Effects of pyrimidine bundle-binding protein-associated splicing factors on the function of hypoxia-induced human retinal microvascular endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Xu Manhong , Wang Linni , Lin Tingting , Ren Xinjun , Ke Yifeng , Hu Liying , Jiao Mingfei , Wang Yong , Wang Qiong , Hong Yaru ,  Li Xiaorong , Dong Lijie

Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, College of Optometry and Ophthalmology, Tianjin 300384, China;

Corresponding author：

Li Xiaorong, Email: xiaorli@163.com

Keywords：

Polypyrimidine tract-binding protein; Retinal neovascularization; Anoxia; Retinal vessels/cytology; Endothelial cells/physiology; Cells, cultured

DOI：

10.3760/cma.j.issn.1005-1015.2020.02.010

Video：

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Objective To observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).Methods A three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.Results The LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).Conclusion PSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.

Citation： Xu Manhong, Wang Linni, Lin Tingting, Ren Xinjun, Ke Yifeng, Hu Liying, Jiao Mingfei, Wang Yong, Wang Qiong, Hong Yaru, Li Xiaorong, Dong Lijie. Effects of pyrimidine bundle-binding protein-associated splicing factors on the function of hypoxia-induced human retinal microvascular endothelial cells. Chinese Journal of Ocular Fundus Diseases, 2020, 36(2): 135-142. doi: 10.3760/cma.j.issn.1005-1015.2020.02.010 Copy

1.	田芳, 东莉洁, 吉洁, 等. 多聚嘧啶序列结合蛋白相关剪接因子对视网膜血管内皮细胞IGF-1/VEGF信号通路的抑制作用[J]. 中华实验眼科杂志, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.Tian F, Dong LJ, Ji J, et al. Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling pathway in retinal vascular endothelial cells[J]. Chin J Exp Ophthalmol, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.
2.	张哲, 刘巨平, 东莉洁, 等. 高糖状态下视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J]. 中华眼底病杂志, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.Zhang Z, Liu JP, Dong LJ, et al. RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition[J]. Chin J Ocul Fundus Dis, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.
3.	牛瑞, 东莉洁, 马腾, 等. 抗血管内皮生长因子药物治疗后视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J]. 中华眼底病杂志, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.Niu R, Dong LJ, Ma T, et al. RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothelial growth factor treatment[J]. Chin J Ocul Fundus Dis, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.
