Microarray reveals complement components are regulated in theserum-deprived rat retinal ganglion cell line

[摘要] Purpose: Glaucoma is a progressive eye disease that leads toblindness due to loss of retinal ganglion cells (RGCs). There aredifficulties in using primary cultures of purified RGC to study thispathophysiology. RGC-5, a transformed not RGC line, expresses severalmarkers characteristic of the RGCs. The aim of this study was togenerate a genome-wide gene expression of RGC-5 following serumdeprivation and to identify candidate genes that may be involved in thesignal transduction pathways.Methods: Apoptosis in the transformed rat RGC-5 was induced by serumdeprivation for 0, 8, 24, 48, and 96 h. Briefly, 400 ng of RNA from eachsample was reverse transcribed and labeled with Cy3 dye. Fragmentedfluorescent cRNA was mixed with hybridization buffer and incubated at 60°C for 16 h. Labeled cRNA was hybridized to Rat GenomeOligonucleotide Arrays. These arrays contain 22,775 transcripts with oneoligonucleotide per transcript (60-mer). Gene expression from scannedimages was quantified and analyzed using ArrayVision software.Reproducibility among triplicate arrays was determined by ANOVAstatistical analysis. Significant differences in gene expression betweenapoptotic and nonapoptotic cells were determined based on p-values.Results: Of the 22,775 transcripts present on the arrays (Agilentrat genome, 60-mer), 713 (8 h), 1,967 (24 h), 1,011 (48 h), and 1,161(96 h) were differentially expressed relative to the 0 h time point(p-values <0.05). Twenty-three transcripts were common to 8, 24, 48,and 96 h and 130 transcripts were common to the 24, 48, and 96 h timepoints. The two most highly upregulated genes were Fdft1 andLgals3 (8 h), C3 and Fcgrt (24 h), C and Lcn2 (48 h), andMgp and C3 (96 h). A subset of the differentially expressed genesidentified in microarray data (Ftl1, C3, C1s, Neu1, Polr2g, Acadm,Nupr1, Gch, Dia1, DNase1, Tgfb2, and Cyr61) were validated usingquantitative real time polymerase chain reaction (QRT-PCR). Here we showthat complement factor H (CFH), the major inhibitor of the alternativecomplement pathway is downregulated in serum-deprived RGC-5. CFH proteinwas detected within RGC-5 cells as well as the rat retina with the aidof immunocytochemistry and confocal microscopy.Conclusions: This study was undertaken to generate a genome-widegene expression profile of RGC-5 after serum deprivation, and toidentify candidate and novel genes that may be involved in the signaltransduction pathways leading to apoptosis. RGC-5 serum deprivationrevealed up-and downregulation in gene expression profiles. The datagathered from this study was the first report that the genes identifiedin microarray data and validated by real-time RT-PCR may play animportant role in RGC-5 cell death. Among the validated genes, C3 andC1s showed significant upregulation of the complement componentpathway. The results further indicate that components of the complementpathway are present in neurons of the rat retina. The data indicatedthat complement factors are likely involved in the pathway leading toganglion cell death in the serum-deprivation paradigm, which may besimilar to the mechanism of cell death in glaucoma.

[学科分类] 生物化学/生物物理