March 2019
Volume 60, Issue 4
Open Access
Retina  |   March 2019
Deletion of miR-182 Leads to Retinal Dysfunction in Mice
Author Affiliations & Notes
  • Kun-Chao Wu
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Xue-Jiao Chen
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Guang-Hui Jin
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Xiao-Yun Wang
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Dan-Dan Yang
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Yan-Ping Li
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Lue Xiang
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Bo-Wen Zhang
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Gao-Hui Zhou
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Chang-Jun Zhang
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Zi-Bing Jin
    Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, State Key Laboratory of Ophthalmology, Optometry and Vision Science, National Center for International Research in Regenerative Medicine and Neurogenetics, Wenzhou Medical University, Wenzhou, China
    Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou, China
  • Correspondence: Zi-Bing Jin, Lab for Stem Cell & Retinal Regeneration, Institute of Stem Cell Research, Division of Ophthalmic Genetics, The Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China; jinzb@mail.eye.ac.cn
Investigative Ophthalmology & Visual Science March 2019, Vol.60, 1265-1274. doi:10.1167/iovs.18-24166
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      Kun-Chao Wu, Xue-Jiao Chen, Guang-Hui Jin, Xiao-Yun Wang, Dan-Dan Yang, Yan-Ping Li, Lue Xiang, Bo-Wen Zhang, Gao-Hui Zhou, Chang-Jun Zhang, Zi-Bing Jin; Deletion of miR-182 Leads to Retinal Dysfunction in Mice. Invest. Ophthalmol. Vis. Sci. 2019;60(4):1265-1274. doi: 10.1167/iovs.18-24166.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: MicroRNA-182 (miR-182) is abundantly expressed in mammalian retinas; however, the association between miR-182 and retinal function remains unclear. In this study, we explored whether miR-182 contributes to functional decline in retinas using a miR-182 depleted mouse.

Methods: Electroretinogram (ERG) amplitudes at different ages were measured in miR-182 knockout (KO) mice. The thickness and lamination of retinas were assessed using a color fundus camera and high-resolution optical coherence tomography. Expression levels of key photoreceptor-specific genes and the miR-183/96/182 cluster (miR-183C) were quantified using quantitative real-time PCR. RNA sequencing and light-induced damage were carried out to observe the changes in the retinal transcriptome and sensitivity to light damage in the miR-182 KO mice.

Results: The ERG recording reveals that the ERG response amplitude decreased both at early and later ages when compared with control littermates. The expression of some key photoreceptor-specific genes was down-regulated with deletion of miR-182 in retina. RNA sequencing indicated that some biological processes of visual system were affected, and the numbers of potential target genes of miR-182 were presented in the mouse retina using bioinformatics analysis. The miR-182 KO mice were characterized by progressively losing the outer segment after being treated with light-damage exposure. The thickness and lamination of retina as well as compensatory expression of miR-183C showed no apparent changes in retina of miR-182 KO mice under normal laboratory lighting condition.

Conclusions: Our findings provided new insights into the relationship between the miR-182 and retinal development and revealed that miR-182 may play a critical role in maintaining retinal function.

