The expression and role of miR-195 in diabetic retinopathy_Chinese Journal of Ocular Fundus Diseases

Authors：

QinShiyue , YinLi ,  YaoYong , 邹健 , 孙超

Department of Ophthalmology, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, China;

Corresponding author：

YaoYong, Email: pard1@126.com

Keywords：

Diabetic retinopathy/psychology; MicroRNAs; High mobility group proteins; Gene transfer techniques

DOI：

10.3760/cma.j.issn.1005-1015.2015.02.006

Video：

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Objective To investigate the expression of miR-195 and the underlying molecular mechanisms of miR-195 regulating HMGB1 in diabetic retinopathy (DR). Methods Extract 5 ml venous blood from DR patients, diabetes mellitus (DM) patients and normal subjects, then extract and perificate plasma total RNA. MicroRNA array and real time polymerase chain reaction (RT-PCR) was used to screen out miRNAs which were expressed with significant differences in the serum of patients with DR. Bioinformatics was employed to predict the miR-195 related to high mobility group box 1 (HMGB1) regulation. Next, miR-195 was down-regulated or up-regulated in umbilical vein endothelial cells through transfection of miR-195 inhibitor and miR-29b mimics respectively.Then we analyzed expression of HMGB1 mRNA and protein by RT-PCR and Western blot. Results MicroRNA array results showed the expression of miR-195 in DR group is decreased by 8.34 times and 11.47 times compared with DM group and the normal group. RT-PCR verification results conforms to the microRNA array results. Compared with the DM group (F=0.034, t=8.057) and the normal group (F=0.370, t=9.522), the expression of miR-195 in DR group were significantly reduced, the differences were statistically significant (P < 0.05). RT-PCR showed that the expression of HMGB1 mRNA was significantly decreased in up-regulation group, compared with blank (F=0.023, t=11.287) and negative control group (F=0.365, t=7.471), the difference was statistically significant (P < 0.05). The expression of HMGB1 mRNA was significantly increased in down-regulation group, compared with blank (F=0.053, t=10.871) and negative control group (F=0.492, t=6.883), the difference was statistically significant (P < 0.05). Western blot showed that the expression of HMGB1 protein was significantly decreased in up-regulation group, compared with blank (F=0.021, t=8.820) and negative control group (F=0.039, t=7.401), the difference was statistically significant (P < 0.05); and significantly increased in down-regulation group, compared with blank (F=0.186, t=10.092) and negative control group (F=0.017, t=12.923), the difference was statistically significant (P < 0.05). Conclusion MiR-195 can inhibit the expression of HMGB1, reduce the inflammation and angiogenesis, thereby delaying or inhibiting the occurrence and development of DR.

Citation： QinShiyue, YinLi, YaoYong. The expression and role of miR-195 in diabetic retinopathy. Chinese Journal of Ocular Fundus Diseases, 2015, 31(2): 134-138. doi: 10.3760/cma.j.issn.1005-1015.2015.02.006 Copy

