Abstract

   Physiological and pathophysiological differences widely exist in paired
   organ systems. However, the molecular basis for these differences
   remains 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
   testes. Ten microRNAs (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.

   Keywords: miRNA, paired organ, gene profile, bioinformatic analysis

Graphical Abstract

   graphic file with name fx1.jpg
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     __________________________________________________________________

   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.

Introduction

   Diseases of the paired organ systems in the human body—including the
   eyes, lungs, and testes—form a large proportion of all diseases. For
   example, retinoblastoma is the most common intraocular tumor in
   children; approximately 1 in 20,000 children are affected by
   retinoblastoma worldwide.[35]^1 Furthermore, various lung diseases have
   become the leading cause of human deaths; among them, lung cancer is
   the leading cause of cancer-related deaths worldwide.[36]^2 Pneumonia
   caused by the coronavirus disease 2019 (COVID-19), of which there was a
   recent breakout in Wuhan, China, is rapidly spreading throughout many
   countries.[37]^3 Moreover, of reproductive system diseases, testicular
   cancer is the most common cancer diagnosed in males under the age of 40
   years.[38]^4

   To be particular, the left and right organs in most paired organ
   systems are asymmetric, and it has been previously reported that not
   only the anatomical structure but also the morbidity of some paired
   organs is different between the left and right sides. Munuera-Gifre
   et al.[39]^5 have demonstrated that retinal lesions are not distributed
   randomly but rather follow a determined pattern. They found that the
   left eye exhibits more microaneurysms/hemorrhages and hard exudates of
   a greater density in the central retina than the right eye. Similarly,
   on evaluating the pathological anatomy of patients with
   COVID-19-related pneumonia, X-ray images showed rapid progression of
   pneumonia and some differences between the left and right lungs.[40]^6
   In a study performed by Vaganée et al.,[41]^7 approximately 78%–93% of
   varicoceles are shown to appear on the left side, and a smaller left
   testis is more frequently observed on comparing patients with
   varicoceles and healthy adolescents. Collectively, there are
   differences in pathophysiological and biological functions between the
   left and right sides of paired organs, but the underlying molecular
   basis causing these differences remains unknown.

   MicroRNAs (miRNAs) are an important class of noncoding RNAs, which are
   widely involved in the post-transcriptional regulation of gene
   expression and play important roles in physiological processes as well
   as the development of various diseases. Recently, miRNAs have been
   found to be widely involved in the development of many diseases
   associated with the eyes, lungs, and testes. For example, it was
   reported that rno-miR-138-5p regulates early diabetic retinopathy by
   promoting cell proliferation via targeting Nova1 (NOVA alternative
   splicing regulator 1) in rat.[42]^8 Besides, hsa-miR-4651 inhibits the
   progression of non-small-cell lung cancer by targeting BRD4
   (bromodomain-containing protein 4). [43]^9 Moreover, hsa-miR-371a-3p
   can be used as an informative biomarker for follow-up in patients with
   testicular germ cell cancer.[44]^10

   We have previously revealed that differentially expressed miRNAs (DEMs)
   exist between the left and right kidneys of normal mice,[45]^11
   providing new insights into the physiological and pathophysiological
   differences between the two sides of kidneys and facilitating the
   diagnosis and treatment of lateralized kidney diseases. Thus far, it is
   unclear whether the miRNA expression profiles are different between two
   sides of the eyes, lungs, and testes. Accordingly, in this study, we
   first identified DEMs in these paired organs of normal mice using RNA
   sequencing (RNA-seq) technology and then performed bioinformatic
   analyses on these miRNAs.

Results

miRNA Expression Profiles in Paired Organ Systems of a Normal C57BL/6J Mouse

   To determine the potential DEMs concerning physiological processes in
   paired organ systems, the miRNA profiles in the eyes, lungs, and testes
   of mice were determined using RNA-seq technology at BGI Genomics
   (Beijing, China). As a result, a total of 1,431 miRNA signals were
   detected in the paired eyes, lungs, and testes. We identified 26 DEMs
   in the eyes (with the expression of 23 miRNAs being higher and 3 being
   lower in the left eyes than in the right ones), 21 DEMs in the lungs
   (with the expression of 15 miRNAs being higher and 6 being lower in the
   left lungs than in the right ones), and 54 DEMs in the testes (with the
   expression of 6 miRNAs being higher and 48 being lower in the left
   testes than in the right ones). The DEMs are shown in [46]Tables S1,
   [47]S2, and [48]S3. Heatmap and dendrogram cluster analyses
   demonstrated that we can well distinguish the left samples from the
   right ones with the help of these DEMs ([49]Figure 1A).