MicroRNAs (miRNAs) are a class of single-stranded, endogenous, and noncoding RNAs of 20 to 24 nucleotides in length that play a critical role in several essential biological processes.13 For their biogenesis, primary RNA transcripts are processed by Drosha/Dgcr8 to generate precursor miRNAs (pre-miRNA) in the nucleus and are subsequently modified by Dicer in the cytoplasm to become mature miRNAs.4,5 Mature miRNAs generally regulate gene expression by base pairing with sequences in the 3′-UTR of target messenger RNAs (mRNAs), which leads to mRNA degradation and translation inhibition.3,6 
MiRNAs have emerged as key players in retinal development710 and are involved in retinal degenerative disease as well.1114 The conditional ablation of Dicer in rods resulted in histological and functional impairments in the retina.15 The cone-specific disruption of Dgcr8 led to a serious loss of outer segments and a significantly reduced light response in cone photoreceptors.16 A transgenic “sponge” mouse model that reduced the activity of the whole miR-183/96/182 cluster (miR-183C) revealed increased sensitivity to light-induced damage in the retina, but no structural or functional defects under normal lighting conditions.17 A mouse line generated by gene-trap in ES cells, in which retinal miR-183C was almost completely lacking, showed age-dependent retinal dystrophy, synaptic defects in photoreceptors, and increased sensitivity to light-induced damage.18 The characterization of the miR-183/96 double knockout (KO) mouse revealed defective cone nuclear polarization and progressive retinal dystrophy.19 A miR-183C KO mouse model generated by homologous recombination presented multiple deficits in sensory systems.20 
miR-182, a member of miR-183C, is one of most abundantly expressed miRNAs in the retinas and is likely important during retinal development.21,22 In our previous study, we generated a miR-182 KO mouse model, but unexpectedly, no apparent histological abnormalities of the retina were observed in this mouse line.23 However, the relationship of miR-182 and retinal function remains unknown. To address this question, we used the miR-182 KO mouse model to determine the association of miR-182 and retinal function. 
We found that the deletion of miR-182 led to significantly decreased ERG responses in mice both at early and later ages. Some photoreceptor-specific genes showed down-regulation at the mRNA level in the miR-182 KO mice. Furthermore, we found that the deletion of miR-182 in mice increased the sensitivity to light damage characterized by a progressive loss of the outer segments. Bioinformatics analyses presented some potential target genes of miR-182 in the retina. Our findings suggest that miR-182 contributes to maintaining retinal function. 
Materials and Methods
Animals
Mice were maintained in the animal resource center of Wenzhou Medical University with a 12-hour light–dark cycle and a standard chow diet. The light intensity for the cages was about 20 lux in the animal resource center. Animal care followed the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All animal experiments and procedures were approved by the Wenzhou Medical University Animal Care and Use Committee. Generation and genotyping of miR-182 KO mice was described in our previous study.23 
Color Fundus Images and Fundus Fluorescein Angiography
A Micron-IV retinal imaging system from Phoenix Research Laboratories (Pleasanton, CA, USA) was used to screen phenotypes in the fundus. Briefly, the pupils were dilated with 0.5% tropicamide for 10 minutes. Then the mice were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg). A drop of Gen Teal lubricant eye gel (Novartis, East Hanover, NJ, USA) was applied on the surface of the cornea to keep it moist. The mice were faced to the camera to capture fundus photographs. For fundus fluorescein angiography, fluorescein AK-FLOUR (Akorn, Lake Forest, IL, USA) at 5 μg/g body weight was intraperitoneally injected, and retinal vascular leakage was assessed using a Micron-IV image microscope. 
High-Resolution Spectral-Domain Optical Coherence Tomography (SD-OCT) Imaging
The pupils were fully dilated with 0.5% tropicamide for 10 minutes. One drop of Gen Teal lubricant eye gel (Novartis) was administered to the eyes before examination. The imaging of the retinal layers was performed using a high-resolution SD-OCT instrument (Micron IV, Phoenix Research Laboratories). A total of 50 pictures were acquired and used to construct each final averaged SD-OCT image. The vertical retinas across the optical nerve head were imaged for each eye. The thicknesses of the different retinal layers were measured using Insight (Pleasanton, CA, USA) software. 
Retinal Immunohistochemistry
The eyeballs were extracted immediately after euthanasia. After removing the cornea and lens, the eyecups were fixed in 4% paraformaldehyde for 2 hours followed by dehydration in 30% sucrose solution and finally were embedded in optimum cutting temperature compound. Sections with a thickness of 12 μm were cut. The slides were rinsed for 10 minutes with 0.01 M PBS and blocked for 1 hour in a solution containing 4% BSA and 0.5% Triton X-100 in PBS. Primary antibodies were diluted in 1% BSA and 0.5% Triton X-100 in PBS. The slides were incubated in primary antibodies overnight at 4°C and then incubated for 1 hour in solutions containing appropriate secondary antibodies. Primary antibodies and dilutions were as follows: mouse antirhodopsin (1:4000; Sigma, St. Louis, MO, USA), rabbit antirecoverin (1:500; Sigma), rabbit anticone arrestin (1:200; Millipore, Burlington, MA, USA), and rabbit anti-PKCα (1:500; Abcam, Cambridge, UK). As secondary antibodies, donkey anti-rabbit-Alexa488 (1:200, Jackson ImmunoResearch, West Grove, PA, USA) and donkey anti-mouse-Alexa594 (1:200, Jackson ImmunoResearch) were used. Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:3000, Invitrogen, Carlsbad, CA, USA). Morphologies of the antibody-stained retina were imaged using a Leica SP8 laser scanning confocal microscope (Leica, Wetzlar, Germany). 
