miR-191 inhibits retinal vascular endothelial cell proliferation and angiogenesis_Chinese Journal of Ocular Fundus Diseases

Authors：

Zhao Qi , An Weiting , Liu Boshi , Xu Manhong , Dong Lijie , Li Xiaorong ,  Han Jindong

Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin 300384, China;

Corresponding author：

Han Jindong, Email: djindonghan@126.com

Keywords：

MicroRNAs; Endothelium, vascular; Transfection; Hyperplasia

DOI：

10.3760/cma.j.cn511434-20210406-00176

Video：

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Objective To observe the inhibitory effect of lentivirus (LV)-mediated miR-191 on the proliferation and angiogenesis of human retinal vascular endothelial cells (hREC) cultured in vitro.Methods The hREC cell lines were cultured in vitro and divided into control group, hypoxia group, LV-empty vector (LV-vector) group, and LV-miR-191 (LV-191) group. The LV-vector group and LV-191 group were transferred to the corresponding lentiviral vector respectively. Flow cytometry was used to detect cell transfection efficiency. Cell Counting Kit-8 (CCK-8) test was used to detect cell proliferation ability. Scarification test and invasion chamber (Transwell) test were used to detect cell migration ability. Matrigel test was used to detect cell lumen formation ability. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the relative expression of miR-191 and relative mRNA expression of its downstream target genes p21, vascular endothelial growth factor (VEGF), cell division protein kinase (CDK) 6, cyclin-D1 (Cyclin D1). Independent sample t test was used for pairwise comparison. Results The results of flow cytometry showed that the transfection efficiency of cells in the control group and the LV-191 group were 0.615% and 99.400%, respectively. The results of CCK-8, scarification, Transwell and Matrigel test showed that, compared with the control group, the number of cell proliferation (t=6.130, 4.606), the cell mobility (t=4.910, 6.702), the number of stained cells on the microporous membrane (t=7.244, 6.724) and the lumen formation ability cells (t=8.345, 9.859) were significantly increased in the hypoxia group and the LV-vector group (P<0.01), while the LV-191 group showed completely opposite performance (t=14.710, 6.245, 5.333, 5.892; P≤0.01). The qPCR test results showed that, compared with the control group and the LV-vector group, the relative expression of miR-191 mRNA in the cells of the LV-191 group was significantly up-regulated (t=44.110, 42.680), the relative expression of Cyclin D1 mRNA (t=29.940, 14.010) and CDK6 mRNA (t=15.200, 7.645) decreased significantly, and the difference were statistically significant (P<0.01); the relative expression of p21 mRNA increased, however, the difference was not statistically significant (t=2.013, 2.755; P>0.05). There was no significant difference in the relative expression of VEGF mRNA in the 4 groups of cells (F=0.966, P>0.05). Conclusions LV-191 can inhibit the proliferation, migration and tubing of hREC by up-regulating p21 and down-regulating CDK6 and Cyclin D1.

Citation： Zhao Qi, An Weiting, Liu Boshi, Xu Manhong, Dong Lijie, Li Xiaorong, Han Jindong. miR-191 inhibits retinal vascular endothelial cell proliferation and angiogenesis. Chinese Journal of Ocular Fundus Diseases, 2022, 38(1): 49-55. doi: 10.3760/cma.j.cn511434-20210406-00176 Copy

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13.	Wang XH, Chen L. MicroRNA-370 suppresses the retinal capillary endothelial cell growth by targeting KDR gene[J]. Bratisl Lek Listy, 2017, 118(4): 202-207. DOI: 10.4149/BLL_2017_040.
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22.	Han J, Li N. Adenoviral vector-mediated delivery of p21^WAF1/CIP1 prevents retinal neovascularization in an oxygen-induced retinopathy model[J]. Curr Eye Res, 2016, 41(8): 1113-1117. DOI: 10.3109/02713683.2015.1090002.