4.	王礼明, 东莉洁, 刘勋, 等. 急性原发性闭角型青光眼急性发作期房水蛋白质组学分析[J]. 中华眼科杂志, 2019, 55(9): 687-694. DOI: 10.3760/cma.j.issn.0412-4081.2019.09.011.Wang LM, Dong LJ, Liu X, et al. Proteomic analysis of aqueous humor in acute primary angle closure glaucoma[J]. Chin J Ophthalmol, 2019, 55(9): 687-694. DOI: 10.3760/cma.j.issn.0412-4081.2019.09.011.
5.	温德佳, 任新军, 东莉洁, 等. 应用iTRAQ蛋白组技术筛选增生性糖尿病视网膜病变防治靶点的研究[J]. 中华眼科杂志, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.Wen DJ, Ren XJ, Dong LJ, et al. New exploration of treatment target for proliferative diabetic retinopathy based on iTRAQ LC-MS/MS Proteomics[J]. Chin J Ophthalmol, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.
6.	Dong L, Zhang X, Fu X, et al. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1[J]. J Biol Chem, 2011, 286(5): 3451-3459. DOI: 10.1074/jbc.M110.168377.
7.	漆晨, 张慧, 林婷婷, 等. 聚嘧啶束结合蛋白相关剪接因子高表达对糖基化终末产物诱导下视网膜色素上皮细胞损伤的保护作用[J]. 中华眼底病杂志, 2020, 36(1): 1-7. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.Qi C, Zhang H, Lin TT, et al. Protective effect of polypyrimidine bundle-binding protein-related splicing factor on retinal pigment epithelial cell injury induced by advanced glycation end products[J]. Chin J Ocul Fundus Dis, 2020, 36(1): 1-7. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.
8.	漆晨, 东莉洁, 乐毅, 等. 多聚嘧啶序列结合蛋白相关剪接因子对体外培养的视网膜色素上皮细胞磷脂酰肌醇3激酶/丝氨酸-苏氨酸蛋白激酶信号通路的调控作用[J]. 中华眼底病杂志, 2015(4): 363-367. DOI: 10.3760/cma.j.issn.1005-1015.2015.04.013.Qi C, Dong LJ, Yue Y, et al. The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in retinal pigment epithelial cells[J]. Chin J Ocul Fundus Dis, 2015(4): 363-367. DOI: 10.3760/cma.j.issn.1005-1015.2015.04.013.
9.	Dong L, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2): 233-243. DOI: 10.1007/s00441-014-2104-5.
10.	Dalkara D, Goureau O1, Marazova K, et al. Let there be light: gene and cell therapy for blindness[J]. Hum Gene Ther, 2016, 27(2): 134-47. DOI: 10.1089/hum.2015.147.
11.	Sengillo JD, Justus S, Tsai YT, et al. Gene and cell-based therapies for inherited retinal disorders: an update[J]. Am J Med Genet C Semin Med Genet, 2016, 172(4): 349-366. DOI: 10.1002/ajmg.c.31534.
12.	Annoni A, Gregori S, Naldini L, et al. Modulation of immune responses in lentiviral vector-mediated gene transfer [J/OL]. Cell Immunol, 2018, 342:103802[2018-04-27]. https://linkinghub.elsevier.com/retrieve/pii/S0008-8749(18)30187-4. DOI:10.1016/j.cellimm.2018.04.012.
13.	刘勃实, 东莉洁, 李筱荣, 等. 慢病毒介导的微小RNA-191对小鼠视网膜新生血管的抑制作用[J]. 中华眼底病杂志, 2019, 35(5): 475-479. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.010.Liu BS, Dong LJ, Li XR, et al. miR-191 inhibits oxygen-induced retinal neovascularization in mice[J]. Chin J Ocul Fundus Dis, 2019, 35(5): 475-479. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.010.