Electroretinogram
A Ganzfeld-field ERG system was used to assess the retinal function of KO mice at postnatal day 30.24 KO mice and control mice were dark-adapted overnight and anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg). The pupils were dilated with 0.5% tropicamide, and gold wire loop electrodes were placed over the corneas. Mice were placed on a 37°C warming pad to maintain body temperature. Needle reference and ground electrodes were inserted into the cheek and tail. For ERG stimulation, a LED light source was used. The scotopic ERG was recorded at −2.2 log cd • s/m2 and −0.3 log cd • s/m2 stimulus intensities with an interstimulus interval of 30 seconds, in which five ERG scans were averaged for dark-adapted ERGs. Photopic ERGs were elicited after a steady background illumination of 30 cd/m2 for 10 minutes. Then 50 signals were averaged for photopic measurements taken at 0.65 log cd • s/m2 in background light with an interstimulus interval of 0.4 seconds. To further observe the progressive changes of retinal function, ERG responses were recorded in 8-month-old mice. The a-wave and b-wave amplitudes in the ERG responses were analyzed. 
RNA Isolation and Real-Time PCR
Whole-mount retinas of mice were dissected and placed in TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The total RNA was extracted using an RNeasy MiNi Kit (74204; Qiagen, Hilden, Germany) and then quantified on a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For analyses of the mRNA expression changes, 2 μg of total RNA was reverse transcribed to cDNA with M-MLV reverse transcriptase and random primer, and 50 ng of cDNA was then mixed with SYBR Green PCR master mix (Roche, Basel, Switzerland) and a target gene primer (Supplementary Table S1) at a final concentration of 1 μM to produce a 20-μl reaction mixture. For the analysis of miRNA levels, TaqMan miRNA probes were adopted, 1 μg total RNA was reverse transcribed to cDNA using M-MLV reverse transcriptase and a stem-loop RT primer, and qPCR was performed using a TaqMan PCR kit. The relative expressions of miRNAs were normalized to U6. All real-time PCR analyses were performed using the Roche Realplex real-time PCR system. 
RNA-Sequencing Analyses
Total RNA was extracted from retinas according to the instruction manual of TRIzol Reagent (Life Technologies). The cDNA library was constructed using an Illumina Hiseq 2500 sequencing platform (Illumina, San Diego, CA, USA). The gene expression levels were estimated using fragments per kilobase of exon per million fragments mapped values by Cufflinks software (https://github.com/cole-trapnell-lab/cufflinks, provided by the Trapnell Lab, University of Washington, Seattle, WA, USA). The differentially expressed gene (DEG, fold change > 2, FDR < 0.05) between the KO mice and the control group were identified; genes with the value of log2FC between −4 and 4 were analyzed. When the value of −log10FDR was greater than 16, it was defined as 16. The DEGs were further assessed by gene ontology (GO) analyses. The potential target genes of miR-182 in the retina were coanalyzed using the TargetScan database and RNA-sequencing results. 
Light-Induced Retinal Damage
The 2-month-old mice were dark-adapted overnight. The pupils were dilated by 0.5% tropicamide for 15 minutes, and then the mice were exposed to 10,000 lux white light for 2 hours in cages packaged with aluminum foil.18 After light exposure, the mice were kept in darkness overnight and then raised in normal dark–light cyclic conditions. To ensure that each individual received effective light stimulus, only one mouse was kept in a bucket once during the light-damage process. OCT and immunohistochemistry were conducted to assess retinal structure changes in mice at 2 and 8 weeks following the light-damage session. 
Statistical Analyses
All of the results are presented as the mean ± SEM, and the statistical significance was assessed using Student's t-test and two-way ANOVA. Statistical analysis was performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA; P < 0.05, P < 0.01, P < 0.001 between the KO mice and the control group). 
Results
Dynamic Expression of miR-182 in Mice Retinas
The seed region ranging from the 2nd to the 8th nucleotide of the miRNAs was primarily responsible for their regulatory function. Multiple alignment analysis suggested that the seed sequence of mature miR-182 was highly conserved in vertebrates (Fig. 1A). Accurate determination of miR-182 expression over time is important to identify its function in the retina. To explore this, we measured the dynamic expression of miRNA-182 in C57/BL6J mouse retinas at multiple time points. The expression curve showed that miR-182 was low in the embryonic stage and significantly increased after birth (Fig. 1B), which indicated that miR-182 is primarily involved in retina development at the postnatal stage. We also evaluated the expressional level of whole members of miR-183C in the retina at postnatal day 10, and the results from qPCR analysis showed that miR-182 was completely ablated in the KO mice, which confirmed the availability of this mouse model (Fig. 1C). The expression of miR-96 and miR-183 was scarcely affected in the KO mice at that time point when compared with wild-type littermates (Fig. 1C). 
Figure 1
 
