key: cord-1008959-u95f31ro authors: Shi, Jiangcheng; Hu, Chengqing; Zhou, Yuan; Cui, Chunmei; Yang, Jichun; Cui, Qinghua title: MicroRNA profiling in paired left and right eyes, lungs, and testes of normal mice date: 2020-07-10 journal: Mol Ther Nucleic Acids DOI: 10.1016/j.omtn.2020.07.006 sha: d83ac4a788c6a2b21202af1f56d3ceffde0d22b6 doc_id: 1008959 cord_uid: u95f31ro Abstract Physiological and pathophysiological differences widely exist in paired organ systems. However, the molecular basis for these differences remain largely unknown. We previously reported that there exist differentially expressed miRNAs (DEMs) in the left and right kidneys of normal mice. Here, we identified the DEMs in the left and right eyes, lungs and testes of normal mice via RNA Sequencing. As a result, we identified 26 DEMs in eyes, with 23 higher and 3 lower in the left eyes compared with right eyes, 21 DEMs in lungs, with 15 higher and 6 lower in the left lungs compared with right lungs; and 54 DEMs in testes, with 6 higher and 48 lower in the left testes compared with right ones. Ten miRNAs were further examined by quantitative PCR assays, and seven of these were confirmed. In addition, correlation analysis was performed between paired organ miRNA expressions and diverse body fluid miRNA expressions. Finally, we explored the functions and networks of DEMs and performed biological process and pathway enrichment analysis of target genes for DEMs, providing insights into the physiological and pathophysiological differences between the two entities of paired organs. Introduction 44 The human body has some paired organs including the eyes, lungs, and testes, and Wuhan, China, is rapidly spreading throughout many countries. 3 Moreover, some 52 reproductive system diseases, such as testicular cancer, which is the most common 53 cancer diagnosed in males under the age of 40 years. 4 To be particular, the left and right organs in most paired organ systems are asymmetric, 55 and it has been previously reported that not only the anatomical structure but also the 56 morbidity of some paired organs is different between the left and right sides. Eduardo 89 To determine the potential DEMs concerning physiological processes in paired organ 90 systems, the miRNA profiles in the eyes, lungs and testes of mice were determined 91 using RNA-Seq technology at BGI Genomics Company (Beijing, China). As a result, 92 a total of 1431 miRNA signals were detected in the paired eyes, lungs and testes. We that we can well distinguish the left samples from the right ones with the help of these 100 DEMs ( Figure 1A ). 101 We further investigated some biological features of these DEMs. We found that 102 DEMs are distributed on both autosomes and sex chromosome (X) ( Figure S1A ). Interestingly, the DEMs in the testes were found to make up a sizeable proportion of 104 the X chromosome (24%, 13/54). No significant differentially distribution was 105 observed. Next, the allocation of DEMs in the three organs between autosomes and 106 sex chromosomes was shown as Figure S1B . Finally, evolutionary conservation is 107 also an important feature to explore the function of miRNAs. To illustrate the 108 evolutionary conservation of these DEMs, we first divided miRNAs into three groups 109 as described previously, 12 using the miRNA family resources provided by miRbase 110 (version 21), 13 namely a mammal-specific group, a vertebrate-specific group and a 111 group for miRNAs represent in other more distal species, such as invertebrate animals. No significant difference in conservation was observed between DEMs and 113 non-DEMs. The distribution of miRNAs in the three organs among the three different 114 groups is shown in Figure S1C . Validation of differentially expressed miRNAs 116 To further validate the accuracy of RNA-Seq, we randomly selected ten miRNAs 117 homologous with human miRNAs for verification. Bulge-loop real-time PCR analysis 118 of the ten selected miRNAs showed that the expression levels of seven miRNAs of 119 these miRNAs are different between the left and right organs in paired eyes, lungs and 120 testes of mice, and this result was consistent with the results of RNA-Seq. We found 121 that mmu-miR-142a-5p and mmu-miR-187-5p have significantly higher expression 122 levels， whereas mmu-miR-203b-3p