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Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues Hairong Shi a, 1, Hongshun Wang a, 1, Jun Yao a, b, Changsong Lin a, Qinjun Wei a, b, Yajie Lu a, b, Xin Cao a, b, * a b
Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2019 Accepted 5 October 2019 Available online xxx
In our previous study, Oxysterol-binding protein-related protein 2 (OSBPL2) was first identified as a new deafness-causative gene contribute to non-syndromic hearing loss. However, the underlying mechanism of OSBPL2-induced hearing loss remains unknown. Here, we used hearing-specific cells and tissues OC1 cells and zebrafish inner ear tissues as models to identify common transcriptome changes in genes and pathways in the absence of human OSBPL2 orthologues by RNA-seq analysis. In total, 2112 differentially expressed genes (DEGs) were identified between wild-type (WT) and Osbpl2/ OC-1 cells, and 877 DEGs were identified between WT and osbpl2b/- zebrafish inner ear tissues. Functional annotation implicated Osbpl2/osbpl2b in lipid metabolism, cell adhesion and the extracellular matrix in both OC-1 cells and zebrafish inner ear tissues. Proteineprotein interaction (PPI) analysis indicated that Osbpl2/osbpl2b were also involved in ubiquitination. Further experiments showed that Osbpl2/ OC-1 cells exhibited an abnormal focal adhesion morphology characterized by inhibited FAK activity and impaired cell adhesion. In conclusion, we identified novel pathways modulated by OSBPL2 orthologues, providing new insight into the mechanism of hearing loss induced by OSBPL2 deficiency. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: OSBPL2 RNA-seq ECM Focal adhesion Hearing loss
1. Introduction Hearing loss (HL) is the most prevalent sensorineural disorder, affecting approximately 1 in 500 newborns [1,2]. The causes of HL are complex and varied which can be caused by genetic mutations or environmental factors, 60% of which are related to the mutations of a single gene. More than 180 chromosomal loci and over 117 genes have been shown to cause non-syndromic forms of deafness (https://hereditaryhearingloss.org/). Thus far, the molecular mechanisms of deafness caused by these mutant genes remain unknown. OSBPL2 is a member of the oxysterol-binding proteins (OSBP) related protein (ORP) family. Previous studies have shown that OSBPL2 is involved in cholesterol metabolism, cell signalling, and
* Corresponding author. Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, China. E-mail address: [email protected] (X. Cao). 1 These authors contributed equally to this work.
cytoskeleton formation [3e6]. OSBPL2 overexpression has been shown to increase the efflux of cholesterol and enhance its intracellular transport [5]. Knockdown of OSBPL2 and its ER anchors, the VAP proteins in HuH7 hepatoma cells increases hydrolysis and decreases the synthesis of triglyceride (TG) [7]. Additionally, OSBPL2 knockout causes changes in the cytoskeleton and the Akt signalling pathway in HuH7 hepatoma cells [8]. Our previous work first described the association of a mutation in human OSBPL2 with autosomal dominant non-syndromic hearing loss (ADNSHL) in a large affected Chinese family [9], which has also been reported in an affected German family and Mongolian family [10,11]. However, the pathogenesis of OSBPL2 deficits in ADNSHL remains unclear. The transcriptome is the link between the genome and the proteome; the genome carries genetic information; and the proteome plays a biological role [12]. Transcriptome analysis has been an important component of the search for various mechanisms of disease because it helps to identify genes that are dysregulated in specific cells or tissues. Collectively, these advantages allow us to obtain an in-depth understanding of molecular mechanisms and
https://doi.org/10.1016/j.bbrc.2019.10.061 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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therefore make it possible to identify novel therapeutic targets that may suggest innovative therapies. In the past several years, highthroughput transcriptome analyses have provided many new insights into hearing loss [13,14]. In the present study, Osbpl2/osbpl2b-knockout mutants of OC1 cells and zebrafish were subjected to perform transcriptome sequencing to investigate the potential role of OSBPL2 in ADNSHL. The transcriptome analysis showed that ECM-receptor interaction and focal adhesion were the most significant pathways in both OC1 cells and zebrafish inner ear tissues. Further experiments implied that Osbpl2/ inhibited the activity of FAK, the rate of cell adhesion and the formation of focal adhesion. In conclusion, our results suggest that OSBPL2 regulates ECM-receptor-interaction and focal adhesion. This work contributes to a deeper understanding of the molecular function of OSBPL2 in auditory cells and provides new insight into the pathogenesis of OSBPL2-deficit implicated in ADNSHL. 2. Materials and methods 2.1. Animals WT Tübingen zebrafish were provided by the YSY Biotech (Nanjing, China). osbpl2b/ zebrafish were obtained via the CRISPR/Cas9 approach, which can correctly delete genes of interest in living organisms without affecting other gene sequences. All fish maintained under a 14 h light/10 h dark cycle with a water temperature of 28 C and were fed Aquatox Fish Diet flakes (Zeigler, China) twice per day. All zebrafish were maintained under standard procedures and using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University.
