Identification of key regulatory pathways in response to long-term zinc supplementation in human primary retinal pigment epithelium: a multi-omics approach. Identification of key regulatory pathways in response to long-term zinc supplementation in human primary retinal pigment epithelium: a multi-omics approach

Purpose: In age-related macular degeneration (AMD), both systemic and local zinc concentrations are decreased. Elevating zinc levels by nutrition or oral supplementation has a positive impact on delaying the progression to end-stage AMD in clinical studies. Here we set out to identify key regulatory pathways involved in this beneficial effect in retinal pigment epithelial cells. Methods: Transcriptome profiles of 5 weeks old primary human fetal retinal pigment epithelial cells from 3 different donors were generated by RNA sequencing using Illumina NextSeq 5000. The samples from the 3 donors represent replicates of each conditions. The sequence reads that passed quality filters were analyzed further for differentially expressed genes (DEG) performing quasi-likelihood F-test in edgeR package (Robinson, McCarthy et al. 2010). Lowly expressed genes were filtered out using the following script was used: > keep 0.05) >= 1. To be considered as differentislly expressed gene, the Benjamini-Hochberg–adjusted P value was less than 0.05. Results: Using our data analysis workflow, we were able to identify over 32,000 transcripts. 826 of these were significantly changed in the three independent samples after apical and 218 after basal zinc supplementation when compared to untreated controls. Apical zinc supplementation upregulated 415 and down-regulated 411 transcripts. Basal zinc supplementation upregulated 157 and down-regulated 59 transcripts. 163 transcripts changed similarly after both apical and basal treatment. Overall design: Transcriptome profile of human primary fetal retinal pigment epithelial cells from 3 donors (n=12) following zinc apical (n=4) or basal (n=3) supplementation compared to untreated cells (n=5)

研究目的：年龄相关性黄斑变性（age-related macular degeneration, AMD）患者的全身及局部锌浓度均出现降低。临床研究显示，通过营养干预或口服补锌提升锌水平，对延缓进展为终末期AMD具有积极作用。本研究旨在明确视网膜色素上皮细胞中介导该有益效应的关键调控通路。

实验方法：本研究采用Illumina NextSeq 5000平台开展RNA测序，获取3名不同供体来源的5周龄人原代胎儿视网膜色素上皮细胞的转录组谱。3名供体的样本分别对应各处理条件的生物学重复。对通过质量过滤的测序读段，使用edgeR软件包（Robinson、McCarthy等，2010）中的拟似然F检验分析差异表达基因（differentially expressed genes, DEG）。采用下述脚本剔除低表达基因：> keep 0.05) >= 1。差异表达基因的判定标准为经Benjamini-Hochberg校正后的P值小于0.05。

实验结果：通过本研究的数据分析流程，共鉴定到超过32000条转录本。与未处理对照组相比，在3份独立样本中，经顶侧补锌处理后有826条转录本发生显著表达变化，经基底侧补锌处理后则有218条。其中，顶侧补锌处理上调了415条转录本、下调411条；基底侧补锌处理上调157条、下调59条。另有163条转录本在顶侧与基底侧补锌处理后均发生相似的表达变化。

实验整体设计：本研究以3名供体来源的人原代胎儿视网膜色素上皮细胞为研究对象（总样本量n=12），设置顶侧补锌组（n=4）、基底侧补锌组（n=3）及未处理对照组（n=5），对各组细胞进行转录组谱分析。