Dynamic expression of miR-182 in mouse retina. (A) Conserved alignment of the seed sequence in mature miR-182. (B) Dynamically expressional pattern of miR-182 in the retina of a wild-type mouse. (C) Retinal expression levels of miR-183C in the miR-182 KO mice at postnatal day 10; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1
 
Dynamic expression of miR-182 in mouse retina. (A) Conserved alignment of the seed sequence in mature miR-182. (B) Dynamically expressional pattern of miR-182 in the retina of a wild-type mouse. (C) Retinal expression levels of miR-183C in the miR-182 KO mice at postnatal day 10; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Deletion of miR-182 Leads to Subtle Changes of Retinal Structure
To assess the retinal structure directly in vivo, a fundus color camera and SD-OCT were used to screen the retinal morphologies of KO mice and their littermates at postnatal day 42. The fundus photographs and fundus fluorescein angiography images showed normal fundus appearances and no leakage both in KO and wild-type mice (Figs. 2A, 2B). The results from the OCT images also presented no apparent changes of retinal lamination (Fig. 2C). The thickness of different retinal layers derived from the OCT images was measured using Insight software. Both the ventral and dorsal retinas were assessed. The statistical analysis showed slightly shorter inner segment and outer segment (IS/OS) layers (Fig. 2D) in the KO mice. The thickness of the other layers were similar between the KO mice and the control group (Supplementary Fig. S1). In addition, the immunostaining results also showed no obvious histological changes in the retinas of the KO mice when compared with the control mice (Supplementary Fig. S2). TUNEL staining was also performed, and no obvious cell death was observed (Supplementary Fig. S2). These results suggest that the deletion of miR-182 has no apparent effect on retinal structure except for the slight shortening of IS/OS in mice. 
Figure 2
 
Structural phenotypes of retina in the miR-182 KO mice. (A) Color fundus photography of control and KO mice. (B) Fundus fluorescent angiography of wild-type control and KO mice. (C) OCT images of control and KO mice along dorsal and ventral orientation. Both the horizontal and vertical scale bars: 100 μm. (D) A two-way ANOVA was used to statistically analyze the thickness of the IS/OS layer. n = 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P <0.001.
Figure 2
 
Structural phenotypes of retina in the miR-182 KO mice. (A) Color fundus photography of control and KO mice. (B) Fundus fluorescent angiography of wild-type control and KO mice. (C) OCT images of control and KO mice along dorsal and ventral orientation. Both the horizontal and vertical scale bars: 100 μm. (D) A two-way ANOVA was used to statistically analyze the thickness of the IS/OS layer. n = 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P <0.001.
Deletion of miR-182 Leads to Decreased ERG Response in Mice
We next determined whether the miR-182 deletion influenced retinal function. First, the mice at postnatal day 30 were adapted in darkness for 24 hours, and the retinal response to a low-light stimulus intensity at −2.2 cd • s/m2 was recorded. The ERGs showed much lower response in the KO mice (Fig. 3A), and the b-wave amplitude significantly decreased (Figs. 3B, 3C). Then a high-stimulus intensity of −0.3 log cd • s/m2 was applied, and the results from the ERG statistical analyses showed that there were significant differences in both a-wave and b-wave amplitudes between the miR-182 KO mice and the wild-type littermates (Figs. 3D–F). The above ERG recordings belong to the scotopic ERGs, which suggested rod photoreceptor function was reduced in mice with miR-182 deletion. 
Figure 3
 
Evaluation of retinal function in miR-182 mice at postnatal day 30. (A) Representative scotopic ERGs (−2.2 cd • s/m2 flashes). (B, C) Plots of a-wave and b-wave amplitudes in scotopic ERG. (D) Representative maximal ERGs (−0.3 log cd • s/m2). (E, F) Plots of a-wave and b-wave amplitudes in maximal ERG. (G) Representative photopic ERG response (0.65 log cd • s/m2). (H, I) Statistical analysis of a-wave and b-wave amplitudes in the photopic ERG. N ≥ 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
 
Evaluation of retinal function in miR-182 mice at postnatal day 30. (A) Representative scotopic ERGs (−2.2 cd • s/m2 flashes). (B, C) Plots of a-wave and b-wave amplitudes in scotopic ERG. (D) Representative maximal ERGs (−0.3 log cd • s/m2). (E, F) Plots of a-wave and b-wave amplitudes in maximal ERG. (G) Representative photopic ERG response (0.65 log cd • s/m2). (H, I) Statistical analysis of a-wave and b-wave amplitudes in the photopic ERG. N ≥ 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Furthermore, the mice stayed in a 30 cd/m2 background illumination for 10 minutes followed by stimulation with an intensity of 0.65 log cd • s/m2. The photopic response of the KO mice also showed decreased a-wave and b-wave amplitudes (Figs. 3G–I), indicating a functional reduction in cone photoreceptors. 
To observe the retinal function at a late stage, we evaluated the ERG responses of 8-month-old KO mice. The b-wave amplitude of both the scotopic and photopic ERG responses in KO mice were significantly decreased (Fig. 4). In addition, we analyzed the ERG response at the mentioned two time points using two-way ANOVA. The results also showed significantly decreased a-wave amplitude under stimulus intensities of −2.2 cd • s/m2 (P < 0.05), −0.3 log cd • s/m2 (P < 0.01) and 0.65 log cd • s/m2 (P < 0.001) at postnatal 30 days as well as −0.3 log cd • s/m2 (P < 0.05) at postnatal 8 months. The b-wave amplitude was apparently reduced under the stimulus intensity of −2.2 cd • s/m2 (P < 0.001), −0.3 log cd • s/m2 (P < 0.01), and 0.65 log cd • s/m2 (P < 0.01) at postnatal 30 days as well as −2.2 cd • s/m2 (P < 0.05) and 0.65 log cd • s/m2 (P < 0.05) at postnatal 8 months. The b-wave latency of the ERG recordings at both the early and late stages showed no apparent changes (Supplementary Fig. S3). We concluded that the deletion of miR-182 could lead to reduction of retinal function in mice. 
Figure 4
 