deemed to be significantly enriched. Additionally, PPI analysis of all DEGs was performed using the STRING database (https://string-db. org/), which contains known and predicted PPIs. The visualization of the PPI network was obtained with Cytoscape software (version 3.4.0). The molecular complex detection (MCODE), a plugin of Cytoscape was used to identify the characteristics of the network. The highly interacting nodes in the clusters were identified by maintaining following parameters: K-core ¼ 4, node score cutoff ¼ 0.3 and max depth up to 100. 2.5. Quantitative real-time PCR validation The RNA-Seq results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis of representative candidate genes for OC-1 cells. The qRT-PCR reaction was performed according to the instructions provided with SYBR Green PCR Master Mix (Takara Biotechnology Co, China). OC-1 cells and zebrafish inner ear tissues of each genotype were analysed three times. RNA was reverse transcribed into cDNA following the instructions for HiScript Q Select RT Super Mix (Vazyme, China). The relative expression levels of the selected genes were calculated by using the 2△△Ct method. b-actin was used as an internal control. The primer sequence used for PCR is shown in Table S1. 2.6. Cell adhesion assay OC-1 cells were seeded into a 96-well plate coated with Matrigel and allowed to attach for 0.5 h. Then, unattached cells were washed off and attached cells were fixed with 4% formaldehyde and stained with crystal violet. Attached cells were then photographed and counted using a microscope. 2.7. Western blotting analysis
2.2. Cell culture WT and Osbpl2/ OC-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) supplemented with 10% Fatal Bovine Serum (FBS, Gibco, USA) at 33 C with 10% CO2 and subcultured at a density of 1:4 every 2 days or at a density of 1:8 every 3 days. Osbpl2/ OC-1 cells were obtained by CRISPR/Cas9 technology. 2.3. Total RNA extraction OC-1 cells and zebrafish inner ear tissues were homogenized in TRIzol reagent (Life Technologies, USA) and total RNA was extracted according to the manufacturer’s instructions. The integrity of the total RNA was examined by 1% agarose gel electrophoresis. In addition, the RNA concentration and purity were measured by using a Nanodrop One (Thermo Fisher, USA). The OD 260/280 absorbance ratios ranged from 1.8 to 2.0 and the OD 260/230 absorbance ratios from 2.0 to 2.5 for all samples.
OC-1 cells were lysed in RIPA buffer (Beyotime, China) with a protease and phosphatase inhibitor cocktail (Beyotime, China) on ice for 30 min and then disrupted using an ultrasonic cell crusher (SCIENTZ, China). The cells were centrifuged at 12000 g for 15 min, and the supernatants were stored at 80 C. Protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime, China). Fifty micrograms of protein for each sample was loaded into a 10% polyacrylamide gel (Bio-Rad, USA), which was pre-run at 70 V and then at 120 V. The proteins were next transferred to a polyvinylidene difluoride membranes using the Trans-Blot Turbo transfer system (Bio-Rad, USA). The membranes were blocked with 5% BSA in TBST, incubated with the diluted primary antibody in 5% BSA in TBST at 4 C and then probed with the appropriate secondary HRP-linked antibody (Sigma-Aldrich, USA) at room temperature (RT) for 1 h. A ChemiDoc imaging system (Bio-Rad, USA) was used to detect chemiluminescence. Band intensities were quantified using Bio-Rad Image Lab software (v 3.5). 2.8. Immunofluorescence staining
2.4. Transcriptome data analyses DESeq2 (v1.22.2), a Bioconductor package of R (v3.8) was used to determine DEGs between WT and Osbpl2//osbpl2b/. An FDR < 0.05 and a |fold change| > 2 were used as the thresholds to determine significant DEGs. All DEGs were mapped to the Gene Ontology (GO) database using DAVID (https://david.ncifcrf.gov/). The calculated P-value was subjected to Bonferroni’s correction, and GO terms with a p-value < 0.05 were deemed to be significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed with clusterProfiler (v3.10.1), a Bioconductor package of R. KEGG terms with a p-value < 0.05 were
Focal adhesion immunofluorescence staining was performed as previously described [15]. Briefly, OC-1 cells were fixed in 4% paraformaldehyde in PBS for 15 min at RT and permeabilized with 0.1% Triton X-100 for 5 min. After incubation with an anti-Paxillin antibody (Abcam, USA) for 3 h at RT, OC-1 cells were incubated with the appropriate Alexa fluorochrome-594 secondary antibodies (Thermo, USA) for 1 h. F-actin was stained with Alexa Fluor 488-conjugated phalloidin (Sigma-Aldrich, USA) for 30 min at RT. Then nuclei were counterstained with DAPI (Sigma-Aldrich, USA) for 3 min. The samples were visualized with Zeiss LSM 700 confocal microscope (ZEISS, Germany).
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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2.9. Statistical analysis
3.2. DEG identification
Data were represented as mean ± standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA and between-group t-test. p-value 0.05 was regarded as statistically significant.
In total, 24560 genes were mapped in the OC-1 reference genome including 894 up regulated genes and 1218 down regulated genes by comparing the WT group and the Osbpl2/ group (Fig. 1A and C). Additionally, 35134 genes were mapped in the zebrafish reference genome including 307 up regulated genes and 570 down regulated genes by comparing the WT group and the osbpl2b/ group (Fig. 1B and C). Interestingly, many of the DEGs were deaf-genes according to The Hereditary Hearing Loss Homepage (https://hereditaryhearingloss.org/). Most of these genes were related to the cytoskeleton and extracellular matrix, such as Myoa1, Myoa7, Col11a2, Col4a6 and Col2a1 in OC-1 cells and col11a2, col4a6 and col2a1 in zebrafish (Table S4). Hierarchical clustering analysis of OC-1 cells indicated that some of the DEGs present similar functions or participate in the same pathway (Fig. 1D). Highly similar results were observed in zebrafish inner ear tissues (Fig. 1E). These results showed that certain groups of genes in OC-1 cells and zebrafish inner ear tissues might exhibit similar functions or be involved in the regulation of the same pathway.
