Influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium cells_Chinese Journal of Ocular Fundus Diseases

Authors：

YuTian ,  XingYiqiao , ChenChangzheng

Department of Ophthalmology, Renmin Hospital of Wuhan University, Wuhan 430060, China;

Corresponding author：

XingYiqiao, Email: xing_yiqiao@aliyun.com

Keywords：

Retinal pigment epithelium/physiopathology; Vascular endothelial growth factor A; RNA interference; Serine proteases; Photic stimulation/adverse effects; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2016.04.016

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells. Methods Cultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000±500) Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively. Results The mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62, 15.09; P<0.05). Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37, 4.52), migration (t=9.56, 12.13), apoptosis (t=23.37, 29.08) and decreased mRNA (t=17.36, 11.32, 7.29, 4.05) and protein levels (t=12.02, 15.28, 4.98, 6.24) of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant (t=6.24, 4.93; P<0.05). Conclusion Knockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.

Citation： YuTian, XingYiqiao, ChenChangzheng. Influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium cells. Chinese Journal of Ocular Fundus Diseases, 2016, 32(4): 413-417. doi: 10.3760/cma.j.issn.1005-1015.2016.04.016 Copy

1.	Black JR, Clark SJ. Age-related macular degeneration:genome-wide association studies to translation[J].Genet Med, 2016,18(4):283-289.DOI:10.1038/gim.2015.70.
2.	Tuo J, Ross RJ, Reed GF, et al. The HtrA1 promoter polymorphism, smoking, and age-related macular degeneration in multiple case-control samples[J]. Ophthalmology, 2008,115(11):1891-1898. DOI:10.1016/j.ophtha.2008.05.021.
3.	Kumar S, Berriochoa Z, Ambati BK, et al. Angiographic features of transgenic mice with increased expression of human serine protease HtrA1 in retinal pigment epithelium[J]. Invest Ophthalmol Vis Sci, 2014,55(6):3842-3850.DOI:10.1167/iovs.13-13111.
4.	Smith BT, Belani S, Ho AC. Ultraviolet and near-blue light effects on the eye[J]. Int Ophthalmol Clin, 2005,45(1):107-115.
5.	Margrain TH, Boulton M, Marshall J, et al. Do blue light filters confer protection against age-related macular degeneration?[J]. Prog Retin Eye Res, 2004,23(5):523-531.
6.	Kernt M, Neubauer AS, Liegl RG, et al. Sorafenib prevents human retinal pigment epithelium cells from light-induced overexpression of VEGF,PDGF and PIGF[J]. Br J Ophthalmol, 2010,94(11):1533-1539.DOI:10.1136/bjo.2010.182162.
7.	蔡善君, 严密, 张军军. 蓝光诱导体外培养的人视网膜色素上皮细胞凋亡[J].中华眼底病杂志, 2005,21(6):384-387. Cai SJ,Yan M,Zhang JJ.Blue-light-induced apoptosis of cultured human retinal pigment epithelial cells in vitro[J].Chin J Ocul Fundus Dis,2005, 21(6):384-387.
8.	Jones A, Kumar S, Zhang N, et al. Increased expression of multifunctional serine protease, HTRA1, in retinal pigment epithelium induces polypoidal choroidal vasculopathy in mice[J]. Proc Natl Acad Sci USA, 2011,108(35):14578-14583.DOI:10.1073/pnas.1102853108.
9.	Nie GY, Hampton A, Li Y, et al. Identification and cloning of two isoforms of human high-temperature requirement factor A3(HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2[J].Biochem J,2003,371(Pt 1):39-48.
10.	Pei X, Ma K, Xu J, et al. Inhibition of cell proliferation and migration after HTRA1 knockdown in retinal pigment epithelial cells[J]. Graefe's Arch Clin Exp Ophthalmol, 2015,253(4):565-572.DOI:10.1007/s00417-014-2901-2.
11.	Supanji, Shimomachi M, Hasan MZ, et al. HtrA1 is induced by oxidative stress and enhances cell senescence through p38 MAPK pathway[J]. Exp Eye Res, 2013,112:79-92.DOI:10.1016/j.exer.2013.04.013.
12.	Mullany SA,Moslemi-Kebria M, Rattan R, et al. Expression and functional significance of HtrA1 loss in endometrial cancer[J]. Clin Cancer Res, 2011,17(3):427-436. DOI:10.1158/1078-0432.CCR-09-3069.
13.	Oka C, Tsujimoto R, Kajikawa M, et al. HtrA1 serine protease inhibits signaling mediated by TGFbeta family proteins[J]. Development, 2004,131(5):1041-1053.
14.	Ng TK, Yam GH, Chen WQ, et al. Interactive expressions of HtrA1 and VEGF in human vitreous humors and fetal RPE cells[J]. Invest Ophthalmol Vis Sci, 2011,52(6):3706-3712.DOI:10.1167/iovs.10-6773.
15.	潘颖喆, 邢怡桥,陈长征,等. 抑制光损伤人视网膜色素上皮细胞血管内皮生长因子A的表达对趋化因子受体3配体表达的影响[J]. 中华眼底病杂志, 2015,31(1):62-66. DOI:10.3760/cma.j.issn.1005-1015.2015.01.016. Pan YZ, Xing YQ, Chen CZ, et al. Down-regulated expression of CC chemokine receptor 3 ligands in ranibizumab treated light-injured human retinal pigment epithelium cells[J]. Chin J Ocul Fundus Dis, 2015, 31(1):62-66. DOI:10.3760/cma.j.issn.1005-1015.2015.01.016.

