Transcriptome sequencing and microRNA-mRNA regulatory network construction in the lens from a Na2SeO3-induced Sprague Dawley rat cataract model

Abstract

Background: A sight-threatening, cataract is a common degenerative disease of the ocular lens. This study aimed to explore the regulatory mechanism of age-related cataract (ARC) formation and progression.

Methods: Cataracts in Sprague Dawley rats were induced by adopting the method that injected selenite subcutaneously in the nape. We performed high-throughput RNA sequencing technology to identify the mRNA and microRNA(miRNA) expression profiles of the capsular membrane of the lens from Na₂SeO₃-induced and saline-injected Sprague Dawley rats. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were carried out to forecast the regulatory and functional role of mRNAs in cataracts by DAVID and Metascape. The protein-protein interaction(PPI) network of differentially expressed mRNA(DEmRNAs) was built via the STRING. Target miRNAs of hub genes were predicted by miRBD and TargetScan. Furthermore, differentially expressed miRNA(DEmiRNAs) were selected as hub genes' targets, validated by quantitative real-time polymerase chain reaction(qRT-PCR), and a DEmiRNA-DEmRNA regulatory network was constructed via Cytoscape.

Result: In total, 329 DEmRNAs including 40 upregulated and 289 downregulated genes were identified. Forty seven DEmiRNAs including 29 upregulated and 18 downregulated miRNAs were detected. The DEmRNAs are involved in lens development, visual perception, and aging-related biological processes. A protein-protein interaction network including 274 node genes was constructed to explore the interactions of DEmRNAs. Furthermore, a DEmiRNA-DEmRNA regulatory network related to cataracts was constructed, including 8 hub DEmRNAs, and 8 key DEmiRNAs which were confirmed by qRT-PCR analysis.

Conclusion: We identified several differentially expressed genes and established a miRNA-mRNA-regulated network in a Na₂SeO₃-induced Sprague Dawley rat cataract model. These results may provide novel insights into the clinical treatment of cataracts, and the hub DEmRNAs and key DEmiRNAs could be potential therapeutic targets for ARC.

Keywords: Cataract; Len; Network; Rat; mRNA; microRNA.

Fig. 1

Establishment of the Na₂SeO₃-induced Sprague Dawley rat cataract model. A The experimental process of Na₂SeO₃-induced Sprague Dawley rat cataract model construction. B The slit-lamp image and HE staining of lenses in control and Na₂SeO₃-induced groups. C Soluble and insoluble proteins of lenses were analyzed in control and Na₂SeO₃-induced groups (n = 3). D Body weight was measured before administration and anesthesia (n = 10)

Fig. 2

Identification of the significant expression changes of RNAs in cataract rat models. A The distribution of the total expression of the miRNA in six lenses. B The distribution of the total expression of the mRNAs in six lenses. C Volcano plots of the DEmiRNAs (red, upregulated; blue, down-regulated). D Volcano plots of the DEmRNAs (red, upregulated; blue, down-regulated). E Heatmaps for DEmiRNAs (red, upregulated; blue, down-regulated). F Heatmaps for DEmRNA (red, upregulated; blue, down-regulated)

Fig. 3

Three types of networks. A Top 20 clusters and their representative enriched terms (colored by cluster ID). B The PPI network of down-related DEmRNAs by the online database STRING. C The PPI network of up-related DEmRNAs by the online database STRING

Fig. 4

Construction of miRNA-mRNA regulatory network. A Venn diagram of DEmiRNAs and predicted miRNAs of down-related mRNA by Targetscan/miRDB. B Venn diagram of DEmiRNAs and predicted miRNAs of up-related mRNA by Targetscan/miRDB. C Construction of the miRNA-mRNA regulatory network including 8 miRNAs and 8 mRNAs (red ellipse, up-regulated DEmRNAs; blue ellipse, down-regulated DEmRNAs; V-shape, DEmiRNAs)

Fig. 5

The expression of key DEmiRNAs in H₂O₂ induced model group compared with the control group by qPCR (n = 3)