3. Results 3.1. Sequencing data quality control The quality control results for the sequencing data of the OC1 cells and zebrafish inner ear tissues are shown in Table S2 and Table S3. After the raw reads with poor quality were removed, the number of clean bases in each sample was over 6 G, and the sequencing error rate of single bases was below 1%. Both the Q20 and Q30 of each sample were>90% and no GC bias was found in all samples. Principal component analysis (PCA) provided an overall view of the gene expression profiles of all the samples. The first principal component (PC1) accounted for 60.0% of the overall variability, and the second principal component (PC2) for 28.3% in OC-1 cells (Fig. S1A). In addition, PC1 accounted for 28.3% of the overall variability and the PC2 for 23.7% in zebrafish inner ear tissues (Fig. S1B). These results showed that the control and knockout gene expression profiles were distinct in OC-1 cells or zebrafish inner ear tissues, and the profiles of the three biological triplicates were grouped together.
3.3. GO and KEGG enrichment analysis The GO analysis was divided into three categories: “biological process,” “cellular component,” and “molecular function”. Eighteen significantly enriched GO terms were identified in OC-1 cells (Fig. 2A) or zebrafish inner ear tissues (Fig. 2B). By comparing the results of the GO analysis of OC-1 cells and zebrafish inner ear tissues, we found that Osbpl2//osbpl2b/ mainly affects lipid metabolism, cell adhesion and the extracellular matrix in OC-1 cells and zebrafish inner ear tissues (Table S5).
Fig. 1. Analysis of DEGs. Volcano plot of DEGs in (A) WT/Osbpl2/ OC-1 cells (n ¼ 3) and (B) WT/osbpl2b// zebrafish inner ear tissues (n ¼ 3). Red dots represent the up-regulated genes. Blue dots represent the genes whose expression was down-regulated. (C) The number of DEGs identified in OC-1 cells and zebrafish inner ear tissues. (D) Heatmap of DEGs in OC-1 cells. (E) Heatmap of DEGs in zebrafish inner ear tissues. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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Fig. 2. Functional annotation of DEGs in OC-1 cells and zebrafish inner ear tissues. (A) GO analysis of DEGs between WT and Osbpl2/ OC-1 cells. (B) GO analysis of DEGs between WT and osbpl2b/ zebrafish inner ear tissues. (C) KEGG pathway analysis of DEGs between WT and Osbpl2/ OC-1 cells. (D) KEGG pathway analysis of DEGs between WT and osbpl2b/ zebrafish inner ear tissues.
To explore the pathways in which these differentially expressed genes participate, we conducted a KEGG analysis. Twelve significantly enriched KEGG pathways were identified in OC-1 cells (Fig. 2C) or zebrafish inner ear tissues (Fig. 2D). We identified two specific pathways that existed in both the OC-1 cells and zebrafish inner tissues: ECM-receptor interaction and focal adhesion. In addition, heatmaps were used to illustrate the DEGs in these pathways. We found that the collagen family, integrin family, immune cell surface antigen molecule family, and platelet-derived growth factor family existed in both OC-1 cells (Fig. S2A) and zebrafish inner ear tissues (Fig. S2B). 3.4. Network affected by the DEGs Proteins may function by binding with other proteins to form a protein complex. To further identify the function of OSBPL2, Cytoscape was used to map the DEG network. Only genes with a confidence cutoff 0.9 were selected for the network analysis. The network of DEGs in OC-1 cells exhibited 727 nodes and 2623 edges (Fig. S3), and the network of DEGs in zebrafish inner ear tissues presented 141 nodes and 332 edges (Fig. S4). Next, we used MOCDE software to screen the top two sub-networks with high scores from the network of cells and tissues. In OC-1 cells, Module 1, which contained 70 nodes and 812 edges (Fig. 3A) was enriched in chemokine signalling and ubiquitin mediated proteolysis. Module 2, which contained 93 nodes and 629 edges (Fig. 3B) was enriched in
protein digestion, absorption and focal adhesion. In zebrafish inner ear tissues, Module 3, which contained 17 nodes and 136 edges (Fig. 3C) was enriched in ECM-receptor interaction and focal adhesion. Module 4, which contained 10 nodes and 45 edges (Fig. 3D) was enriched in ubiquitin mediated proteolysis. PPI analysis indicated that Osbpl2/osbpl2b was not only associated with cell adhesion, but also was associated with ubiquitination. 3.5. Verification of RNA-seq results by quantitative real-time PCR To verify the accuracy of the RNA-seq results, we conducted real-time PCR analysis of selected genes in OC-1 cells (Fig. 4A) and zebrafish inner ear tissues (Fig. 4B). The selected genes were associated with ECM-receptor interaction, focal adhesion, and ubiquitin mediated proteolysis pathways in OC-1 cells and zebrafish inner ear tissues. The results showed that the trend of the differential expression of these genes was consistent with the RNAseq data. 3.6. Osbpl2/ decreases focal adhesion formation and inhibits FAK activity To investigate the effect of Osbpl2/ on the focal adhesion and ECM-receptor interaction, the activity of FAK (the key protein of focal adhesion pathway and ECM-receptor interaction) (Fig. 4C) was measured in WT/Osbpl2/ OC-1 cells. The results showed that
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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Fig. 3. PPI analysis of DEGs in OC-1 cells and zebrafish inner ear tissues. (A) Module 1 identified by the MCODE algorithm in OC-1 cells. (B) Module 2 identified by the MCODE algorithm in OC-1 cells. (C) Module 3 identified by the MCODE algorithm in zebrafish inner ear tissues. (D) Module 4 identified by the MCODE algorithm in zebrafish inner ear tissues. (Red nodes represent the up regulated proteins, and blue nodes represent the down regulated proteins). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
