Bioinformatics analysis of transcriptome sequencing of early hypoxia damage in photoreceptor 661W cell line_Chinese Journal of Ocular Fundus Diseases

Authors：

Yang Qixiang , Shi Pingling , Lu Cong , Song Hao ,  Song Zongming

Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital, Henan Eye Hospital, Henan Eye Institution, Zhengzhou 450003, China;

Corresponding author：

Song Zongming, Email: szmeyes@126.com

Keywords：

Photoreceptor cells; Anoxia; Transcriptome; Computational biology

DOI：

10.3760/cma.j.cn511434-20201009-00483

Video：

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Objective To analyze the early changes of gene expression levels and signaling pathways in 661W cell line under hypoxic conditions and to find potential functional target genes.Methods The cultured mouse 661W cells were divided into hypoxia treatment group and normoxia control group. Cells in the hypoxia treatment group were cultured in a three-gas incubator with volume fraction of 1% and 5% CO₂ at 37 ℃. Cells in the normoxia control group were cultured in an incubator at 37 ℃ with volume fraction of 5% CO₂. High-throughput sequencing technology was used to sequence the transcriptome of 661W cell treated with hypoxia and normoxia for 4 hours to screen for differentially expressed genes (DEG). Clustering heat map analysis, gene ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network (PPI) analysis were performed. The reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the accuracy of the sequencing results.Results A total of 506 differentially expressed genes were screened, including 459 up-regulated genes and 47 down-regulated genes. GO functional enrichment analysis showed that the main biological processes of DEG were the cell's response to hypoxia, glycolysis, negative regulation of cell proliferation and apoptosis. hypoxia inducible factor (HIF)-1α pathway, glycolysis, Forkhead box O (FoxO) pathway, Insulin signaling pathway and Adenosine 5'-monophosphate-activated protein kinase (AMPK) pathway were involved in the above process. PPI analysis results showed that hub genes related to hypoxia were Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10 and Fbxo27. The RT-PCR results showed that the relative expression levels of 15 DEG mRNA in the hypoxic treatment group were higher than that of the normoxic control group, and the difference was statistically significant (P＜0.05). The mRNA expression levels of N-myc downstream-regulated gene-1 (Ndrg1), Mt1, and vascular endothelial growth factor A (VEGFA) were time-dependent on hypoxia.Conclusions Under hypoxia, DEG is mainly related to glucose metabolism, cell response to hypoxia, regulation of proliferation and apoptosis. HIF-1α pathway, glycolysis, FoxO pathway and AMPK pathway are involved in the early changes of 661W cells under hypoxia. Aldoa, Aldoc, Gpi1, Hk2, Hk1, Pfkl, Pfkp, Vhl, Fbxo10, Fbxo27 may play key roles in the response of 661W cells to hypoxia. Ndrg1, Mt1 and VEGFA could be potential functional target genes for the study of ischemia and hypoxia-related fundus diseases.

Citation： Yang Qixiang, Shi Pingling, Lu Cong, Song Hao, Song Zongming. Bioinformatics analysis of transcriptome sequencing of early hypoxia damage in photoreceptor 661W cell line. Chinese Journal of Ocular Fundus Diseases, 2021, 37(3): 214-223. doi: 10.3760/cma.j.cn511434-20201009-00483 Copy