Figure 1.

   [50]Figure 1
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   Heatmap of DEMs

   Significantly DEMs between the left and right eyes, lungs, and testes.

   We further investigated some biological features of these DEMs. We
   found that DEMs are distributed on both autosomes and the X chromosome
   ([52]Figure S1A). Interestingly, the DEMs in the testes were found to
   make up a sizeable proportion of the X chromosome (24%; 13/54). No
   significant differentially expressed distribution was observed. Next,
   the allocation of DEMs in the three organs between autosomes and sex
   chromosomes was shown in [53]Figure S1B. Finally, evolutionary
   conservation is also an important feature to explore the function of
   miRNAs. To illustrate the evolutionary conservation of these DEMs, we
   first divided miRNAs into three groups as described previously,[54]^12
   using the miRNA family resources provided by miRbase (v.21);[55]^13
   namely, a mammal-specific group, a vertebrate-specific group and a
   group for miRNAs represented in other more distal species such as
   invertebrate animals. No significant difference in conservation was
   observed between DEMs and non-DEMs. The distribution of miRNAs in the
   three organs among the three different groups is shown in
   [56]Figure S1C.

Validation of DEMs

   To further validate the accuracy of RNA-seq, we randomly selected 10
   miRNAs homologous with human miRNAs for verification. Bulge-loop
   real-time PCR analysis of the 10 selected miRNAs showed that the
   expression levels of seven of these miRNAs are different between the
   left and right organs in paired eyes, lungs, and testes of mice, and
   this result was consistent with the results of RNA-seq. We found that
   mmu-miR-142a-5p and mmu-miR-187-5p have significantly higher expression
   levels, whereas mmu-miR-203b-3p has a lower expression level in the
   left eyes than in the right eyes ([57]Figures 2A–2C). Further, we found
   that the expression levels of mmu-miR-107-5p and mmu-miR-122b-5p are
   significantly higher, whereas that of mmu-miR-100-3p is lower in the
   left lungs than in the right lungs ([58]Figures 2D–2F). Moreover, the
   expression level of mmu-miR-204-5p was confirmed to be lower in left
   testes than in right ones ([59]Figure 2H). Finally, the real-time PCR
   results of the remaining three miRNAs were not consistent with the
   RNA-seq results. The trend observed with regard to the expression level
   of mmu-miR-148a-5p in the left and right lungs after PCR analysis was
   in contrast to that observed with RNA-seq ([60]Figure 2G). Further, we
   observed no significant difference in the expression level of
   mmu-miR-532-3p in paired testes ([61]Figure 2I). In addition, the
   expression level of mmu-miR-297a-5p in the eyes was not detected
   successfully, which may have resulted because of the degradation of
   some RNA during the procedure. Overall, the real-time PCR data
   supported the accuracy of RNA-seq data.

Figure 2.

   [62]Figure 2
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   Validation of DEMs in the Paired Organ Systems of C57BL/6J Mice

   (A–C) The expression levels of mmu-miR-142a-5p (A), mmu-miR-187-5p (B),
   and mmu-miR-203b-5p (C) in the eyes. (D–G) The expression levels of
   mmu-miR-100-3p (D), mmu-miR-107-5p (E), mmu-miR-122b-5p (F), and
   mmu-miR-148a-5p (G) in the lungs. (H and I) The expression levels of
   mmu-miR-204-5p (H) and mmu-miR-532-3p (I) in the testes. The data were
   normalized to the left organ (ns = 10–15; mean ± SEM). ∗p < 5e−2,
   versus left organ.

Comparison of miRNA Expression Profiles in Paired Organs with Those in
Various Body Fluids