knockout of Osbpl2 significantly reduced FAK phosphorylation at Tyr397 in OC-1 cells. In contrast, there was no difference in levels of total FAK in WT/Osbpl2/ OC-1 cells (Fig. 4D). The real-time PCR results for total FAK are consistent with western blot (Fig. S5). FAK localizes to sites of transmembrane integrin receptor clustering and facilitates focal adhesion formation [16]. As expected, compared with WT OC-1 cells, the rate of cell adhesion in Osbpl2/ cells was decreased significantly (Fig. 4E and F). In addition, a significant decrease in focal adhesion was observed in Osbpl2/ cells (Fig. 4G). These results demonstrated that Osbpl2/ inhibits FAK activity and decreases focal adhesion formation. Interestingly, Antoniades et al. has reported the important role of FAK in ciliogenesis [17,18]. Therefore, it is deduced that FAK may plays an important role in OSBPL2-induced hearing loss. 4. Discussion In a previous, we successfully generated Osbpl2/ OC-1 cells and osbpl2b/ zebrafish by using CRISPR/Cas9 gene editing technology (results not shown) based on Rattus norvegicus Osbpl2 and Danio rerio osbpl2b, which are highly homologous to Homo sapiens OSBPL2 (Fig. S6). Here, we performed a series of transcriptome analyses of Osbpl2/ OC-1 cells and osbpl2b/ zebrafish inner tissues and partite experiments to provide new evidence regarding
how OSBPL2 deficiency contributes to hearing loss. In the present study, we first performed a sequencing quality assessment. Over 99% of the sequence reads passed the QC step, and the mapping rate of the sequence reads matched with the rat and zebrafish genomes was very high. In addition, more than 60 M reads were obtained from most of the samples, which indicated that the sequencing depth was deep enough for this study. To verify the findings of the transcriptome analysis, qRT-PCR was used to quantify the expression of the five DEGs in OC-1 cells and zebrafish inner ear tissues. The above results demonstrated the reliability of the transcriptome data. In addition, GO analysis showed that Osbpl2//osbpl2b/ mainly affected lipid metabolism, cell adhesion and the extracellular matrix in the cells and zebrafish inner ear tissues, and KEGG pathway analysis indicated that ECM-receptor interaction and focal adhesion were the most significant pathways involved in both Osbpl2/ OC-1 cells and osbpl2b/ zebrafish inner ear tissues. PPI analysis also indicated that most of the hub genes in sub-networks were associated with ECM and focal adhesion. Hence, transcriptome analysis revealed that ECM-receptor interaction and focal adhesion might be involved in the mechanism of OSBPL2-induced hearing loss. OSBPL2 was previously reported to participate in lipid/cholesterol transport, triglyceride metabolism and adrenocortical steroid hormone production [5,6]. In addition, a recent study showed that
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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Fig. 4. Osbpl2/ inhibits FAK activity and focal adhesion formation. (A) mRNA expression of Itgb7, Col6a1, Pdgfc, Ubac2 and Cd9 in WT/Osbpl2/ OC-1 cells. (B) mRNA expression of itgb7, col6a1, pdgfbb, uba3 and cd8a in WT/osbpl2b/- zebrafish inner ear tissues. mRNA expression was normalized to Actin (Rattus norvegicus)/actin (Danio rerio) (mean ± SEM, n ¼ 3), *p < 0.05, **p < 0.01. (C) Schematic diagram of the “focal adhesion” pathway showing all identified DEGs. Red squares represent up-regulated genes, green squares represent down-regulated genes and blue squares represent mix-regulated genes. (D) Knockout of Osbpl2 reduced FAK Y397 phosphorylation in OC-1 cells. (E) Knockout of Osbpl2 reduced cell adhesion to Matrigel in OC-1 cells. The experiments were performed in triplicate. (F) The relative adhesion ability of cells in WT and Osbpl2/ OC1 cells, **p < 0.01. (G) Knockout of Osbpl2 reduced the formation of focal adhesion in OC-1 cells. Green: F-actin (stained with phalloidin); Red: Focal adhesion (stained with anti-paxillin); Blue: nucleus (stained with DAPI). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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OSBPL2 can regulate the cellular actin cytoskeleton [8]. Consistent with this finding, the present study first showed that ECMreceptor-interaction and focal adhesion might be significant in the development of hearing loss. Extracellular matrix (ECM) is mainly composed of collagen, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminin and other glycoproteins. It not only acts as a scaffold for tissue formation, but also interacts with cells to regulate cell adhesion, migration, proliferation, apoptosis, survival and differentiation. Excessive ECM deposition can lead to pathological fibrosis, increasing the risk of cancer. On the other hand, increasingly ECM breakdown causes tissue destruction, a process that is mainly mediated by proteinases [19,20]. Additionally, many studies have indicated that collagen family ECM components including COL2A1, COL4A6, COL11A1 and COL11A2 are associated with hearing loss [21e24]. At focal adhesion sites, integrin and proteoglycan mediate adhesion to the actin cytoskeleton, which is composed of scaffolding molecules, GTPases, and enzymes such as kinases, phosphatases, proteases, and lipases. Focal adhesion maturation insufficiency is implicated in glomerular pathologies and schizophrenia [25,26]. Furthermore, there is a strong correlation between focal adhesion activation status and neurolathyrism [27]. In addition to the above diseases, focal adhesion is related to hearing loss. Cadherins are adhesion molecules and are often involved in calcium-dependent cell adhesion. Under mechanical stimulation, cadherin 23 (CDH23) and protocadherin 15 (PCDH15) can gap hair-cell mechanosensitive channels to initiate sensory perception [28]. The central mediator of ECM-receptor interaction and focal adhesion is the non-receptor-bound tyrosine kinase FAK, and the phosphorylation of FAK at Tyr397 has been demonstrated to be a key step in the assembly of focal adhesion complexes (Fig. 4C). In the present study, we observed that knockout of OSBPL2 significantly reduced FAK phosphorylation at Tyr397 in OC-1 cells (Fig. 4D). Additionally, the number of focal adhesions was significantly decreased in OC-1 cells (Fig. 4G). Bioinformatics and experimental results revealed that the OSBPL2 participates in ECMreceptor interaction and focal adhesion. Interestingly, previous studies had suggested that FAK activity is closely related to hearing loss: FAK activity is critical for ciliogenesis and ciliary function by disturbing the association between basal bodies and the actin cytoskeleton [17,18]. Considering our results and reported findings, we infer that knocking out of OSBPL2 disrupts the ECM-receptor interactions and focal adhesion pathways, thus reducing FAK activity, which may affect the formation of cilia, eventually inducing hearing loss. In summary, we identified potential pathway and gene expression changes common to tissue and cell models after OSBPL2 orthologues knockout, which provides a direction forward in the attempt to understand the mechanism of hearing loss induced by OSBPL2 deficiency.
