3p in uveal melanoma cells significantly decreased compared tomelanocyte cells (t=13.2, P<0.001). After the transfection, the MTS assay showed that the relative number of cells transfected with miR-199a-3p (23.8%±1.7%) was significantly lower than those transfected with NC (t=78.1, P<0.001). The recovery expression level of miR-199a-3p inhibited colony formation, and also induced G1-phase arrest in uveal melanoma cells (t= -8.5, P=0.001). Furthermore, it was found that miR-199a-3p could deregulate the protein level of cyclindependent protein kinases (CDK2, CDK4) and the transcription factor (E2F1) (t=10.3, P=0.001; t=9.9, P=0.001; t=10.4, P<0.001). Conclusion: miR-199a-3p inhibits the proliferation of uveal melanoma cells by preventing the cell cycle process.
Lane AM, Kim IK, Gragoudas ES. Survival rates in patients after treatment for metastasis from uveal melanoma. JAMA Ophthalmol, 2018, 136(9): 981-986. DOI: 10.1001/
jamaophthalmol. 2018.2466.
[3]
Sato T, Han F, Yamamoto A. The biology and management of uveal melanoma. Curr Oncol Rep, 2008, 10(5): 431-438.
He L, Hannon GJ. MicroRNAs: Small RNAs with a big role in gene regulation. Nat Rev Genet, 2004, 5(7): 522-531. DOI: 10.1038/nrg1379.
[6]
Kumar MS, Lu J, Mercer KL, et al. Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat Genet, 2007, 39(5): 673-677. DOI: 10.1038/ng2003.
[7]
Yan D, Zhou X, Chen X, et al. MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-Met. Invest Ophthalmol Vis Sci, 2009, 50(4): 1559-1565. DOI: 10.1167/iovs.08-2681.
[8]
Chen X, Wang J, Shen H, et al. Epigenetics, microRNAs, and carcinogenesis: Functional role of microRNA-137 in uveal melanoma. Invest Ophthalmol Vis Sci, 2011, 52(3): 1193-1199. DOI: 10.1167/iovs.10-5272.
[9]
Callegari E, D'Abundo L, Guerriero P, et al. miR-199a-3p modulates MTOR and PAK4 pathways and inhibits tumor growth in a hepatocellular carcinoma transgenic mouse model.
Han Y, Kuang Y, Xue X, et al. NLK, a novel target of miR-199a- 3p, functions as a tumor suppressor in colorectal cancer. Biomed Pharmacother, 2014, 68(5): 497-505. DOI: 10.1016/j.biopha. 2014.05.003.
[11]
Cui Y, Wu F, Tian D, et al. miR-199a-3p enhances cisplatinsensitivity of ovarian cancer cells by targeting ITGB8. Oncol Rep, 2018, 39(4): 1649-1657. DOI: 10.3892/or.2018.6259.
Zhang B, Pan X, Cobb GP, et al. microRNAs as oncogenes and tumor suppressors. Dev Biol, 2007, 302(1): 1-12. DOI: 10.1016/j.ydbio.2006.08.028.
[15]
Li J, Smith AR, Marquez RT, et al. MicroRNA-383 acts as a tumor suppressor in colorectal cancer by modulating CREPT/ RPRD1B expression. Mol Carcinog, 2018, 57(10): 1408-1420. DOI: 10.1002/mc.22866.
[16]
Liang C, Shi S, Meng Q, et al. MiR-29a, targeting caveolin 2 expression, is responsible for limitation of pancreatic cancer metastasis in patients with normal level of serum CA125. Int J Cancer, 2018, 143(11): 2919-2931. DOI: 10.1002/ijc.31654.
[17]
Yan D, Dong Xda E, Chen X, et al. MicroRNA-1/206 targets c-Met and inhibits rhabdomyosarcoma development. J Biol Chem, 2009, 284(43): 29596-29604. DOI: 10.1074/jbc.M109.
02
0511.
[18]
Herr I, Sähr H, Zhao Z, et al. MiR-127 and miR-376a act as tumor suppressors by in vivo targeting of COA1 and PDIA6 in giant cell tumor of bone. Cancer Lett, 2017, 409: 49-55. DOI: 10.1016/j.canlet.2017.08.029.
[19]
Yu F, Liu JB, Wu ZJ, et al. Tumor suppressive microRNA-124a inhibits stemness and enhances gefitinib sensitivity of non-small cell lung cancer cells by targeting ubiquitin-specific protease 14. Cancer Lett, 2018, 427: 74-84. DOI: 10.1016/j.canlet.2018.04. 022.
[20]
Viallard JF, Lacombe F, Belloc F, et al. [Molecular mechanisms controlling the cell cycle: Fundamental aspects and implications for oncology. Cancer Radiother, 2001, 5(2): 109-129.
[21]
Vermeulen K, Van Bockstaele DR, Berneman ZN. The cell cycle: A review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif, 2003, 36(3): 131-149.
[22]
Leone G, DeGregori J, Yan Z, et al. E2F3 activity is regulated during the cell cycle and is required for the induction of S phase. Genes Dev, 1998, 12(14): 2120-2130.
[23]
An HX, Beckmann MW, Reifenberger G, et al. Gene amplification and overexpression of CDK4 in sporadic breast carcinomas is associated with high tumor cell proliferation. Am
Wölfel T, Hauer M, Schneider J, et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science, 1995, 269(5228): 1281-1284.
[25]
Rao PH, Houldsworth J, Dyomina K, et al. Chromosomal and genge amplofication in diffuse large B-cell lymphoma. Blood, 1998, 92(1): 234-240.