   In the past decade, circulating miRNAs have been proposed to be useful
   in diagnostics and prognosis as biomarkers for diseases,[64]^14 and
   their expression profiles have been correlated with those of miRNAs in
   tissues. Here, we compared the association of miRNA expression profiles
   in the left and right organs of mice with those in mouse body fluids,
   homologous miRNA expression profiles in rat arterial plasma and venous
   plasma, and homologous miRNA expression profiles in diverse human body
   fluids. As a result, we found that the expression profiles in the left
   and right organs of mice are highly correlated with those in the
   plasma, bile, and urine of mice (rho > 0.60, p < 1e−50 in plasma; rho >
   0.50, p < 1e−20 in bile; and rho > 0.3, p < 1e−4 in urine; [65]Figures
   3A–3C). Particularly, the miRNA expression profiles in the left and
   right lungs showed higher correlations with the expression profiles in
   the three body fluids of mice, but the paired testes showed lower
   correlations; this finding was consistent with the previous findings in
   humans.[66]^15 Interestingly, we found a significant difference in
   correlation coefficients between miRNA expression profiles in left and
   right lungs and those in rat arterial plasma (p= 3.6e−2, matched-pair t
   test, [67]Figure 3D), but there was no difference between miRNA
   expression profiles in left and right lungs and those in rat venous
   plasma ([68]Figure 3E). On evaluating the correlations between miRNA
   expression profiles in the left and right organs of mice and homologous
   miRNA expression profiles in diverse human body fluids, the patterns of
   correlations observed between paired organs in mice and diverse human
   body fluids were similar to those observed between paired organs and
   body fluids in mice ([69]Figures 3F–3I). Collectively, we demonstrated
   that the expression profiles of miRNA in the left and right organs in
   mice are significantly correlated with those in various body fluids
   ([70]Table S4).

Figure 3.

   [71]Figure 3
   [72]Open in a new tab

   Comparison of miRNA Expression Profiles in Paired Organs with miRNA
   Expression Profiles in Various Body Fluids

   (A–I) Expression correlation between paired organ miRNAs and miRNAs in
   plasma (A), bile (B), and urine (C) of mice; homologous miRNAs in
   arterial plasma (D) and venous plasma (E) of rats; and homologous
   miRNAs in plasma (F), serum (G), bile (H), and urine (I) of humans.
   Asterisk indicates significant difference in correlation coefficients
   between miRNA expression profiles in the left and right lungs and miRNA
   expression profile in arterial plasma of rats (∗p < 5e−2), according to
   matched-pair t test.

Functional Enrichment Analysis of the DEMs

   Using the TAM v.2.0 tool, we found that the homologous human miRNAs
   with higher expression levels in the left eyes, left lungs, and right
   testes are significantly enriched in 9, 26, and 28 functional terms,
   respectively. The miRNAs with higher expression levels in the left eyes
   were mainly involved in the following functions: skeletal muscle cell
   differentiation, inflammation, response to hypoxia, T cell
   differentiation, smooth muscle cell proliferation, cytokine secretion,
   hematopoiesis, apoptosis, tumor suppressor miRNAs ([73]Figure 4A). This
   suggested that the DEMs in the left eyes may contribute to the
   development of eye muscles and defense against disease. Besides, the
   highly expressed miRNAs in the left lungs were mainly enriched in
   functional terms such as adipogenesis, inflammation, lipid metabolism,
   insulin resistance, regulation of stem cell, circadian rhythm
   ([74]Figure 4B). Moreover, the top five functional terms associated
   with highly expressed miRNAs in the right testes were neural stem cell
   differentiation, cell death, tumor suppressor miRNAs, aging, and
   adipocyte differentiation ([75]Figure 4C). Notably, the functional
   terms associated with disease defense such as inflammation, tumor
   suppressor miRNAs, T cell differentiation, and immune response were
   found to be enriched in all these organs. Surprisingly, we did not
   observe any significant functional terms for the miRNAs with higher
   expression levels in the right eyes, right lungs, and left testes.

Figure 4.

   [76]Figure 4
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   Functional Enrichment Analysis of the DEMs

   (A–C) Enriched biological functions associated with the DEMs
   upregulated in the left eyes (A), left lungs (B), and right testes (C).
   The length of each bar is the value of −log[10] (p value).

Functional Similarity Network of the DEMs

   We calculated the functional similarity of DEMs by implementing
   the MISIM algorithm, which is based on the miRNA disease association
   dataset in the Human MicroRNA Disease Database (HMDD). Finally, we
   filtered miRNAs with a similarity higher than 0.2 and constructed a
   functional similarity network using Cytoscape software.