Declaration of competing interest The authors declare no conflict of interest exists.
Acknowledgements We gratefully acknowledge generous funding from the National Natural Science Foundation of China (31571302 and 81771000), the Key Research and Development Program of Jiangsu Province (Social Development, BE2016762) and the Key Project of Science and Technology Innovation of Nanjing Medical University (2017NJMUCX001) to Xin Cao.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.061. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.061. References [1] N. Hilgert, R.J. Smith, G. Van Camp, Function and expression pattern of nonsyndromic deafness genes, Curr. Mol. Med. 9 (2009) 546e564. [2] M.L. Marazita, L.M. Ploughman, B. Rawlings, et al., Genetic epidemiological studies of early-onset deafness in the U.S. school-age population, Am. J. Med. Genet. 46 (1993) 486e491. [3] V.M. Olkkonen, A. Koponen, A. Arora, OSBP-related protein 2 (ORP2): unraveling its functions in cellular lipid/carbohydrate metabolism, signaling and Factin regulation, J. Steroid Biochem. Mol. Biol. 192 (2019) 105298. [4] A. Koponen, A. Arora, K. Takahashi, et al., ORP2 interacts with phosphoinositides and controls the subcellular distribution of cholesterol, Biochimie 158 (2019) 90e101. [5] R. Hynynen, S. Laitinen, R. Kakela, et al., Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis, Biochem. J. 390 (2005) 273e283. [6] S. Laitinen, M. Lehto, S. Lehtonen, et al., ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism, J. Lipid Res. 43 (2002) 245e255. [7] H. Kentala, S.G. Pfisterer, V.M. Olkkonen, et al., Sterol liganding of OSBPrelated proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure, Steroids 99 (2015) 248e258. [8] H. Kentala, A. Koponen, A.M. Kivela, et al., Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation, FASEB J. 32 (2018) 1281e1295. [9] G. Xing, J. Yao, B. Wu, et al., Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by whole-exome sequencing, Genet. Med. 17 (2015) 210e218. [10] M. Thoenes, U. Zimmermann, I. Ebermann, et al., OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67), Orphanet J. Rare Dis. 10 (2015) 15. [11] N. Wu, H. Husile, L. Yang, et al., A novel pathogenic variant in OSBPL2 linked to hereditary late-onset deafness in a Mongolian family, BMC Med. Genet. 20 (2019) 43. [12] S.Q. Shen, X.W. Yan, P.T. Li, et al., Analysis of differential gene expression by RNA-seq data in ABCG1 knockout mice, Gene 689 (2019) 24e33. [13] S. Yang, Q. Cai, J. Bard, et al., Variation analysis of transcriptome changes reveals cochlear genes and their associated functions in cochlear susceptibility to acoustic overstimulation, Hear. Res. 330 (2015) 78e89. [14] J.C. Maass, R. Gu, T. Cai, et al., Transcriptomic analysis of mouse cochlear supporting cell maturation reveals large-scale changes in notch responsiveness prior to the onset of hearing, PLoS One 11 (2016) e0167286. [15] Y. Fan, G. Abrahamsen, R. Mills, et al., Focal adhesion dynamics are altered in schizophrenia, Biol. Psychiatry 74 (2013) 418e426. [16] D.J. Sieg, C.R. Hauck, D.D. Schlaepfer, Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration, J. Cell Sci. 112 (Pt 16) (1999) 2677e2691. [17] I. Antoniades, P. Stylianou, N. Christodoulou, et al., Addressing the functional determinants of FAK during ciliogenesis in multiciliated cells, J. Biol. Chem. 292 (2017) 488e504. [18] I. Antoniades, P. Stylianou, P.A. Skourides, Making the connection: ciliary adhesion complexes anchor basal bodies to the actin cytoskeleton, Dev. Cell 28 (2014) 70e80. [19] H. Bao, T.K. Sin, G. Zhang, Activin A induces leiomyoma cell proliferation, extracellular matrix (ECM) accumulation and myofibroblastic transformation of myometrial cells via p38 MAPK, Biochem. Biophys. Res. Commun. 504 (2018) 447e453. [20] C. Bonnans, J. Chou, Z. Werb, Remodelling the extracellular matrix in development and disease, Nat. Rev. Mol. Cell Biol. 15 (2014) 786e801. [21] T. Lopponen, J. Korkko, T. Lundan, et al., Childhood-onset osteoarthritis, tall stature, and sensorineural hearing loss associated with Arg75-Cys mutation in procollagen type II gene (COL2A1), Arthritis Rheum. 51 (2004) 925e932. [22] S. Rost, E. Bach, C. Neuner, et al., Novel form of X-linked nonsyndromic hearing loss with cochlear malformation caused by a mutation in the type IV collagen gene COL4A6, Eur. J. Hum. Genet. 22 (2014) 208e215. [23] K.T. Booth, J.W. Askew, Z. Talebizadeh, et al., Splice-altering variant in COL11A1 as a cause of nonsyndromic hearing loss DFNA37, Genet. Med. 21 (2018) 948e954. [24] W.T. McGuirt, S.D. Prasad, A.J. Griffith, et al., Mutations in COL11A2 cause nonsyndromic hearing loss (DFNA13), Nat. Genet. 23 (1999) 413e419.
