The effect of <i>NDRG1</i> gene on the angiogenesis ability of retinal endothelial cells in vitro_Chinese Journal of Ocular Fundus Diseases

Authors：

Cao Jingjing , Li Hui , Kou Zhenyu , Wu Guijia , Dong Lijie ,  Jiao Mingfei

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：

Jiao Mingfei, Email: jiaomingfei2004@163.com

Keywords：

NDRG1; Retinal vascular endothelial cell; Cell proliferation; Cell migration; Cell tube formation; Neovascularization; Cell experiment

DOI：

10.3760/cma.j.cn511434-20230529-00240

Video：

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Objective To observe the effects of NDRG1 on proliferation, migration and lumen formation of retinal vascular endothelial cells (RF/6A cells) in monkeys under high glucose condition. Methods RF/6A cells were divided into normal group, mannitol group, high glucose group, small interfering RNA (siRNA) negative control group without target gene (siRNA group), 30 nmol/L siRNA down-regulated NDRG1 genome (siNDRG1 group) and 50 nmol/L siNDRG1 group. Normal group cells were cultured conventionally. The mannitol group was added with 25 mmol/L mannitol, and the high-glucose group was added with 25 mmol/L glucose. In the siRNA group, 25 mmol/L glucose was added, and then blank siRNA was added for induction. The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1, respectively. All cells were incubated for 24 h for follow-up experiments. Cell proliferation was observed by 4', 6-diaminidine 2-phenylindole staining. Cell counting kit-8 staining was used to detect cell activity. The expression level of NDRG1 mRNA and protein was detected by Western blot and real-time quantitative polymerase chain reaction. Cell migration was observed by cell scratch assay. Cell lumen formation assay was used to detect lumen formation. The two-tailed Student t test was used to compare the two groups. One-way analysis of variance was used to compare groups. Results There were significant differences in cell proliferation rate (t=36.659, 57.645) mobility rate (t=24.745, 33.638) and lumen formation number (t=41.276, 22.867) between high glucose group and normal group and mannitol group (P＜0.01). Compared with normal group and mannitol group, the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased, with statistical significance (t=46.145, 21.541, 36.738, 32.976; P＜0.001). Compared with the siRNA negative group, the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased, and the differences were statistically significant (t=44.275, 40.7577, 57.167, 25.877; P＜0.01). Compared with normal group and siRNA group, cell mobility in 30 nmol/LsiNDRG1 group was increased, and the difference was statistically significant (t=57.562, 49.522; P＜0.01). Compared with normal group and siRNA group, the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision, and the difference was statistically significant (t=63.446, 42.742; P＜0.01). Conclusion Down-regulation of NDRG1 gene can improve the activity, migration and lumen formation of RF/6A cells under hyperglycemia.

Citation： Cao Jingjing, Li Hui, Kou Zhenyu, Wu Guijia, Dong Lijie, Jiao Mingfei. The effect of NDRG1 gene on the angiogenesis ability of retinal endothelial cells in vitro. Chinese Journal of Ocular Fundus Diseases, 2024, 40(7): 538-544. doi: 10.3760/cma.j.cn511434-20230529-00240 Copy

