The effects of down-regulation of Claudin-3 on the cultured retinal ganglion cells in vitro_Chinese Journal of Ocular Fundus Diseases

Authors：

Cao Di , Zhang Xiaomei , Lu Jing , Wang Xiao , Liu Ruyuan , Liu Qiuhui ,  Luo Yan , Lyu Lin

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Guangzhou 510060, China;

Corresponding author：

Luo Yan, Email: luoyan2@mail.sysu.edu.cn

Keywords：

Retinal ganglion cells/physiology; Claudins; RNA, small interfering; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2017.02.013

Video：

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Objective To study the effect of down-regulation of Claudin-3 mediated by adeno-associated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro. Methods RGCs isolated from mouse eyes were divided into normal control group, AAV-shScramble group, and AAV-shClaudin-3 group. The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding. Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection. Immunofluorescent staining of β-tubulin was used to measure the length of RGCs′ axon. 4′, 6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells. The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction. The protein levels of Claudin-3, vascular endothelial growth factor (VEGF), Bcl-2 and Caspase-3 was determined by Western blot. Results The positive transfection rate was more than 50% in both AAV-shScramble group and AAV-shClaudin-3 group. The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAV-shScramble group (F=22 363.274,P＜0.05). Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage, tapering, and nucleolus formation of apoptotic bodies. The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408, 160.533;P＜0.05). The protein levels of Claudin-3, VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671, 420.552, 62.669;P＜0.05), while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P＜0.05). Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3, reduces the expression of VEGF and Bcl-2, accelerates RGCs' apoptosis and inhibit the RGCs' axon growth.

Citation： Cao Di, Zhang Xiaomei, Lu Jing, Wang Xiao, Liu Ruyuan, Liu Qiuhui, Luo Yan, Lyu Lin. The effects of down-regulation of Claudin-3 on the cultured retinal ganglion cells in vitro. Chinese Journal of Ocular Fundus Diseases, 2017, 33(2): 166-171. doi: 10.3760/cma.j.issn.1005-1015.2017.02.013 Copy

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12.	Hubenia OV, Minchenko DO, Minchenko OH, et al. Changing of the expression of VEGF genes encoded important regulators of angiogenesis and neurogenesis under hypoxic and ischemic conditions[J]. Lik Sprava, 2012(7): 103-107.
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1. Li CP, Wang SH, Wang WQ, et al. Long Noncoding RNA-Sox2OT knockdown alleviates diabetes mellitus-induced retinal ganglion cell (RGC) injury[J]. Cell Mol Neurobiol, 2017, 37(2): 361-369. DOI: 10.1007/s10571-016-0380-1.
2. Wang AL, Carroll RC, Nawy S.Down-regulation of the RNA editing enzyme ADAR2 contributes to RGC death in a mouse model of glaucoma[J/OL]. PloS One, 2014, 9(3): 91288 [2014-03-07]. . DOI: 10.1371/ journal.pone.0091288.
3. Luo Y, Xiao W, Zhu X, et al. Differential expression of claudins in retinas during normal development and the angiogenesis of oxygen-induced retinopathy[J]. Invest Ophthalmol Vis Sci, 2011, 52(10): 7556-7564. doi: 10.1167/iovs.11-7185.
4. Haines RJ, Beard RS Jr3, Chen L, et al. Interleukin-1β mediates β-catenin-driven downregulation of claudin-3 and barrier dysfunction in Caco2 cells[J]. Dig Dis Sci, 2016, 61(8): 2252-2261. DOI: 10.1007/s10620-016-4145-y.
5. Mansoori B, Sandoghchian Shotorbani S, Baradaran B.RNA interference and its role in cancer therapy[J]. Adv Pharm Bull, 2014, 4(4): 313-321. DOI: 10.5681/apb.2014.046.
6. Flotte TR.Adeno-associated virus-mediated gene transfer for lung diseases[J]. Human Gene Ther, 2005, 16(6): 643-648.
7. Romano G.Current development of adeno-associated viral vectors[J]. Drug News Perspect, 2005, 18(5): 311-316.
8. Morin PJ.Claudin proteins in human cancer: promising new targets for diagnosis and therapy [J]. Cancer Res, 2005, 65(21): 9603-9606.
9. Chen Q, Xu T, Li D, et al. JNK/PI3K/Akt signaling pathway is involved in myocardial ischemia/reperfusion injury in diabetic rats: effects of salvianolic acid A intervention[J]. Am J Transl Res, 2016, 8(6): 2534-2548.
10. Ilisso CP, Sapio L, Delle Cave D, et al. S-Adenosylmethionine Affects ERK1/2 and Stat3 pathways and induces apotosis in osteosarcoma cells[J]. J Cell Physiol, 2016, 231(2): 428-435. DOI: 10.1002/jcp.25089.
11. Wang YC, Sanchez-Mendoza EH, Doeppner TR, et al. Post-acute delivery of memantine promotes post-ischemic neurological recovery, peri-infarct tissue remodeling, and contralesional brain plasticity[J]. J Cereb Blood Flow Metab, 2017, 37(3): 980-993. DOI: 10.1177/0271678X16648971.
12. Hubenia OV, Minchenko DO, Minchenko OH, et al. Changing of the expression of VEGF genes encoded important regulators of angiogenesis and neurogenesis under hypoxic and ischemic conditions[J]. Lik Sprava, 2012(7): 103-107.
13. Welberg L.Adult neurogenesis: uncoupling the roles of VEGF[J]. Nat Rev Neurosci, 2011, 12(5): 247. DOI: 10.1038/nrn3028.
14. Jin KL, Mao XO, Greenberg DA. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia[J]. Proc Natl Acad Sci USA, 2000, 97(18): 10242-10247.

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