Assessment of retinal function in the aged miR-182 KO mice. (A–C) Statistical analysis of a-wave and b-wave amplitudes in the scotopic ERG response between control and KO mice. (D–F) Comparison for the maximal ERG response. (G–I) Assessment of photopic ERG response. N = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
 
Assessment of retinal function in the aged miR-182 KO mice. (A–C) Statistical analysis of a-wave and b-wave amplitudes in the scotopic ERG response between control and KO mice. (D–F) Comparison for the maximal ERG response. (G–I) Assessment of photopic ERG response. N = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
miR-182 Deletion Influences Expression of Photoreceptor-Specific Genes
After assessing the retinal function in the KO mouse, we further evaluated the expression levels of multiple photoreceptor-specific genes, which are critical for light response in the retina. Total RNA was extracted from retinas at postnatal days 7 and 42, and the expression levels of Rho, Pde6b, Prph2, Opn1mw, Opn1sw, and Gnat2 were then measured. 
At postnatal day 7, Prph2, and Opn1sw were decreased nearly 50% in the retinas of KO mice, whereas the other genes showed similar expression levels to those in the wild-type mice (Figs. 5A–F). Furthermore, we found that most of the evaluated genes, including Rho, Prph2, Opn1mw, Opn1sW, and Gnat2, were significantly down-regulated in the KO mice at postnatal day 42 (Figs. 5A–F). Although there was no significant difference in the expression of Pde6b, it had a decreasing expression trend. Thus, we concluded that deletion of miR-182 could result in down-regulation of some phototransduction genes at the RNA level. 
Figure 5
 
Expressional changes of photoreceptor-specific genes in KO mice. (A–C) Expressional levels of the rod photoreceptor-specific genes at postnatal day 7 and day 42. (D–F) Assessment of cone-specific genes at postnatal day 7 and day 42; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
 
Expressional changes of photoreceptor-specific genes in KO mice. (A–C) Expressional levels of the rod photoreceptor-specific genes at postnatal day 7 and day 42. (D–F) Assessment of cone-specific genes at postnatal day 7 and day 42; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Retinal Transcriptome Analyses in miR-182 KO Mice
To identify which genes or gene regulation networks in the retina were affected, we performed whole-retinal RNA-seq transcriptome analysis at postnatal day 7. In total, 119 DEGs between the KO mice and control group were identified (Fig. 6A), in which 8 genes were linked to retinal degenerative disorders in humans. Gpm6, Nyx, and Trpm1 are associated with congenital stationary night blindness.2527 Cdh3 and Cfh are linked to macular dystrophy.28,29 Mkks, Mfrp, and Rgr are reported in Bardet-Bied1 syndrome, microphthalmos, and retinitis pigmentosa, respectively.3032 
Figure 6
 
RNA-sequencing and bioinformatics analyses in the retinas. (A) Retinal DEGs between the KO mice and the control group. The red plots represent the up-regulated genes, and the blue plots represent the down-regulated genes. (B) Biological process analysis. Blue bars represent the highest ranked BP items depending on the DEGs. The red plots represent the gene count number that was included in the related biological process based on DEGs. The upper scale bar represents the gene count number, and the lower scale bar shows the P values of BP items. (C) Target prediction of miR-182 in the retina by the TargetScan database and RNA-sequencing analyses. Only the conserved prediction target genes by the TargetScan database were analyzed.
Figure 6
 