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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[25] C. Schell, M. Rogg, M. Suhm, et al., The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E4621eE4630. [26] M.A. Wozniak, K. Modzelewska, L. Kwong, et al., Focal adhesion regulation of cell behavior, Biochim. Biophys. Acta 1692 (2004) 103e119. [27] R.Y. Tan, G.Y. Xing, G.M. Zhou, et al., Plant toxin beta-ODAP activates integrin
beta 1 and focal adhesion: a critical pathway to cause neurolathyrism, Sci. Rep. 7 (2017) 40677. [28] A. Jaiganesh, Y. Narui, R. Araya-Secchi, et al., Beyond cell-cell adhesion: sensational cadherins for hearing and balance, Cold Spring Harb Perspect Biol 10 (2018).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues Hairong Shi a, 1, Hongshun Wang a, 1, Jun Yao a, b, Changsong Lin a, Qinjun Wei a, b, Yajie Lu a, b, Xin Cao a, b, * a b
Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 October 2019 Accepted 5 October 2019 Available online xxx
In our previous study, Oxysterol-binding protein-related protein 2 (OSBPL2) was first identified as a new deafness-causative gene contribute to non-syndromic hearing loss. However, the underlying mechanism of OSBPL2-induced hearing loss remains unknown. Here, we used hearing-specific cells and tissues OC1 cells and zebrafish inner ear tissues as models to identify common transcriptome changes in genes and pathways in the absence of human OSBPL2 orthologues by RNA-seq analysis. In total, 2112 differentially expressed genes (DEGs) were identified between wild-type (WT) and Osbpl2/ OC-1 cells, and 877 DEGs were identified between WT and osbpl2b/- zebrafish inner ear tissues. Functional annotation implicated Osbpl2/osbpl2b in lipid metabolism, cell adhesion and the extracellular matrix in both OC-1 cells and zebrafish inner ear tissues. Proteineprotein interaction (PPI) analysis indicated that Osbpl2/osbpl2b were also involved in ubiquitination. Further experiments showed that Osbpl2/ OC-1 cells exhibited an abnormal focal adhesion morphology characterized by inhibited FAK activity and impaired cell adhesion. In conclusion, we identified novel pathways modulated by OSBPL2 orthologues, providing new insight into the mechanism of hearing loss induced by OSBPL2 deficiency. © 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: OSBPL2 RNA-seq ECM Focal adhesion Hearing loss
1. Introduction Hearing loss (HL) is the most prevalent sensorineural disorder, affecting approximately 1 in 500 newborns [1,2]. The causes of HL are complex and varied which can be caused by genetic mutations or environmental factors, 60% of which are related to the mutations of a single gene. More than 180 chromosomal loci and over 117 genes have been shown to cause non-syndromic forms of deafness (https://hereditaryhearingloss.org/). Thus far, the molecular mechanisms of deafness caused by these mutant genes remain unknown. OSBPL2 is a member of the oxysterol-binding proteins (OSBP) related protein (ORP) family. Previous studies have shown that OSBPL2 is involved in cholesterol metabolism, cell signalling, and
* Corresponding author. Department of Medical Genetics, School of Basic Medical Science, Nanjing Medical University, Nanjing, China. E-mail address: [email protected] (X. Cao). 1 These authors contributed equally to this work.