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15.	傅祖值.糖尿病[M]//叶任高, 陆再英.内科学.北京:人民卫生出版社, 2004:787-809.
16.	Wilkinson CP, Ferris FI, Klein RE, et al.Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales[J].Ophthalmology, 2003, 110(9):1677-1682.
17.	van Beijnum JR, Dings RP, van der Linden E, et al.Gene expression of tumor angiogenesis dissected:specific targeting of colon cancer angiogenic vasculature[J].Blood, 2006, 108(7):2339-2348.
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23.	Yang X, Yin J, Yu J, et al.MiRNA-195 sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w[J].Oncol Rep, 2012, 27(1):250-257.
24.	Flavin RJ, Smyth PC, Laios A, et al.Potentially important microRNA cluster on chromosome 17p13.1 in primary peritoneal carcinoma[J].Mod Pathol, 2009, 22(2):197-205.
25.	Zhang QQ, Xu H, Huang MB, et al.MicroRNA-195 plays a tumorsuppressor role in human glioblastoma cells by targeting signaling pathways involved in cellular proliferation and invasion[J].Neuro Oncol, 2012, 14(3):278-287.
26.	Mao JH, Zhou RP, Peng AF, et al.MicroRNA-195 suppresses osteosarcoma cell invasion and migration in vitro by targeting FASN[J].Oncol Lett, 2012, 4(5):1125-1129.
27.	Wang R, Zhao N, Li S, et al.MicroRNA-195 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF, VAV2 and CDC42[J].Hepatology, 2013, 58(2):642-653.
28.	Ulloa L, Messmer D.High-mobility group box 1(HMGB1)protein:friend and foe[J].Cytokine Growth Factor Rev, 2006, 17(3):189-201.
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32.	El-Asrar AM, Nawaz MI, Kangave D, et al.High-mobility group box-1 and biomarkers of inflammation in the vitreous from patients with proliferative diabetic retinopathy[J].Mol Vis, 2011, 17:1829-1838.
33.	Biscetti F, Straface G, De Cristofaro R, et al.High-mobility group box-1 protein promotes angiogenesis after peripheral ischemia in diabetic mice through a VEGF-dependent mechanism[J]. Diabetes, 2010, 59(6):1496-1505.
34.	程满根, 李佩霞, 胡向阳.人脐静脉内皮细胞体外培养的影响因素[J].苏州医学院学报, 1990, 10(3):224-227.
35.	Pearso JD.EndothelIal cell biology[J].Radiology, 199l, 179(1):9-14.
36.	Jiang J, Xia X, Xu H, et al.Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF-lalpha and VEGF[J].Cell Physiol, 2009, 218(1):66-74.
37.	Xia X, Xiong S, Xu H, et al.Suppression of retinal neovascularization by shRNA targeting HIF-lalpha[J].Curr Eye Res, 2008, 33(10):892-902.