1. Hou XR, Miao H, Tao Y, et al. Expression of cytokines on the iris of patients with neovascular glaucoma[J]. Acta Ophthalmol, 2015, 93(2): 100-104. DOI: 10.1111/aos.12510.
2. Miao H, Hou X, Hwang DK, et al. Vascular endothelial growth factor, basic fibroblast growth factor, and pigment epithelium-derived factor expression in the neovascular iris in retinal diseases[J/OL]. J Ophthalmol, 2018, 2018: 8025951[2018-04-11]. https://pubmed.ncbi.nlm.nih.gov/29850214/. DOI:10.1155/2018/8025951.
3. Trzybulska D, Vergadi E, Tsatsanis C. miRNA and other non-coding RNAs as promising diagnostic markers[J]. EJIFCC, 2018, 29(3): 221-226.
4. Han S, Kong YC, Sun B, et al. microRNA-218 inhibits oxygen-induced retinal neovascularization via reducing the expression of roundabout 1[J]. Chin Med J (Engl), 2016, 129(6): 709-715. DOI: 10.4103/0366-6999.178013.
5. Nagpal N, Kulshreshtha R. miR-191: an emerging player in disease biology[J/OL]. Front Genet, 2014, 5: 99[2014-04-23]. https://pubmed.ncbi.nlm.nih.gov/24795757/. DOI: 10.3389/fgene.2014.00099.
6. 张璐, 李妍, 胡竹林. 慢病毒转染在眼科疾病中的应用[J]. 国际眼科纵览, 2018, 42(4): 227-231. DOI: 10.3760/cma.j.issn.1673-5803.2018.04.003.Zhang L, Li Y, Hu ZL. Application of lentiviral transfection in ophthalmic diseases[J]. Int Rev Ophthalmol, 2018, 42(4): 227-231. DOI: 10.3760/cma.j.issn.1673-5803.2018.04.003.
7. Annabi B, Lee YT, Turcotte S, et al. Hypoxia promotes murine bone-marrow-derived stromal cell migration and tube formation[J]. Stem Cells, 2003, 21(3): 337-347. DOI: 10.1634/stemcells.21-3-337.
8. Nebenfuehr S, Kollmann K, Sexl V. The role of CDK6 in cancer[J]. Int J Cancer, 2020, 147(11): 2988-2995. DOI: 10.1002/ijc.33054.
9. Mansilla SF, de la Vega MB, Calzetta NL, et al. CDK-Independent and PCNA-dependent functions of p21 in DNA replication[J]. Genes (Basel), 2020, 11(6): 593. DOI: 10.3390/genes11060593.
10. Lim S, Kaldis P. CDKs, Cyclins and CKIs: roles beyond cell cycle regulation[J]. Development, 2013, 140(15): 3079-3093. DOI: 10.1242/dev.091744.
11. Pichler M, Stiegelbauer V, Vychytilova-Faltejskova P, et al. Genome-wide miRNA analysis identifies miR-188-3p as a novel prognostic marker and molecular factor involved in colorectal carcinogenesis[J]. Clin Cancer Res, 2017, 23(5): 1323-1333. DOI: 10.1158/1078-0432.CCR-16-0497.
12. Yan R, Yang T, Zhai H, et al. MicroRNA-150-5p affects cell proliferation, apoptosis, and EMT by regulation of the BRAF(V600E) mutation in papillary thyroid cancer cells[J]. J Cell Biochem, 2018, 119(11): 8763-8772. DOI: 10.1002/jcb.27108.
13. Wang XH, Chen L. MicroRNA-370 suppresses the retinal capillary endothelial cell growth by targeting KDR gene[J]. Bratisl Lek Listy, 2017, 118(4): 202-207. DOI: 10.4149/BLL_2017_040.
14. Diao Y, Jin B, Huang L, et al. MiR-129-5p inhibits glioma cell progression in vitro and in vivo by targeting TGIF2[J]. J Cell Mol Med, 2018, 22(4): 2357-2367. DOI: 10.1111/jcmm.13529.
15. Wang YF, Yang HY, Shi XQ, et al. Upregulation of microRNA-129-5p inhibits cell invasion, migration and tumor angiogenesis by inhibiting ZIC2 via downregulation of the Hedgehog signaling pathway in cervical cancer[J]. Cancer Biol Ther, 2018, 19(12): 1162-1173. DOI: 10.1080/15384047.2018.1491497.
16. Tian F, Yu C, Wu M, et al. MicroRNA-191 promotes hepatocellular carcinoma cell proliferation by has_circ_0000204/miR-191/KLF6 axis[J/OL]. Cell Prolif, 2019, 52(5): e12635[2019-07-13]. https://pubmed.ncbi.nlm.nih.gov/31334580/. DOI: 10.1111/cpr.12635.
17. Gu Y, Ampofo E, Menger MD, et al. miR-191 suppresses angiogenesis by activation of NF-κB signaling[J]. FASEB J, 2017, 31(8): 3321-3333. DOI: 10.1096/fj.201601263R.
18. Lena AM, Mancini M, Rivetti di Val Cervo P, et al. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation[J]. Biochem Biophys Res Commun, 2012, 423(3): 509-514. DOI: 10.1016/j.bbrc.2012.05.153.
19. Colamaio M, Borbone E, Russo L, et al. miR-191 down-regulation plays a role in thyroid follicular tumors through CDK6 targeting[J/OL]. J Clin Endocrinol Metab, 2011, 96(12): E1915-1924[2011-09-28]. https://pubmed.ncbi.nlm.nih.gov/21956418/. DOI: 10.1210/jc.2011-0408.
20. Di Leva G, Piovan C, Gasparini P, et al. Estrogen mediated-activation of miR-191/425 cluster modulates tumorigenicity of breast cancer cells depending on estrogen receptor status[J/OL]. PLoS Genet, 2013, 9(3): e1003311[2013-03-07]. https://pubmed.ncbi.nlm.nih.gov/23505378/. DOI: 10.1371/journal.pgen.1003311.
21. Kim JH, Lee BJ, Kim JH, et al. Rosmarinic acid suppresses retinal neovascularization via cell cycle arrest with increase of p21(WAF1) expression[J]. Eur J Pharmacol, 2009, 615(1-3): 150-154. DOI: 10.1016/j.ejphar.2009.05.015.
22. Han J, Li N. Adenoviral vector-mediated delivery of p21^WAF1/CIP1 prevents retinal neovascularization in an oxygen-induced retinopathy model[J]. Curr Eye Res, 2016, 41(8): 1113-1117. DOI: 10.3109/02713683.2015.1090002.

Chinese Journal of Ocular Fundus Diseases

miR-191 inhibits retinal vascular endothelial cell proliferation and angiogenesis

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