1. 田芳, 东莉洁, 吉洁, 等. 多聚嘧啶序列结合蛋白相关剪接因子对视网膜血管内皮细胞IGF-1/VEGF信号通路的抑制作用[J]. 中华实验眼科杂志, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.Tian F, Dong LJ, Ji J, et al. Inhibition of PTB-associated splicing factor on IGF-1/VEGF signaling pathway in retinal vascular endothelial cells[J]. Chin J Exp Ophthalmol, 2016, 34(1): 11-16. DOI: 10.3760/cma.j.issn.2095-0160.2016.01.003.
2. 张哲, 刘巨平, 东莉洁, 等. 高糖状态下视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J]. 中华眼底病杂志, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.Zhang Z, Liu JP, Dong LJ, et al. RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition[J]. Chin J Ocul Fundus Dis, 2018, 34(4): 377-381. DOI: 10.3760/cma.j.issn.1005-1015.2018.04.014.
3. 牛瑞, 东莉洁, 马腾, 等. 抗血管内皮生长因子药物治疗后视网膜血管内皮细胞基因表达谱的RNA-Seq分析[J]. 中华眼底病杂志, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.Niu R, Dong LJ, Ma T, et al. RNA-Seq analysis of gene expression profiling in human retinal vascular endothelial cells after anti-vascular endothelial growth factor treatment[J]. Chin J Ocul Fundus Dis, 2018, 34(3): 275-280. DOI: 10.3760/cma.j.issn.1005-1015.2018.03.016.
4. 王礼明, 东莉洁, 刘勋, 等. 急性原发性闭角型青光眼急性发作期房水蛋白质组学分析[J]. 中华眼科杂志, 2019, 55(9): 687-694. DOI: 10.3760/cma.j.issn.0412-4081.2019.09.011.Wang LM, Dong LJ, Liu X, et al. Proteomic analysis of aqueous humor in acute primary angle closure glaucoma[J]. Chin J Ophthalmol, 2019, 55(9): 687-694. DOI: 10.3760/cma.j.issn.0412-4081.2019.09.011.
5. 温德佳, 任新军, 东莉洁, 等. 应用iTRAQ蛋白组技术筛选增生性糖尿病视网膜病变防治靶点的研究[J]. 中华眼科杂志, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.Wen DJ, Ren XJ, Dong LJ, et al. New exploration of treatment target for proliferative diabetic retinopathy based on iTRAQ LC-MS/MS Proteomics[J]. Chin J Ophthalmol, 2019, 55(10): 769-776. DOI: 10.3760/cma.j.issn.0412-4081.2019.10.008.
6. Dong L, Zhang X, Fu X, et al. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1[J]. J Biol Chem, 2011, 286(5): 3451-3459. DOI: 10.1074/jbc.M110.168377.
7. 漆晨, 张慧, 林婷婷, 等. 聚嘧啶束结合蛋白相关剪接因子高表达对糖基化终末产物诱导下视网膜色素上皮细胞损伤的保护作用[J]. 中华眼底病杂志, 2020, 36(1): 1-7. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.Qi C, Zhang H, Lin TT, et al. Protective effect of polypyrimidine bundle-binding protein-related splicing factor on retinal pigment epithelial cell injury induced by advanced glycation end products[J]. Chin J Ocul Fundus Dis, 2020, 36(1): 1-7. DOI: 10.3760/cma.j.issn.1005-1015.2020.01.
8. 漆晨, 东莉洁, 乐毅, 等. 多聚嘧啶序列结合蛋白相关剪接因子对体外培养的视网膜色素上皮细胞磷脂酰肌醇3激酶/丝氨酸-苏氨酸蛋白激酶信号通路的调控作用[J]. 中华眼底病杂志, 2015(4): 363-367. DOI: 10.3760/cma.j.issn.1005-1015.2015.04.013.Qi C, Dong LJ, Yue Y, et al. The regulation of PTB-associated splicing factor on phosphatidylinositol 3 kinase/Akt signaling pathway in retinal pigment epithelial cells[J]. Chin J Ocul Fundus Dis, 2015(4): 363-367. DOI: 10.3760/cma.j.issn.1005-1015.2015.04.013.
9. Dong L, Nian H, Shao Y, et al. PTB-associated splicing factor inhibits IGF-1-induced VEGF upregulation in a mouse model of oxygen-induced retinopathy[J]. Cell Tissue Res, 2015, 360(2): 233-243. DOI: 10.1007/s00441-014-2104-5.
10. Dalkara D, Goureau O1, Marazova K, et al. Let there be light: gene and cell therapy for blindness[J]. Hum Gene Ther, 2016, 27(2): 134-47. DOI: 10.1089/hum.2015.147.
11. Sengillo JD, Justus S, Tsai YT, et al. Gene and cell-based therapies for inherited retinal disorders: an update[J]. Am J Med Genet C Semin Med Genet, 2016, 172(4): 349-366. DOI: 10.1002/ajmg.c.31534.
12. Annoni A, Gregori S, Naldini L, et al. Modulation of immune responses in lentiviral vector-mediated gene transfer [J/OL]. Cell Immunol, 2018, 342:103802[2018-04-27]. https://linkinghub.elsevier.com/retrieve/pii/S0008-8749(18)30187-4. DOI:10.1016/j.cellimm.2018.04.012.
13. 刘勃实, 东莉洁, 李筱荣, 等. 慢病毒介导的微小RNA-191对小鼠视网膜新生血管的抑制作用[J]. 中华眼底病杂志, 2019, 35(5): 475-479. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.010.Liu BS, Dong LJ, Li XR, et al. miR-191 inhibits oxygen-induced retinal neovascularization in mice[J]. Chin J Ocul Fundus Dis, 2019, 35(5): 475-479. DOI: 10.3760/cma.j.issn.1005-1015.2019.05.010.

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Chinese Journal of Ocular Fundus Diseases

Effects of pyrimidine bundle-binding protein-associated splicing factors on the function of hypoxia-induced human retinal microvascular endothelial cells

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