RNA-sequencing and bioinformatics analyses in the retinas. (A) Retinal DEGs between the KO mice and the control group. The red plots represent the up-regulated genes, and the blue plots represent the down-regulated genes. (B) Biological process analysis. Blue bars represent the highest ranked BP items depending on the DEGs. The red plots represent the gene count number that was included in the related biological process based on DEGs. The upper scale bar represents the gene count number, and the lower scale bar shows the P values of BP items. (C) Target prediction of miR-182 in the retina by the TargetScan database and RNA-sequencing analyses. Only the conserved prediction target genes by the TargetScan database were analyzed.
The biological progress of GO analysis for these identified DEGs was determined using the DAVID program.33 The results from the bioinformatics analysis showed that two GO terms, visual perception (GO: 0007601) and sensory perception of light stimulus (GO: 0050953), presented the highest ranks (Fig. 6B), which suggested that some biological processes of the visual system may be affected in the KO mice. 
Furthermore, the candidate target genes of miR-182 were computationally analyzed using TargetScan 7.1 (http://www.targetscan.org/vert_71/, provided by the Whitehead Institute for Biomedical Research, Cambridge, MA, USA) as well as the retinal RNA-seq dataset in which multiple parameters were considered, including expressional fold changes of the genes, FDR value, and PCT score. Five candidate genes, namely, Arrdc3, Ezr, Gja3, Fign, and Stard13, showed significant increases in the retinas of miR-182 KO mice at postnatal day 7 (Fig. 6C). Among these genes, Arrdc3 encodes an arrestin domain-containing protein and then recruits the NEDD4E3 ligase,34 which is critical for nervous system development and has been reported as a downstream target of miR-183C.17 Ezr has been reported as a target of miR-184 and can affect the phagocytosis in retinal pigment epithelium cells of humans.35 
Light-Induced Retinal Damage
Previous studies have shown that the retinal susceptibility to light damage increased in mice following ablation of whole members of miR-183C.17,18 To determine whether the single miR-182 molecule influences the retinal response to light damage, we exposed adult miR-182 KO mice and their normal littermates to 10,000 lux cool, white fluorescent light and then evaluated the dynamic changes of thickness and lamination in the retinas (Fig. 7A). OCT images suggested that there were no apparent thickness changes in different retinal layers at 2 weeks following the light-damage session (Supplementary Fig. S4). At 8 weeks following the light damage, we found that the thickness of the IS/OS layer was significantly decreased in the KO mice (Fig. 7B). Other retinal layers showed similar thicknesses to the wild-type mice. 
Figure 7
 
Structural changes in retinas after light-induced damage. (A) Experimental schema of the light-induced damage. (B) Retinal OCT images at 8 weeks following the light damage. (C) Immunostaining of the dorsal retina after light-induced damage in the miR-182 KO mice. (D) Statistical analysis for the thickness of dorsal IS/OS layer. (E) Immunostaining of ventral retina after light-induced damage in the miR-182 KO mice. (F) Statistical analysis for the thickness of dorsal IS/OS layer. The red color represents antirhodopsin, and green color represents antirecoverin. The horizontal and vertical scale bars in the OCT images: 100 μm. The scale bar in the immunostaining images: 50 μm. For the thickness comparison, the middle region of dorsal and ventral retina was selected; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7
 