1. Pillai RS, Bhattacharyya SN, Filipowicz W.Repression of protein synthesis by miRNAs:how many mechanisms?[J]. Trends Cell Biol, 2007, 17(3):118-126.
2. Liu J.Control of protein synthesis and mRNA degradation by microRNAs[J].Curr Opin Cell Biol, 2008, 20(2):214-221.
3. Chekulaeva M, Filipowicz W. Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells[J].Curr Opin Cell Biol, 2009, 21(3):452-460.
4. Reinhart BJ, Slack FJ, Basson M, et al.The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans[J]. Nature, 2000, 403(6772):901-906.
5. Brennecke J, Cohen SM. Towards a complete description of the microRNA complement of animal genomes[J].Genome Biol, 2003, 4(9):228.
6. Baehrecke EH.MiRNAs:micro managers of programmed cell death[J].Curr Biol, 2003, 13(12):473-475.
7. Michael MZ, O'Connor SM, van Holst Pellekaan NG, et al.Reduced accumulation of specific microRNAs in colorectal neoplasia[J].Mol Cancer Res, 2003, 1(12):882-891.
8. Schickel R, Boyerinas B, Park SM, et al.MicroRNAs:key players in the immune system, differentiation, tumorigenesis and cell death[J].Oncogene, 2008, 27(45):5959-5974.
9. Wang Y, Wang X, Zhang J, et al.MicroRNAs involved in the EGFR/PTEN/AKT pathway in gliomas[J].Neurooncol, 2012, 106(2):217-224.
10. Zhao C, Dong J, Jiang T, et al.Early second-trimester serum miRNAprofiling predicts gestational diabetes mellitus.PLoS One.2011, 6(8):23925. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023925..
11. Kovacs B, Lumayag S, Cowan C, et al.MicroRNAs in early diabetic retinopathy in streptozotocin-induced diabetic rats[J].Invest Opthalmol Vis Sci, 2011, 52(7):4402-4409.
12. Feng B, Chen S, McArthur K, et al.MiR-146a-mediated extracellular matrix protein production in chronic diabetes complications[J].Diabetes, 2011, 60(11):2975-2984.
13. Kuehbacher A, Urbich C, Zeiher AM, et al.Role of Dicer and Drosha for endothelial microRNA expression and angiogenesis[J].Circ Res, 2007, 101(1):59-68.
14. McArthur K, Feng B, Wu Y, et al. MicroRNA-200b regulates vascular endothelial growth factor-mediated alterations in diabetic retinopathy[J].Diabetes, 2011, 60 (4):1314-1323.
15. 傅祖值.糖尿病[M]//叶任高, 陆再英.内科学.北京:人民卫生出版社, 2004:787-809.
16. Wilkinson CP, Ferris FI, Klein RE, et al.Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales[J].Ophthalmology, 2003, 110(9):1677-1682.
17. van Beijnum JR, Dings RP, van der Linden E, et al.Gene expression of tumor angiogenesis dissected:specific targeting of colon cancer angiogenic vasculature[J].Blood, 2006, 108(7):2339-2348.
18. Scanlan MJ, Welt S, Gordon CM, et al.Cancer-related seroligical recognition of human colon cancer:identification of potential diagnostic and immunotherapeutic targets[J].Cancer Res, 2002, 62(5):4041-4047.
19. 高华, 史伟云, 谢立信, 等.雷帕霉素缓释片防治兔高危角膜移植免疫排斥反应和新生血管增殖的研究[J].中华眼科杂志, 2006, 42(1):6-11.
20. Hui W, Yuntao L, Lun L, et al.MicroRNA-195 inhibits the proliferation of human glioma cells by directly targeting cyclin D1 and cyclin E1.PLoS One, 2013, 8(1):54932.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054932..
21. Xu T, Zhu Y, Xiong Y, et al.MicroRNA-195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells[J].Hepatology, 2009, 50(1):113-121.
22. Liu L, Chen L, Xu Y, et al.MicroRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells[J].Biochem Biophys Res Commun, 2010, 400(2):236-240.
23. Yang X, Yin J, Yu J, et al.MiRNA-195 sensitizes human hepatocellular carcinoma cells to 5-FU by targeting BCL-w[J].Oncol Rep, 2012, 27(1):250-257.
24. Flavin RJ, Smyth PC, Laios A, et al.Potentially important microRNA cluster on chromosome 17p13.1 in primary peritoneal carcinoma[J].Mod Pathol, 2009, 22(2):197-205.
25. Zhang QQ, Xu H, Huang MB, et al.MicroRNA-195 plays a tumorsuppressor role in human glioblastoma cells by targeting signaling pathways involved in cellular proliferation and invasion[J].Neuro Oncol, 2012, 14(3):278-287.
26. Mao JH, Zhou RP, Peng AF, et al.MicroRNA-195 suppresses osteosarcoma cell invasion and migration in vitro by targeting FASN[J].Oncol Lett, 2012, 4(5):1125-1129.
27. Wang R, Zhao N, Li S, et al.MicroRNA-195 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF, VAV2 and CDC42[J].Hepatology, 2013, 58(2):642-653.
28. Ulloa L, Messmer D.High-mobility group box 1(HMGB1)protein:friend and foe[J].Cytokine Growth Factor Rev, 2006, 17(3):189-201.
29. Oh IK, Kim SW, Oh J, et al.Inflammatory and angiogenic factors in the aqueous humor and the relationship to diabetic retinopathy[J].Curr Eye Res, 2010, 35(12):1116-1127.
30. Lutty GA, Cao JT, McLeod DS.Relationship of polymorphonuclear leukocytes to capillary dropout in the human diabetic choroid[J].Am J Pathol, 1997, 151(3):707-714.
31. Soro-Paavonen A, Watson AM, Li J, et al.Receptor for advanced glycation end products(RAGE)deficiency attenuates the development of atherosclerosis in diabetes[J].Diabetes, 2008, 57(9):2461-2469.
32. El-Asrar AM, Nawaz MI, Kangave D, et al.High-mobility group box-1 and biomarkers of inflammation in the vitreous from patients with proliferative diabetic retinopathy[J].Mol Vis, 2011, 17:1829-1838.
33. Biscetti F, Straface G, De Cristofaro R, et al.High-mobility group box-1 protein promotes angiogenesis after peripheral ischemia in diabetic mice through a VEGF-dependent mechanism[J]. Diabetes, 2010, 59(6):1496-1505.
34. 程满根, 李佩霞, 胡向阳.人脐静脉内皮细胞体外培养的影响因素[J].苏州医学院学报, 1990, 10(3):224-227.
35. Pearso JD.EndothelIal cell biology[J].Radiology, 199l, 179(1):9-14.
36. Jiang J, Xia X, Xu H, et al.Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF-lalpha and VEGF[J].Cell Physiol, 2009, 218(1):66-74.
37. Xia X, Xiong S, Xu H, et al.Suppression of retinal neovascularization by shRNA targeting HIF-lalpha[J].Curr Eye Res, 2008, 33(10):892-902.

Chinese Journal of Ocular Fundus Diseases

The expression and role of miR-195 in diabetic retinopathy

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