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18.	Zhao T, Meng Y, Wang Y, et al. NDRG1 regulates osteosarcoma cells via mediating the mitochondrial function and CSCs differentiation[J]. J Orthop Surg Res, 2021, 16(1): 364. DOI: 10.1186/s13018-021-02503-5.
19.	Huang H. Matrix metalloproteinase-9 (MMP-9) as a cancer biomarker and MMP-9 biosensors: recent advances[J]. Sensors (Basel), 2018, 18(10): 3249. DOI: 10.3390/s18103249.
20.	Wei M, Zhang Y, Yang X, et al. Claudin-2 promotes colorectal cancer growth and metastasis by suppressing NDRG1 transcription[J/OL]. Clin Transl Med, 2021, 11(12): e667[2021-12-01]. https://pubmed.ncbi.nlm.nih.gov/34965023/. DOI: 10.1002/ctm2.667.
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1. Klein BE. Overview of epidemiologic studies of diabetic retinopathy[J]. Ophthalmic Epidemiol, 2007, 14(4): 179-183. DOI: 10.1080/09286580701396720.
2. Yau JW, Rogers SL, Kawasaki R, et al. Global prevalence and major risk factors of diabetic retinopathy[J]. Diabetes Care, 2012, 35(3): 556-564. DOI: 10.2337/dc11-1909.
3. Boyer DS, Yoon YH, Belfort R, et al. Three-year, randomized, sham-controlled trial of dexamethasone intravitreal implant in patients with diabetic macular edema[J]. Ophthalmology, 2014, 121(10): 1904-1914. DOI: 10.1016/j.ophtha.2014.04.024.
4. Li X, Zhang P, Wang B, et al. NDRG1 negatively regulates proliferation and milk bio-synthesis of bovine epithelial cells via the mTOR signaling pathway[J]. Res Vet Sci, 2019, 124: 158-165. DOI: 10.1016/j.rvsc.2019.03.007.
5. Park KC, Paluncic J, Kovacevic Z, et al. Pharmacological targeting and the diverse functions of the metastasis suppressor, NDRG1, in cancer[J]. Free Radic Biol Med, 2020, 157: 154-175. DOI: 10.1016/j.freeradbiomed.2019.05.020.
6. Lin Y, Wang L, Luo W, et al. CYLD promotes apoptosis of nasopharyngeal carcinoma cells by regulating NDRG1[J]. Cancer Manag Res, 2020, 12: 10639-10649. DOI: 10.2147/CMAR.S268216.
7. Chekmarev J, Azad MG, Richardson DR. The oncogenic signaling disruptor, NDRG1: molecular and cellular mechanisms of activity[J]. Cells, 2021, 10(9): 2382. DOI: 10.3390/cells10092382.
8. Toffoli S, Delaive E, Dieu M, et al. NDRG1 and CRK-Ⅰ/Ⅱ are regulators of endothelial cell migration under intermittent hypoxia[J]. Angiogenesis, 2009, 12(4): 339-354. DOI: 10.1007/s10456-009-9156-2.
9. Dong L, Zhang Z, Liu X, et al. RNA sequencing reveals BMP4 as a basis for the dual-target treatment of diabetic retinopathy[J]. J Mol Med (Berl), 2021, 99(2): 225-240. DOI: 10.1007/s00109-020-01995-8.
10. Zhang Y, Lv X, Hu Z, et al. Protection of Mcc950 against high-glucose-induced human retinal endothelial cell dysfunction[J/OL]. Cell Death Dis, 2017, 8(7): e2941[2017-07-20]. https://pubmed.ncbi.nlm.nih.gov/28726778/. DOI: 10.1038/cddis.2017.308.
11. Rezzola S, Belleri M, Gariano G, et al. In vitro and ex vivo retina angiogenesis assays[J]. Angiogenesis, 2014, 17(3): 429-442. DOI: 10.1007/s10456-013-9398-x.
12. Kong H, Zhao H, Chen T, et al. Targeted P2X7/NLRP3 signaling pathway against inflammation, apoptosis, and pyroptosis of retinal endothelial cells in diabetic retinopathy[J]. Cell Death Dis, 2022, 13(4): 336. DOI: 10.1038/s41419-022-04786-w.
13. Watari K, Shibata T, Nabeshima H, et al. Impaired differentiation of macrophage lineage cells attenuates bone remodeling and inflammatory angiogenesis in NDRG1 deficient mice[J/OL]. Sci Rep, 2016, 6: 19470[2016-01-18]. https://pubmed.ncbi.nlm.nih.gov/26778110/. DOI: 10.1038/srep19470.
14. Li D, Mei H, Qi M, et al. FOXD3 is a novel tumor suppressor that affects growth, invasion, metastasis and angiogenesis of neuroblastoma[J]. Oncotarget, 2013, 4(11): 2021-2044. DOI: 10.18632/oncotarget.1579.
15. Zhang G, Qin Q, Zhang C, et al. NDRG1 signaling is essential for endothelial inflammation and vascular remodeling[J]. Circ Res, 2023, 132(3): 306-319. DOI: 10.1161/CIRCRESAHA.122.321837.
16. Matsushima K, Yang D, Oppenheim JJ. Interleukin-8: an evolving chemokine[J/OL]. Cytokine, 2022, 153: 155828[2022-03-02]. https://pubmed.ncbi.nlm.nih.gov/35247648/. DOI: 10.1016/j.cyto.2022.155828.
17. Lai LC, Su YY, Chen KC, et al. Down-regulation of NDRG1 promotes migration of cancer cells during reoxygenation[J/OL]. PLoS One, 2011, 6(8): e24375[2011-08-30]. https://pubmed.ncbi.nlm.nih.gov/21912630/. DOI: 10.1371/journal.pone.0024375.
18. Zhao T, Meng Y, Wang Y, et al. NDRG1 regulates osteosarcoma cells via mediating the mitochondrial function and CSCs differentiation[J]. J Orthop Surg Res, 2021, 16(1): 364. DOI: 10.1186/s13018-021-02503-5.
19. Huang H. Matrix metalloproteinase-9 (MMP-9) as a cancer biomarker and MMP-9 biosensors: recent advances[J]. Sensors (Basel), 2018, 18(10): 3249. DOI: 10.3390/s18103249.
20. Wei M, Zhang Y, Yang X, et al. Claudin-2 promotes colorectal cancer growth and metastasis by suppressing NDRG1 transcription[J/OL]. Clin Transl Med, 2021, 11(12): e667[2021-12-01]. https://pubmed.ncbi.nlm.nih.gov/34965023/. DOI: 10.1002/ctm2.667.
21. Takita S, Wada Y, Kawamura S. Effects of NDRG1 family proteins on photoreceptor outer segment morphology in zebrafish[J/OL]. Sci Rep, 2016, 6: 36590[2016-11-04]. https://pubmed.ncbi.nlm.nih.gov/27811999/. DOI: 10.1038/srep36590.

Chinese Journal of Ocular Fundus Diseases

The effect of NDRG1 gene on the angiogenesis ability of retinal endothelial cells in vitro

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