Structural changes in retinas after light-induced damage. (A) Experimental schema of the light-induced damage. (B) Retinal OCT images at 8 weeks following the light damage. (C) Immunostaining of the dorsal retina after light-induced damage in the miR-182 KO mice. (D) Statistical analysis for the thickness of dorsal IS/OS layer. (E) Immunostaining of ventral retina after light-induced damage in the miR-182 KO mice. (F) Statistical analysis for the thickness of dorsal IS/OS layer. The red color represents antirhodopsin, and green color represents antirecoverin. The horizontal and vertical scale bars in the OCT images: 100 μm. The scale bar in the immunostaining images: 50 μm. For the thickness comparison, the middle region of dorsal and ventral retina was selected; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
To confirm these phenotypes, we performed cryosections of the eyeballs and further evaluated the retinas by using immunofluorescence staining. Consistent with the results of the OCT images, the IS/OS layers were much shorter in the KO mice (Figs. 7C, 7E). Further statistical analysis demonstrated that the thickness of the IS/OS layer was significantly decreased in the KO mice (Figs. 7D, 7F). Thus, we concluded that miR-182 deletion may contribute to increased light-damage sensitivity in mice retinas. 
Discussion
The results presented here demonstrated that the deletion of miR-182 could lead to significant retinal dysfunction and down-regulation of some photoreceptor-specific genes in mice. In addition, the miR-182-depleted mice also showed an increased sensitivity to acute light exposure. We observed significant shorter IS/OS in the KO mice, but the whole retina thickness was not changed significantly, suggesting that miR-182 ablation affected the IS/OS layers and not other layers. 
Busskamp et al.16 evaluated the expression pattern of miRNAs in cone photoreceptors of adult wild-type mice and found that miR-182 represented 64% of all miRNA reads. Sundermeier et al.15 reported significant down-regulation of miR-182 in a rod-specific Dicer KO mouse line. Saxena et al.36 showed that the expression of miR-182 was significantly decreased in a retinal degeneration animal model (Sprague-Dawley rats) after light exposure. Impairments in retinal function were also described in the miR-183C KO or miR-183/96 KO mouse lines.19,20 All of these studies indicated that miR-182 may be involved in the process of retinal development. In our study, we initially evaluated the association between miR-182 and retinal function and found that both scotopic and photopic ERG responses in the miR-182 KO mice were apparently decreased, which suggested that miR-182 may be involved in maintaining retinal function. In previous reports of miR-183C KO and miR-183/96 mice, photopic ERG response were extinguished, which seemed more serious than that in the miR-182 KO mice, suggesting that single miR-183 or miR-96 may be critical for the healthy function of photoreceptors. 
The level of miR-183C was regulated by different light levels and could be up-regulated in the light-adapted retina in a mouse.37 Zhu et al.17 and Lumayag et al.18 reported that the absence of three miRNAs of miR-183C could lead to substantial retinal impairments in mice treated with acute light exposure. We also found that retinal susceptibility to light exposure increased in the miR-182 KO mice; however, the degeneration process was relatively slow and mainly located at the OS layer in the miR-182 KO mice, which suggested that miR-182 may protect photoreceptors from light damage. 
Unexpectedly, ablation of miR-182 hardly affected retinal structure. miR-182, miR-96, and miR-183 were members of miR-183C, whose seed sequences were highly similar (Supplementary Fig. S5A). In addition, the target-predicted program derived from the TargetScan database indicated that most candidate target genes were coregulated by them (Supplementary Fig. S5B). We wondered whether a compensatory expression of miR-96 and miR183 contributed to maintaining retinal structure. However, the expression of miR-183 and miR-96 showed no obvious changes in the miR-182 KO mice at the time point of postnatal day 10 (Fig. 1C). Recently, characterization of a mouse line with ablation of microglia showed increases in dystrophic presynaptic termini, but no influence on the retinal structure and the survival of retinal neurons.38 Thus, whether ablation of miR-182 causes atrophy of some ultrastructures in the retina should be further assessed. 
We preliminarily evaluated the gene networks regulated by miR-182 in the retina. Notably, multiple DEGs between the miR-182 KO mice and controls were related to some inherited retinal degeneration diseases. We also identified some potential target genes of miR-182 by bioinformatics analyses. The regulatory relationships between miR-182 and its target genes require further study to explore the mechanism of retinal dysfunction with deletion of miR-182. 
In summary, our findings provided evidence that miR-182 is primarily key for the maintenance of retinal function, highlighting an important role of miR-182 in the mammalian retina. 
Acknowledgments
The authors thank the Specific Pathogen Free animal facility at Wenzhou Medical University for supporting this project. The authors also appreciate Naoharu Iwai for providing materials. 
Supported by the National Key Research and Development Program of China (2017YFA0105300), the National Natural Science Foundation of China (81500741, 81870690, 81800857), the Zhejiang Provincial Natural Science Foundation of China (LQ14B020005, LD18H120001LD), the Zhejiang Provincial Key R&D Program (2015C03029), the Wenzhou Science and Technology Innovation Team Project (C20150004), and the Ministry of Education 111 project (D16011). 
Disclosure: K.-C. Wu, None; X.-J. Chen, None; G.-H. Jin, None; X.-Y. Wang, None; D.-D. Yang, None; Y.-P. Li, None; L. Xiang, None; B.-W. Zhang, None; G.-H. Zhou, None; C.-J. Zhang, None; Z.-B. Jin, None 
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Figure 1
 
Dynamic expression of miR-182 in mouse retina. (A) Conserved alignment of the seed sequence in mature miR-182. (B) Dynamically expressional pattern of miR-182 in the retina of a wild-type mouse. (C) Retinal expression levels of miR-183C in the miR-182 KO mice at postnatal day 10; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 1
 
Dynamic expression of miR-182 in mouse retina. (A) Conserved alignment of the seed sequence in mature miR-182. (B) Dynamically expressional pattern of miR-182 in the retina of a wild-type mouse. (C) Retinal expression levels of miR-183C in the miR-182 KO mice at postnatal day 10; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
 
Structural phenotypes of retina in the miR-182 KO mice. (A) Color fundus photography of control and KO mice. (B) Fundus fluorescent angiography of wild-type control and KO mice. (C) OCT images of control and KO mice along dorsal and ventral orientation. Both the horizontal and vertical scale bars: 100 μm. (D) A two-way ANOVA was used to statistically analyze the thickness of the IS/OS layer. n = 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P <0.001.
Figure 2
 
Structural phenotypes of retina in the miR-182 KO mice. (A) Color fundus photography of control and KO mice. (B) Fundus fluorescent angiography of wild-type control and KO mice. (C) OCT images of control and KO mice along dorsal and ventral orientation. Both the horizontal and vertical scale bars: 100 μm. (D) A two-way ANOVA was used to statistically analyze the thickness of the IS/OS layer. n = 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P <0.001.
Figure 3
 