   Among the network of DEMs in the eyes ([78]Figure 5A), miR-150 and
   miR-187 have been extensively studied, Shi et al.[79]^16 have reported
   that the deletion of mmu-miR-150 can significantly increase retinal
   pathological angiogenesis in high fat diet (HFD)-induced type 2
   diabetic mice. In another study, the researchers revealed that
   rno-miR-187 upregulation alleviates oxidative stress injury in
   retina tissues of rat with ocular hypertension,[80]^17 which is closely
   associated with the development of retinal disease. Further, among the
   network of DEMs in the lungs ([81]Figure 5B), miR-107, miR-125a,
   miR-148a, and miR-100 showed high similarity; and in literature
   research, we found that they play a role in many common lung diseases,
   such as lung cancer, small-cell lung cancer, and non-small-cell lung
   cancer. Among them, miR-125a is mostly associated with lung diseases
   ([82]Table 1 shows the detailed associations of miR-125a with lung
   diseases). The results indicated that these miRNAs may have a
   synergistic effect in the occurrence and development of lung diseases.
   Finally, as shown in the network of DEMs in the testes ([83]Figure 5C),
   many functionally similar miRNAs play important roles in the
   pathophysiological progress in the testes. In the study by Abhari
   et al.,[84]^18 hsa-miR-100 and hsa-let-7b in oligospermic patients were
   found to be possibly associated with the susceptibility and progression
   of infertility. Similarly, miR-191 and miR-10b were also reported to be
   associated with spermatogenesis,[85]^19^,[86]^20 and mmu-miR-10b was
   reported to be highly expressed in mouse spermatogonial stem cells
   (SSCs) in vitro and in enhanced SSC proliferation.[87]^10 Moreover,
   hsa-miR-302 was inferred to act as an oncogene in human testicular germ
   cell tumors by inducing the expression of SPRY4 and activating the
   mitogen-activated protein kinase (MAPK)/ERK pathway while inhibiting
   apoptosis by increasing survivin expression.[88]^21

Figure 5.

   [89]Figure 5
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   Functional Similarity Network of the DEMs

   (A–C) Each red/green node represents an upregulated miRNA in the
   left/right eye (A), the left/right lung (B), and the left/right testis
   (C), respectively.

Table 1.

   Detailed Information for the Associations of miR-125a with Various Lung
   Diseases
   Lung Diseases Evidence PubMed Identifier (PMID)
   Lung neoplasms the results demonstrated that the panel of miRNA
   biomarkers (miR-125a-5p, miR-25, and miR-126) had the potential for the
   early detection of lung cancer; [91]25639977
   Non-small-cell lung carcinoma these preliminary data suggest that serum
   miR-125a-5p, miR-145, and miR-146a may be useful noninvasive biomarkers
   for the clinical diagnosis of non-small-cell lung cancer [92]25755772
   Lung neoplasms our results provide compelling evidence that
   miR-125a-5p, an epidermal growth factor-signaling-regulated miRNA, may
   function as a metastatic suppressor [93]19702827
   Non-small-cell lung carcinoma hsa-miR-125a-5p was poorly expressed in
   lung cancer cells, and it could enhance lung cancer cell invasion by
   upregulating hsa-miR-125a-5p [94]20719190
   Lung neoplasms miRNA hsa-miR-125a-3p activates p53 and induces
   apoptosis in lung cancer cells [95]24044511
   Non-small-cell lung carcinoma hsa-miR-125a-5p could upregulate Rock-1
   and enhance invasion in lung cancer cells [96]20723344
   Small-cell lung carcinoma chemotherapy-regulated miRNA-125-HER2 pathway
   as a novel therapeutic target for trastuzumab-mediated cellular
   cytotoxicity in small-cell lung cancer [97]25833836
   Non-small-cell lung carcinoma miR-125a-3p targets MTA1 to suppress
   non-small-cell lung cancer cell proliferation, migration, and invasion
   [98]25998575
   Lung carcinoma miRNA-125a-5p plays a role as a tumor suppressor in lung
   carcinoma cells by directly targeting STAT3 [99]28631574
   Non-small-cell lung carcinoma miR-125a-3p:Hsa-miR-125a-3p and
   hsa-miR-125a-5p are downregulated in non-small-cell lung cancer
   [100]20569443
   Non-small-cell lung carcinoma we showed that, compared to adjacent
   non-neoplastic lung tissues, the expressions of miR-125a-5p and let-7e
   were decreased in lung adenocarcinomas and squamous-cell lung
   carcinomas samples [101]22618509
   Non-small-cell lung carcinoma differential expression of miR-125a-5p
   and let-7e predicts the progression and prognosis of non-small-cell
   lung cancer [102]24945821
   Lung carcinoma miRNA expression profiles of granulocytic
   myeloid-derived suppressor cells from mice bearing Lewis lung carcinoma
   [103]27748875
   [104]Open in a new tab

   All these findings further our understanding of the miRNA functions in
   paired-organ-related diseases and provide a basis for the diagnosis,
   treatment, and prevention of these diseases.