1. Black JR, Clark SJ. Age-related macular degeneration:genome-wide association studies to translation[J].Genet Med, 2016,18(4):283-289.DOI:10.1038/gim.2015.70.
2. Tuo J, Ross RJ, Reed GF, et al. The HtrA1 promoter polymorphism, smoking, and age-related macular degeneration in multiple case-control samples[J]. Ophthalmology, 2008,115(11):1891-1898. DOI:10.1016/j.ophtha.2008.05.021.
3. Kumar S, Berriochoa Z, Ambati BK, et al. Angiographic features of transgenic mice with increased expression of human serine protease HtrA1 in retinal pigment epithelium[J]. Invest Ophthalmol Vis Sci, 2014,55(6):3842-3850.DOI:10.1167/iovs.13-13111.
4. Smith BT, Belani S, Ho AC. Ultraviolet and near-blue light effects on the eye[J]. Int Ophthalmol Clin, 2005,45(1):107-115.
5. Margrain TH, Boulton M, Marshall J, et al. Do blue light filters confer protection against age-related macular degeneration?[J]. Prog Retin Eye Res, 2004,23(5):523-531.
6. Kernt M, Neubauer AS, Liegl RG, et al. Sorafenib prevents human retinal pigment epithelium cells from light-induced overexpression of VEGF,PDGF and PIGF[J]. Br J Ophthalmol, 2010,94(11):1533-1539.DOI:10.1136/bjo.2010.182162.
7. 蔡善君, 严密, 张军军. 蓝光诱导体外培养的人视网膜色素上皮细胞凋亡[J].中华眼底病杂志, 2005,21(6):384-387. Cai SJ,Yan M,Zhang JJ.Blue-light-induced apoptosis of cultured human retinal pigment epithelial cells in vitro[J].Chin J Ocul Fundus Dis,2005, 21(6):384-387.
8. Jones A, Kumar S, Zhang N, et al. Increased expression of multifunctional serine protease, HTRA1, in retinal pigment epithelium induces polypoidal choroidal vasculopathy in mice[J]. Proc Natl Acad Sci USA, 2011,108(35):14578-14583.DOI:10.1073/pnas.1102853108.
9. Nie GY, Hampton A, Li Y, et al. Identification and cloning of two isoforms of human high-temperature requirement factor A3(HtrA3), characterization of its genomic structure and comparison of its tissue distribution with HtrA1 and HtrA2[J].Biochem J,2003,371(Pt 1):39-48.
10. Pei X, Ma K, Xu J, et al. Inhibition of cell proliferation and migration after HTRA1 knockdown in retinal pigment epithelial cells[J]. Graefe's Arch Clin Exp Ophthalmol, 2015,253(4):565-572.DOI:10.1007/s00417-014-2901-2.
11. Supanji, Shimomachi M, Hasan MZ, et al. HtrA1 is induced by oxidative stress and enhances cell senescence through p38 MAPK pathway[J]. Exp Eye Res, 2013,112:79-92.DOI:10.1016/j.exer.2013.04.013.
12. Mullany SA,Moslemi-Kebria M, Rattan R, et al. Expression and functional significance of HtrA1 loss in endometrial cancer[J]. Clin Cancer Res, 2011,17(3):427-436. DOI:10.1158/1078-0432.CCR-09-3069.
13. Oka C, Tsujimoto R, Kajikawa M, et al. HtrA1 serine protease inhibits signaling mediated by TGFbeta family proteins[J]. Development, 2004,131(5):1041-1053.
14. Ng TK, Yam GH, Chen WQ, et al. Interactive expressions of HtrA1 and VEGF in human vitreous humors and fetal RPE cells[J]. Invest Ophthalmol Vis Sci, 2011,52(6):3706-3712.DOI:10.1167/iovs.10-6773.
15. 潘颖喆, 邢怡桥,陈长征,等. 抑制光损伤人视网膜色素上皮细胞血管内皮生长因子A的表达对趋化因子受体3配体表达的影响[J]. 中华眼底病杂志, 2015,31(1):62-66. DOI:10.3760/cma.j.issn.1005-1015.2015.01.016. Pan YZ, Xing YQ, Chen CZ, et al. Down-regulated expression of CC chemokine receptor 3 ligands in ranibizumab treated light-injured human retinal pigment epithelium cells[J]. Chin J Ocul Fundus Dis, 2015, 31(1):62-66. DOI:10.3760/cma.j.issn.1005-1015.2015.01.016.

Chinese Journal of Ocular Fundus Diseases

Influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium cells

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content