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1. Holekamp NM, Shui YB, Beebe DC. Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation[J]. Am J Ophthalmology, 2005, 139(2): 302-310. DOI: 10.1016/j.ajo.2004.09.046.
2. Budzynski E, Wangsa-Wirawan N, Padnick-Silver L, et al. Intraretinal pH in diabetic cats[J]. Curr Eye Res, 2005, 30(3): 229-240. DOI: 10.1080/02713680590934067.
3. Sayyad Z, Sirohi K, Radha V, et al. 661W is a retinal ganglion precursor-like cell line in which glaucoma-associated optineurin mutants induce cell death selectively[J/OL]. Sci Rep, 2017, 7(1): 16855[2017-12-04]. https://www.nature.com/articles/s41598-017-17241-0. DOI: 10.1038/s41598-017-17241-0.
4. Liu X, Xie J, Yang L, et al. Bone marrow mesenchymal stem cells enhance autophagy and help protect cells under hypoxic and retinal detachment conditions[J]. J Cell Mol Med, 2020, 24(6): 3346-3358. DOI: 10.1111/jcmm.15008.
5. Li N, Gao S, Wang J, et al. Anti-apoptotic effect of interleukin-17 in a mouse model of oxygen-induced retinopathy[J/OL]. Exp Eye Res, 2019, 187: 107743[2019-10-01]. https://doi.org/10.1016/j.exer.2019.107743. DOI: 10.1016/j.exer.2019.107743.
6. Li N, Zhu Y, Wang J, et al. Müller cells derived neurotrophin-3 inhibits hypoxia-induced photoreceptor apoptosis via the TrkC/ERK pathway[J]. Cytotechnology, 2020, 72(1): 47-56. DOI: 10.1007/s10616-019-00356-9.
7. Tsui L, Fong TH, Wang IJ. The effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) on cell viability under hypoxia[J]. Mol Vis, 2013, 19: 2260-2273.
8. Inoue Y, Shimazawa M, Nakamura S, et al. Protective effects of placental growth factor on retinal neuronal cell damage[J]. J Neurosci Res, 2014, 92(3): 329-337. DOI: 10.1002/jnr.23316.
9. Shelby SJ, Angadi PS, Zheng QD, et al. Hypoxia inducible factor 1α contributes to regulation of autophagy in retinal detachment[J]. Exp Eye Res, 2015, 137: 84-93. DOI: 10.1016/j.exer.2015.06.016.
10. Waller R, Woodroofe MN, Wharton SB, et al. Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role[J]. J Neuroimmunol, 2016, 299: 139-146. DOI: 10.1016/j.jneuroim.2016.09.010.
11. Li D, Luo L, Xu M, et al. AMPK activates FOXO3a and promotes neuronal apoptosis in the developing rat brain during the early phase after hypoxia-ischemia[J]. Brain Res Bull, 2017, 132: 1-9. DOI: 10.1016/j.brainresbull.2017.05.001.
12. Zhang MH, Fang XS, Guo JY, et al. Effects of AMPK on apoptosis and energy metabolism of gastric smooth muscle cells in rats with diabetic gastroparesis[J]. Cell Biochem Biophys, 2019, 77(2): 165-177. DOI: 10.1007/s12013-019-00870-9.
13. 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.
14. 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[2016-11-01]. https://pubmed.ncbi.nlm.nih.gov/27811999/. DOI: 10.1038/srep36590.
15. Hu CB, Sui BD, Wang BY, et al. NDRG2 suppression as a molecular hallmark of photoreceptor-specific cell death in the mouse retina[J/OL]. Cell Death Discov, 2018, 4: 32[2018-09-12]. https://pubmed.ncbi.nlm.nih.gov/30245855/. DOI: 10.1038/s41420-018-0101-2.
16. Ide T, Kitajima Y, Miyoshi A, et al. Tumor-stromal cell interaction under hypoxia increases the invasiveness of pancreatic cancer cells through the hepatocyte growth factor/c-Met pathway[J]. Int J Cancer, 2006, 119(12): 2750-2759. DOI: 10.1002/ijc.22178.
17. Trisciuoglio D, Desideri M, Ciuffreda L, et al. Bcl-2 overexpression in melanoma cells increases tumor progression-associated properties and in vivo tumor growth[J]. J Cell Physiol, 2005, 205(3): 414-421. DOI: 10.1002/jcp.20413.
18. Jiang Y, Zhou J, Zou D, et al. Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia[J]. EBioMedicine, 2019, 48: 36-48. DOI: 10.1016/j.ebiom.2019.09.037.
19. Noda K, Ishida S, Shinoda H, et al. Hypoxia induces the expression of membrane-type 1 matrix metalloproteinase in retinal glial cells[J]. Invest Ophthalmol Vis Sci, 2005, 46(10): 3817-3824. DOI: 10.1167/iovs.04-1528.
20. Kurihara T, Westenskow PD, Gantner ML, et al. Hypoxia-induced metabolic stress in retinal pigment epithelial cells is sufficient to induce photoreceptor degeneration[J/OL]. Elife, 2016, 5: e14319[2016-03-15]. https://pubmed.ncbi.nlm.nih.gov/26978795/. DOI: 10.7554/eLife.14319.
21. Barben M, Schori C, Samardzija M, et al. Targeting Hif1a rescues cone degeneration and prevents subretinal neovascularization in a model of chronic hypoxia[J/OL]. Mol Neurodegener, 2018, 13(1): 12[2018-03-07]. https://pubmed.ncbi.nlm.nih.gov/29514656/. DOI: 10.1186/s13024-018-0243-y.
22. Kim JH, Kim J, Kim DH, et al. SCFhFBH1 can act as helicase and E3 ubiquitin ligase[J]. Nucleic Acids Res, 2004, 32(8): 2287-2297. DOI: 10.1093/nar/gkh534.
23. Jeong YT, Rossi M, Cermak L, et al. FBH1 promotes DNA double-strand breakage and apoptosis in response to DNA replication stress[J]. J Cell Biol, 2013, 200(2): 141-149. DOI: 10.1083/jcb.201209002.
24. Chiorazzi M, Rui L, Yang Y, et al. Related F-box proteins control cell death in caenorhabditis elegans and human lymphoma[J]. Proc Natl Acad Sci USA, 2013, 110(10): 3943-3948. DOI: 10.1073/pnas.1217271110.
25. Li Y, Bouchlaka MN, Wolff J, et al. FBXO10 deficiency and BTK activation upregulate BCL2 expression in mantle cell lymphoma[J]. Oncogene, 2016, 35(48): 6223-6234. DOI: 10.1038/onc.2016.155.
26. Xu X, Prough RA, Samuelson DJ. Differential 12-O-Tetradecanoylphorbol-13-acetate- induced activation of rat mammary carcinoma susceptibility Fbxo10 variant promoters via a PKC-AP1 pathway[J]. Mol Carcinog, 2015, 54(2): 134-147. DOI: 10.1002/mc.22081.
27. Yoshida Y, Yasuda S, Fujita T, et al. Ubiquitination of exposed glycoproteins by SCF (FBXO27) directs damaged lysosomes for autophagy[J]. Proc Natl Acad Sci USA, 2017, 114(32): 8574-8579. DOI: 10.1073/pnas.1702615114.

Chinese Journal of Ocular Fundus Diseases

Bioinformatics analysis of transcriptome sequencing of early hypoxia damage in photoreceptor 661W cell line

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