cytoskeleton formation [3e6]. OSBPL2 overexpression has been shown to increase the efflux of cholesterol and enhance its intracellular transport [5]. Knockdown of OSBPL2 and its ER anchors, the VAP proteins in HuH7 hepatoma cells increases hydrolysis and decreases the synthesis of triglyceride (TG) [7]. Additionally, OSBPL2 knockout causes changes in the cytoskeleton and the Akt signalling pathway in HuH7 hepatoma cells [8]. Our previous work first described the association of a mutation in human OSBPL2 with autosomal dominant non-syndromic hearing loss (ADNSHL) in a large affected Chinese family [9], which has also been reported in an affected German family and Mongolian family [10,11]. However, the pathogenesis of OSBPL2 deficits in ADNSHL remains unclear. The transcriptome is the link between the genome and the proteome; the genome carries genetic information; and the proteome plays a biological role [12]. Transcriptome analysis has been an important component of the search for various mechanisms of disease because it helps to identify genes that are dysregulated in specific cells or tissues. Collectively, these advantages allow us to obtain an in-depth understanding of molecular mechanisms and
https://doi.org/10.1016/j.bbrc.2019.10.061 0006-291X/© 2019 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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therefore make it possible to identify novel therapeutic targets that may suggest innovative therapies. In the past several years, highthroughput transcriptome analyses have provided many new insights into hearing loss [13,14]. In the present study, Osbpl2/osbpl2b-knockout mutants of OC1 cells and zebrafish were subjected to perform transcriptome sequencing to investigate the potential role of OSBPL2 in ADNSHL. The transcriptome analysis showed that ECM-receptor interaction and focal adhesion were the most significant pathways in both OC1 cells and zebrafish inner ear tissues. Further experiments implied that Osbpl2/ inhibited the activity of FAK, the rate of cell adhesion and the formation of focal adhesion. In conclusion, our results suggest that OSBPL2 regulates ECM-receptor-interaction and focal adhesion. This work contributes to a deeper understanding of the molecular function of OSBPL2 in auditory cells and provides new insight into the pathogenesis of OSBPL2-deficit implicated in ADNSHL. 2. Materials and methods 2.1. Animals WT Tübingen zebrafish were provided by the YSY Biotech (Nanjing, China). osbpl2b/ zebrafish were obtained via the CRISPR/Cas9 approach, which can correctly delete genes of interest in living organisms without affecting other gene sequences. All fish maintained under a 14 h light/10 h dark cycle with a water temperature of 28 C and were fed Aquatox Fish Diet flakes (Zeigler, China) twice per day. All zebrafish were maintained under standard procedures and using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University.
deemed to be significantly enriched. Additionally, PPI analysis of all DEGs was performed using the STRING database (https://string-db. org/), which contains known and predicted PPIs. The visualization of the PPI network was obtained with Cytoscape software (version 3.4.0). The molecular complex detection (MCODE), a plugin of Cytoscape was used to identify the characteristics of the network. The highly interacting nodes in the clusters were identified by maintaining following parameters: K-core ¼ 4, node score cutoff ¼ 0.3 and max depth up to 100. 2.5. Quantitative real-time PCR validation The RNA-Seq results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis of representative candidate genes for OC-1 cells. The qRT-PCR reaction was performed according to the instructions provided with SYBR Green PCR Master Mix (Takara Biotechnology Co, China). OC-1 cells and zebrafish inner ear tissues of each genotype were analysed three times. RNA was reverse transcribed into cDNA following the instructions for HiScript Q Select RT Super Mix (Vazyme, China). The relative expression levels of the selected genes were calculated by using the 2△△Ct method. b-actin was used as an internal control. The primer sequence used for PCR is shown in Table S1. 2.6. Cell adhesion assay OC-1 cells were seeded into a 96-well plate coated with Matrigel and allowed to attach for 0.5 h. Then, unattached cells were washed off and attached cells were fixed with 4% formaldehyde and stained with crystal violet. Attached cells were then photographed and counted using a microscope. 2.7. Western blotting analysis
2.2. Cell culture WT and Osbpl2/ OC-1 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) supplemented with 10% Fatal Bovine Serum (FBS, Gibco, USA) at 33 C with 10% CO2 and subcultured at a density of 1:4 every 2 days or at a density of 1:8 every 3 days. Osbpl2/ OC-1 cells were obtained by CRISPR/Cas9 technology. 2.3. Total RNA extraction OC-1 cells and zebrafish inner ear tissues were homogenized in TRIzol reagent (Life Technologies, USA) and total RNA was extracted according to the manufacturer’s instructions. The integrity of the total RNA was examined by 1% agarose gel electrophoresis. In addition, the RNA concentration and purity were measured by using a Nanodrop One (Thermo Fisher, USA). The OD 260/280 absorbance ratios ranged from 1.8 to 2.0 and the OD 260/230 absorbance ratios from 2.0 to 2.5 for all samples.
OC-1 cells were lysed in RIPA buffer (Beyotime, China) with a protease and phosphatase inhibitor cocktail (Beyotime, China) on ice for 30 min and then disrupted using an ultrasonic cell crusher (SCIENTZ, China). The cells were centrifuged at 12000 g for 15 min, and the supernatants were stored at 80 C. Protein concentrations were determined by using a BCA Protein Assay Kit (Beyotime, China). Fifty micrograms of protein for each sample was loaded into a 10% polyacrylamide gel (Bio-Rad, USA), which was pre-run at 70 V and then at 120 V. The proteins were next transferred to a polyvinylidene difluoride membranes using the Trans-Blot Turbo transfer system (Bio-Rad, USA). The membranes were blocked with 5% BSA in TBST, incubated with the diluted primary antibody in 5% BSA in TBST at 4 C and then probed with the appropriate secondary HRP-linked antibody (Sigma-Aldrich, USA) at room temperature (RT) for 1 h. A ChemiDoc imaging system (Bio-Rad, USA) was used to detect chemiluminescence. Band intensities were quantified using Bio-Rad Image Lab software (v 3.5). 2.8. Immunofluorescence staining
2.4. Transcriptome data analyses DESeq2 (v1.22.2), a Bioconductor package of R (v3.8) was used to determine DEGs between WT and Osbpl2//osbpl2b/. An FDR < 0.05 and a |fold change| > 2 were used as the thresholds to determine significant DEGs. All DEGs were mapped to the Gene Ontology (GO) database using DAVID (https://david.ncifcrf.gov/). The calculated P-value was subjected to Bonferroni’s correction, and GO terms with a p-value < 0.05 were deemed to be significantly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed with clusterProfiler (v3.10.1), a Bioconductor package of R. KEGG terms with a p-value < 0.05 were
Focal adhesion immunofluorescence staining was performed as previously described [15]. Briefly, OC-1 cells were fixed in 4% paraformaldehyde in PBS for 15 min at RT and permeabilized with 0.1% Triton X-100 for 5 min. After incubation with an anti-Paxillin antibody (Abcam, USA) for 3 h at RT, OC-1 cells were incubated with the appropriate Alexa fluorochrome-594 secondary antibodies (Thermo, USA) for 1 h. F-actin was stained with Alexa Fluor 488-conjugated phalloidin (Sigma-Aldrich, USA) for 30 min at RT. Then nuclei were counterstained with DAPI (Sigma-Aldrich, USA) for 3 min. The samples were visualized with Zeiss LSM 700 confocal microscope (ZEISS, Germany).