Evaluation of retinal function in miR-182 mice at postnatal day 30. (A) Representative scotopic ERGs (−2.2 cd • s/m2 flashes). (B, C) Plots of a-wave and b-wave amplitudes in scotopic ERG. (D) Representative maximal ERGs (−0.3 log cd • s/m2). (E, F) Plots of a-wave and b-wave amplitudes in maximal ERG. (G) Representative photopic ERG response (0.65 log cd • s/m2). (H, I) Statistical analysis of a-wave and b-wave amplitudes in the photopic ERG. N ≥ 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
 
Evaluation of retinal function in miR-182 mice at postnatal day 30. (A) Representative scotopic ERGs (−2.2 cd • s/m2 flashes). (B, C) Plots of a-wave and b-wave amplitudes in scotopic ERG. (D) Representative maximal ERGs (−0.3 log cd • s/m2). (E, F) Plots of a-wave and b-wave amplitudes in maximal ERG. (G) Representative photopic ERG response (0.65 log cd • s/m2). (H, I) Statistical analysis of a-wave and b-wave amplitudes in the photopic ERG. N ≥ 5. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
 
Assessment of retinal function in the aged miR-182 KO mice. (A–C) Statistical analysis of a-wave and b-wave amplitudes in the scotopic ERG response between control and KO mice. (D–F) Comparison for the maximal ERG response. (G–I) Assessment of photopic ERG response. N = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
 
Assessment of retinal function in the aged miR-182 KO mice. (A–C) Statistical analysis of a-wave and b-wave amplitudes in the scotopic ERG response between control and KO mice. (D–F) Comparison for the maximal ERG response. (G–I) Assessment of photopic ERG response. N = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
 
Expressional changes of photoreceptor-specific genes in KO mice. (A–C) Expressional levels of the rod photoreceptor-specific genes at postnatal day 7 and day 42. (D–F) Assessment of cone-specific genes at postnatal day 7 and day 42; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
 
Expressional changes of photoreceptor-specific genes in KO mice. (A–C) Expressional levels of the rod photoreceptor-specific genes at postnatal day 7 and day 42. (D–F) Assessment of cone-specific genes at postnatal day 7 and day 42; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
 
RNA-sequencing and bioinformatics analyses in the retinas. (A) Retinal DEGs between the KO mice and the control group. The red plots represent the up-regulated genes, and the blue plots represent the down-regulated genes. (B) Biological process analysis. Blue bars represent the highest ranked BP items depending on the DEGs. The red plots represent the gene count number that was included in the related biological process based on DEGs. The upper scale bar represents the gene count number, and the lower scale bar shows the P values of BP items. (C) Target prediction of miR-182 in the retina by the TargetScan database and RNA-sequencing analyses. Only the conserved prediction target genes by the TargetScan database were analyzed.
Figure 6
 
RNA-sequencing and bioinformatics analyses in the retinas. (A) Retinal DEGs between the KO mice and the control group. The red plots represent the up-regulated genes, and the blue plots represent the down-regulated genes. (B) Biological process analysis. Blue bars represent the highest ranked BP items depending on the DEGs. The red plots represent the gene count number that was included in the related biological process based on DEGs. The upper scale bar represents the gene count number, and the lower scale bar shows the P values of BP items. (C) Target prediction of miR-182 in the retina by the TargetScan database and RNA-sequencing analyses. Only the conserved prediction target genes by the TargetScan database were analyzed.
Figure 7
 
Structural changes in retinas after light-induced damage. (A) Experimental schema of the light-induced damage. (B) Retinal OCT images at 8 weeks following the light damage. (C) Immunostaining of the dorsal retina after light-induced damage in the miR-182 KO mice. (D) Statistical analysis for the thickness of dorsal IS/OS layer. (E) Immunostaining of ventral retina after light-induced damage in the miR-182 KO mice. (F) Statistical analysis for the thickness of dorsal IS/OS layer. The red color represents antirhodopsin, and green color represents antirecoverin. The horizontal and vertical scale bars in the OCT images: 100 μm. The scale bar in the immunostaining images: 50 μm. For the thickness comparison, the middle region of dorsal and ventral retina was selected; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7
 
Structural changes in retinas after light-induced damage. (A) Experimental schema of the light-induced damage. (B) Retinal OCT images at 8 weeks following the light damage. (C) Immunostaining of the dorsal retina after light-induced damage in the miR-182 KO mice. (D) Statistical analysis for the thickness of dorsal IS/OS layer. (E) Immunostaining of ventral retina after light-induced damage in the miR-182 KO mice. (F) Statistical analysis for the thickness of dorsal IS/OS layer. The red color represents antirhodopsin, and green color represents antirecoverin. The horizontal and vertical scale bars in the OCT images: 100 μm. The scale bar in the immunostaining images: 50 μm. For the thickness comparison, the middle region of dorsal and ventral retina was selected; n = 3. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
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