has a lower expression level in the left eyes than 123 in the right eyes (Figure 2A-C) . Further, we found that the expression levels of 124 mmu-miR-107-5p and mmu-miR-122b-5p are significantly higher, whereas that of 125 mmu-miR-100-3p is lower in the left lungs than in the right lungs ( Figure 2D -F). Moreover, the expression level of mmu-miR-204-5p was confirmed to be lower in left 127 testes than in the right ones ( Figure 2H ). Finally, the real-time PCR results of the 128 remaining three miRNAs were not consistent with the RNA-Seq results. The trend 129 observed with regard to the expression level of mmu-miR-148a-5p in the left and 130 right lungs after PCR analysis was contrasting to that observed on RNA-Seq ( Figure 131 2G). Further, we observed no significant difference in the expression level of 132 mmu-miR-532-3p in paired testes ( Figure 2I ). In addition, the expression level of 133 mmu-miR-297a-5p in the eyes was not detected successfully, which may have 134 resulted because of the degradation of some RNA during the procedure. Overall, the 135 real-time PCR data supported the accuracy of RNA-Seq data. functional terms associated with highly expressed miRNAs in the right testes were neural stem cell differentiation, cell death, tumor suppressor miRNAs, aging, and 177 adipocyte differentiation ( Figure 4C ). Notably, the functional terms associated with 178 disease defense such as inflammation, tumor suppressor miRNAs, T-cell 179 differentiation and immune response were found to be enriched in all these organs. 180 Surprisingly, we did not observe any significant functional terms for the miRNAs 181 with higher expression levels in the right eyes, right lungs and left testes. The detailed procedure has been described in a previous study. 11 In brief, the relative The authors declare no competing interests. Table S1 . Differentially expressed miRNAs between the left and right eyes. Table S2 . Differentially expressed miRNAs between the left and right lungs. Table S3 . Differentially expressed miRNAs between the left and right testes. We showed that, compared to adjacent non-neoplastic lung tissues, the expressions of miR-125a-5p and let-7e were decreased in AD and SCC samples Use of preclinical 355 models to improve treatment of retinoblastoma Enhancing global access to cancer medicines Characteristics of and Public Health Responses to 360 the Coronavirus Disease 2019 Outbreak in China Incidence and incidence trends of the most frequent cancers in adolescent and 363 young adult Americans, including "nonmalignant/noninvasive" tumors Analysis of the location of 367 retinal lesions in central retinographies of patients with Type 2 diabetes Pathological 370 findings of COVID-19 associated with acute respiratory distress syndrome Testicular asymmetry in healthy adolescent boys Transcriptome analysis 441 reveals non-identical microRNA profiles between arterial and venous plasma Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods 445 for exRNA Isolation MicroRNA set analysis MISIM v2.0: a web 449 server for inferring microRNA functional similarity based on microRNA-disease 450 associations HMDD v3.0: a 452 database for experimentally supported human microRNA-disease associations Cytoscape: a software environment for integrated models of biomolecular interaction 456 networks miRTarBase update 2018: a resource for experimentally validated microRNA-target 459 interactions Gene ontology: tool for the unification of biology This work was supported by grants from the Natural Science Foundation of China 342 (81670462, 81970440, and 81921001 to Q.C.), grants from the Peking University percentage of the DEMs on autosomes and sex chromosomes in each organ. (C) The 511 distribution of DEMs in different conserved groups. Note that miRNAs in the more 512 conserved group (e.g., vertebrate-specific group) do not include the miRNAs present 513 in the less conserved groups (e.g., mammal-specific group). Cui and colleagues identified differentially expressed miRNAs in paired left and right eyes, lungs, and testes of normal mice via RNA sequencing. The results were further confirmed by quantitative PCR assays. They revealed biological characteristics of these miRNAs, providing insights into the physiological and pathophysiological differences in paired organ systems.