Target Analysis for DEMs

   Given that the main function of miRNA is to regulate the expression of
   target genes to participate in various physiological and
   pathophysiological activities, we conducted a biological process and
   pathway enrichment analysis for the target genes of DEMs. The results
   showed that these target genes are significantly enriched in various
   biological processes. The target genes of DEMs in the left eyes were
   most enriched in cell adhesion ([105]Figure 6A), which plays an
   important role in the pathogenesis of various eye
   diseases.[106]^22^,[107]^23 Similarly, the target genes of DEMs in the
   right eyes were enriched in the positive regulation of epithelial cell
   proliferation, DNA repair, and B cell activation, among other processes
   ([108]Figure 6B). Interestingly, the target genes of DEMs in the left
   and right lungs were both enriched in tumor-related processes, such as
   regulation of tumor necrosis factor production, p53 signaling pathway,
   and DNA double-strand break repair ([109]Figures 6C and 6D).
   Furthermore, the enrichment results in the right testes showed that the
   target genes were associated with urogenital system development, and
   the target genes in both sides were enriched in the physiological
   process of behavior regulation, including subentries such as learning
   and memory ([110]Figures 6E and 6F). Taken together, the results of
   enrichment analysis indicated that the target genes regulated by these
   DEMs in paired organs have various functions in diverse physiological
   processes and pathways.

Figure 6.

   [111]Figure 6
   [112]Open in a new tab

   Target Analysis for the DEMs

   (A–F) Biological process and pathway enrichment for target genes of the
   DEMs in the left (A) and right (B) eyes, left (C) and right (D) lungs,
   and left (E) and right (F) testes. The color of each point represents
   the value of −log[10] (p value), and the different sizes of each point
   correspond to the number of genes represented in the term. The rich
   factor is the percentage of all the target genes found in the given
   ontology term.

Discussion

   Many organs in the human body exist in pairs; e.g., the eyes, lungs,
   kidneys and testes. The anatomical structures of these organs under
   different physiological and pathological conditions, as well as the
   incidence rates of pathologies in many pairs of organs, are different.

   Anisometropia is a condition in which the refractive errors differ
   between the two eyes.[113]^24 Although there is substantial evidence of
   the axial nature of anisometropia,[114]25, [115]26, [116]27 little is
   known about the biology basis. Further, pulmonary agenesis is an
   uncommon congenital anomaly resulting from the failure of pulmonary
   development beyond the carina, and unilateral lesions constitute 70% of
   cases.[117]^28 However, the details of the reasons of the case are
   still unknown. In addition, varicoceles mostly present at the beginning
   of puberty and are observed in approximately 14%–20% of adolescents. It
   appears on the left side in 78%–93% of all cases.[118]^29^,[119]^30
   Again, the molecular bases behind all these paired organ pathologies
   are unclear and need to be further elucidated.

   miRNAs represent an important class of noncoding RNA molecules and
   accomplish a remarkable variety of biological
   functions.[120]^31^,[121]^32 Here, we identified DEMs in multiple
   paired organs such as the eyes, lungs, and testes using RNA-seq
   technology in normal C57BL/6J mice. Subsequently, seven out of 10 DEMs
   were validated by quantitative PCR assays, confirming the accuracy of
   RNA-seq analysis results. Moreover, we indicated intriguing expression
   correlations between paired organs and diverse body fluids. In
   addition, functional enrichment analysis and network biology analysis
   demonstrated that DEMs are clearly involved in the corresponding
   paired-organ-related diseases. Finally, we also conducted a biological
   process and pathway enrichment analysis for the target genes of the
   DEMs, suggesting that the DEMs play important roles in various
   physiological processes and pathways by regulating the target genes.

   We have, for the first time, detected the miRNA profiles in the paired
   organ systems of normal mice. These findings can provide some new
   insights into understanding the physiological and functional
   differences between the left and right sides of paired organs, and they
   can also help promote the diagnosis and treatment of some related
   diseases.

Materials and Methods

Experimental Mice

   In this study, 8- to 12-week-old male C57BL/6J mice (weight, 22–25 g)
   were used. All animals were bred and housed locally at 24°C ± 2°C and
   were acclimated to standard laboratory conditions (12-h:12-h light:dark
   cycle) with free access to rodent feed and water. All animal care and
   experimental protocols complied with the Animal Management Rules of the
   Ministry of Health of the People’s Republic of China and the Guide for
   the Care and Use of the Laboratory Animals of Peking University. All
   animal protocols were approved by the Animal Research Committee of the
   Peking University Health Science Center.

