Abstract
Purpose :
Small non-coding RNAs (sncRNAs), including miRNAs, snoRNAs, snRNAs, and tRNAs, have been detected in various biofluids, and their presence in exosomes suggests a potential role for sncRNAs in intercellular communication and signaling. The composition of sncRNAs in tear fluid is not well-established. The aims of this study are (i) to create a reference database of the sncRNAs in human tear fluid samples and (ii) to examine sex-specific differences.
Methods :
Tear samples were collected from 30 healthy subjects using Schirmer strips. RNA was isolated using the miRNeasy Serum/Plasma Kit, and cDNA libraries were prepared using the QIAseq miRNA Library kit. Quality and quantity were verified using Bioanalyzer and Qubit. Pooled libraries were run on the Novaseq6000 sequencing system, and cleaned reads were aligned to the human genome (hg38) using STAR v2.7.10a. Local alignment was employed to consider sncRNA isoforms, and mapped sncRNAs were then quantified using miRBase for miRNAs and GENCODE for snoRNAs, snRNAs, and tRNAs. Sex-specific differences were evaluated using DESeq2.
Results :
A total of 331 miRNAs, 195 snoRNAs, 16 snRNAs, and 13 tRNAs were reliably detected in the tear fluid (mean read counts >5 and present in at least 50% of the samples). The most abundant sncRNAs in each category include let-7b-5p, miR-184, and let-7g-5p (miRNAs); SNORD63, SNORD69, and SNORD104 (snoRNAs); RNU12, RNU5B-1, and RNU4-2 (snRNAs); MT-TS2, MT-TL1, and MT-TI (tRNAs). Several sncRNA families were found to be more prevalent in tear fluid. These sncRNA families included let-7 (15 members), miR-30 (10), miR-181 (8), SNORD116 (11), SNORD14 (5), and RNA variant U1 (4 members). Significant sex-specific differences were observed in the levels of 9 miRNAs, 56 snoRNAs, 3 snRNAs, and 2 tRNAs. miR-138-5p had the greatest increase in expression in males compared to females (3.62-fold, adj. p-value = 0.0045), while SNORD118 had the greatest decrease (0.33-fold, adj. p-value = 0.0017).
Conclusions :
A comprehensive profile of sncRNAs in tear fluid is still unknown. This pilot study lays the foundation for our long-term goal of creating a reference database of sncRNAs in tear fluid. Characterizing sex-specific alterations will further our understanding of differences in disease prevalence between the sexes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.