The Alteration of M6A-tagged Transcript Profiles in The Retina of Rats After Traumatic Optic Neuropathy

Messager RNA (mRNA) can be modified in a variety of ways, among which the modification of N6-methyladenosine (m6A) is one of the most common ones. Recent studies have found that the m6A modification in mRNA could functionally regulate the splicing, localization, translation and stability of mRNA, which might be closely related to multiple diseases. However, the roles of m6A modification in traumatic optic neuropathy (TON) are unknown. Herein, we detected the expression of m6A-related genes via quantitative real-time PCR (qRT-PCR) and performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) as well as RNA-sequencing to analyze the alteration profiles of m6A modification after TON. The results showed that the expression of m6A-related genes (METTL3, WTAP, FTO and ALKBH5) were all upregulated after TON. In all, 2810 m6A peaks were differentially upregulated and 689 m6A peaks were downregulated. In addition, the hypermethylated and hypomethylated profiles of mRNA transcripts were also identified. To sum up, our study revealed the differentially expressed m6A modification in the early stage of TON, which may provide novel insights into the mechanism and treatment of TON. Overall design: RNA-seq: Twelve hours after operation, rats were anesthetized with pentobarbital sodium and were transcardially perfused with 20 ml of 4°C PBS. The retinal tissues were rapidly dissected and stored in liquid nitrogen. According to the manufacturer's protocol, total RNAs from the retina of each rat were isolated using TRIzol reagent (Invitrogen). Three replicates were conducted for sham-operated and TON model group. To attain 60 µg of total RNA, retinal samples from 4 rats were integrated into one centrifuge Tubes. The concentration and quality of total RNAs were assessed using NanoDrop ND-1000(Thermo Fisher Scienti?c). The integrity of RNA was evaluated by denaturing agarose gel electrophoresis. The extraction of mRNA was conducted using NEBNext rRNA Depletion Kit (New England Biolabs, Hertfordshire, UK) following the manufacturer's protocol. Bioanalyzer 2100 system (Agilent Technologies, CA, USA) was used for quality control and quantification of the RNA libraries, and Illumina Hiseq4000 platforms (Illumina, CA, USA) was used for double-ended sequencing of RNA. Paired-end reads were harvested from Illumina HiSeq 4000 sequencer and were quality controlled by Q30. After 3' adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3), the high quality clean reads were aligned to the reference genome (UCSC hg19) with hisat2 software (v2.0.4) (Kechin et al., 2017). Then, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified (Trapnell et al., 2010). MeRIP-seq, data analysis and bioinformatics At 12h after operation, rats were anesthetized with pentobarbital sodium and were transcardially perfused with 20 ml of 4°C PBS. The retina was then rapidly dissected and stored in liquid nitrogen. Total RNAs from the retinal tissue of each rat were isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. In this study, three biological replicates were conducted in sham-operated and TON model group, retinal tissue samples from 4 rats were combined into one sample to attain 60 µg of total RNA. mRNA extraction was performed using NEBNext rRNA Depletion Kit (New England Biolabs) following the manufacturer's protocol. Briefly, m6A RNA immunoprecipitation was performed with the GenSeqTM m6A RNA IP Kit (GenSeq Inc., shanghai, China) by following the manufacturer's instructions. Both the input sample without immunoprecipitation and the m6A IP samples were used for RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs). The quality control (QC) of the libraries was evaluated using BioAnalyzer 2100 system (Agilent Technologies). Library sequencing was performed on an Illumina Hiseq4000 platforms under 150bp pair-ended resolution. Cutadapt software (V1.9.3) was used to remove low quality reads and obtain clean reads (Kechin et al., 2017). The clean reads from all samples were then aligned to Ensembl reference genome by HISAT2 software (v2.0.4) (Kim et al., 2015). Model-based Analysis of ChIP-Seq(MACS) software (v1.4.2) was used to identify methylated genes in each sample (Zhang et al., 2008). DiffReps software was used for identification of differential methylated genes (Shen et al., 2013). The differentially methylated peaks (fold changes = 2.0 and p < 0.05) between sham-operated group and TON model group were analyzed by exomePeak and annotated accordingly by the latest Ensembl database. Gene ontology (GO) database (www.geneontology.org)and the latest Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/kegg)were used to perform GO analysis and pathway enrichment analysis on differentially methylated mRNA genes. Statistical analyses Data are expressed as mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism Version 8.3 software (GraphPad Software LLC, CA, USA). Student's t-test (paired) was used for comparing the statistical significance between two groups. For each analysis, P< 0.05 was considered as statistically significant and marked with *.