RNA-Seq Analysis for the Development of the miRNA Profile

   In this study, three left entities and three right entities of the
   eyes, lungs, and testes from normal mice were used for RNA-seq
   analysis. The genome-wide expression profiles of miRNAs were obtained
   from BGI Genomics (Beijing, China). The total RNAs were purified by gel
   cutting to separate the RNAs of the target fragment range, and library
   preparation was performed following BGI Genomics’s standard procedure.
   Shortly, the adapters were ligated into the 3′ end of RNA, and reverse
   transcriptase (RT) primers with unique molecular indices (UMIs) were
   added into the system, which were used for synthesizing the
   single-strand cDNA by reverse transcription. After cDNA amplification,
   target fragment separation, library quantification, and pooling
   cyclization, quantify control was performed on the constructed library,
   and then miRNA expression was quantified by counting the types of UMI
   tags. Finally, 1,431 miRNA signals were detected using the BGISEQ-500
   sequencer, and DEMs were identified based on the p value (unpaired
   student’s test, cutoff = 0.05) and fold change (FC) calculated by
   dividing the right expression value by the left one, where “NA”
   indicates that the expression value in the left organ is zero and is
   also considered to be differentially expressed. The expression profile
   data can be obtained from the GEO database (GEO: [122]GSE146262).

RNA Extraction

   Total RNA was extracted using the RNApure High-Purity Total RNA Rapid
   Extraction Kit (BioTeke, Beijing, China) according to the
   manufacturer’s instructions. After DNase digestion, total RNA was
   eluted with 30–80 μL RNase-free water and stored at −80°C. The purity
   and integrality of extracted total RNA were determined by the ratio of
   absorbance at 260 nm to that at 280 nm and via gel imaging.

Bulge-Loop Real-Time RT-PCR

   For quantitative evaluation of miRNAs, bulge-loop real-time RT-PCR was
   performed. The detailed procedure has been described in a previous
   study.[123]^11 In brief, the relative expression level of each miRNA
   was first normalized to small nuclear RNA U6 and then normalized to
   left group data values using 2^−DDCt methodology. All the bulge-loop RT
   primers were purchased from RiboBio (Guangzhou, China), and the
   real-time PCR analysis was performed according to the manufacturer’s
   instructions.[124]^11

Bioinformatic Analysis for miRNA Expression Profiles

   Correlation analyses were performed between miRNA expression profiles
   in paired organs and those in mouse body fluids obtained from the GEO
   database (GEO: [125]GSE111750 and [126]GSE109655), homologous miRNA
   expression profiles in rat arterial plasma and venous plasma,[127]^33
   and homologous miRNA expression profiles in diverse human body fluids
   from small RNA-seq data across diverse biofluids[128]^34 using the
   Spearman rank correlation test. The TAM v.2.0[129]^35 tool was used to
   determine the enriched biological functions of the DEMs, and the
   MISIM[130]^36 tool was used to calculate the miRNA functional
   similarity of the DEMs based on the miRNA-disease association dataset
   in the HMDD database (v.3.0).[131]^37 As both TAM and MISIM are
   designed for human miRNAs, here the mouse miRNAs were transformed into
   human orthologous miRNAs. The functional similarity networks were then
   constructed using Cytoscape software (v.3.6.1).[132]^38 With regard to
   target analysis, we obtained the target genes of DEMs that have been
   experimentally verified from the miRTarBase database.[133]^39 Following
   this, biological process and pathway enrichment analyses were conducted
   with the following ontology sources: GO Biological Processes;[134]^40
   Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway;[135]^41 and
   Reactome Gene Sets using Metascape,[136]^42^,[137]^43 a web server for
   gene annotation and analysis. Furthermore, the terms were collected and
   grouped into clusters based on their membership similarities by the
   server, and the most statistically significant term within a cluster
   was chosen to represent the cluster.

Statistical Analysis

   Data are presented as mean ± SEM. Statistical significance of
   differences between groups was analyzed by Student’s t test.
   Correlation analysis was performed by the Spearman rank correlation
   test. In all analyses, p < 0.05 was considered to be significant.

Author Contributions

   Q.C. and J.Y. conceived the study. Q.C. and J.Y. supervised the
   experiments. J.S., Y.Z., and C.C. performed bioinformatic analysis.
   C.H. conducted the biological experiments. All authors wrote the
   manuscript.

Conflicts of Interest

   The authors declare no competing interests.

Acknowledgments