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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2.9. Statistical analysis
3.2. DEG identification
Data were represented as mean ± standard error of the mean (SEM). Statistical significance was determined using one-way ANOVA and between-group t-test. p-value 0.05 was regarded as statistically significant.
In total, 24560 genes were mapped in the OC-1 reference genome including 894 up regulated genes and 1218 down regulated genes by comparing the WT group and the Osbpl2/ group (Fig. 1A and C). Additionally, 35134 genes were mapped in the zebrafish reference genome including 307 up regulated genes and 570 down regulated genes by comparing the WT group and the osbpl2b/ group (Fig. 1B and C). Interestingly, many of the DEGs were deaf-genes according to The Hereditary Hearing Loss Homepage (https://hereditaryhearingloss.org/). Most of these genes were related to the cytoskeleton and extracellular matrix, such as Myoa1, Myoa7, Col11a2, Col4a6 and Col2a1 in OC-1 cells and col11a2, col4a6 and col2a1 in zebrafish (Table S4). Hierarchical clustering analysis of OC-1 cells indicated that some of the DEGs present similar functions or participate in the same pathway (Fig. 1D). Highly similar results were observed in zebrafish inner ear tissues (Fig. 1E). These results showed that certain groups of genes in OC-1 cells and zebrafish inner ear tissues might exhibit similar functions or be involved in the regulation of the same pathway.
3. Results 3.1. Sequencing data quality control The quality control results for the sequencing data of the OC1 cells and zebrafish inner ear tissues are shown in Table S2 and Table S3. After the raw reads with poor quality were removed, the number of clean bases in each sample was over 6 G, and the sequencing error rate of single bases was below 1%. Both the Q20 and Q30 of each sample were>90% and no GC bias was found in all samples. Principal component analysis (PCA) provided an overall view of the gene expression profiles of all the samples. The first principal component (PC1) accounted for 60.0% of the overall variability, and the second principal component (PC2) for 28.3% in OC-1 cells (Fig. S1A). In addition, PC1 accounted for 28.3% of the overall variability and the PC2 for 23.7% in zebrafish inner ear tissues (Fig. S1B). These results showed that the control and knockout gene expression profiles were distinct in OC-1 cells or zebrafish inner ear tissues, and the profiles of the three biological triplicates were grouped together.
3.3. GO and KEGG enrichment analysis The GO analysis was divided into three categories: “biological process,” “cellular component,” and “molecular function”. Eighteen significantly enriched GO terms were identified in OC-1 cells (Fig. 2A) or zebrafish inner ear tissues (Fig. 2B). By comparing the results of the GO analysis of OC-1 cells and zebrafish inner ear tissues, we found that Osbpl2//osbpl2b/ mainly affects lipid metabolism, cell adhesion and the extracellular matrix in OC-1 cells and zebrafish inner ear tissues (Table S5).
Fig. 1. Analysis of DEGs. Volcano plot of DEGs in (A) WT/Osbpl2/ OC-1 cells (n ¼ 3) and (B) WT/osbpl2b// zebrafish inner ear tissues (n ¼ 3). Red dots represent the up-regulated genes. Blue dots represent the genes whose expression was down-regulated. (C) The number of DEGs identified in OC-1 cells and zebrafish inner ear tissues. (D) Heatmap of DEGs in OC-1 cells. (E) Heatmap of DEGs in zebrafish inner ear tissues. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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Fig. 2. Functional annotation of DEGs in OC-1 cells and zebrafish inner ear tissues. (A) GO analysis of DEGs between WT and Osbpl2/ OC-1 cells. (B) GO analysis of DEGs between WT and osbpl2b/ zebrafish inner ear tissues. (C) KEGG pathway analysis of DEGs between WT and Osbpl2/ OC-1 cells. (D) KEGG pathway analysis of DEGs between WT and osbpl2b/ zebrafish inner ear tissues.
To explore the pathways in which these differentially expressed genes participate, we conducted a KEGG analysis. Twelve significantly enriched KEGG pathways were identified in OC-1 cells (Fig. 2C) or zebrafish inner ear tissues (Fig. 2D). We identified two specific pathways that existed in both the OC-1 cells and zebrafish inner tissues: ECM-receptor interaction and focal adhesion. In addition, heatmaps were used to illustrate the DEGs in these pathways. We found that the collagen family, integrin family, immune cell surface antigen molecule family, and platelet-derived growth factor family existed in both OC-1 cells (Fig. S2A) and zebrafish inner ear tissues (Fig. S2B). 3.4. Network affected by the DEGs Proteins may function by binding with other proteins to form a protein complex. To further identify the function of OSBPL2, Cytoscape was used to map the DEG network. Only genes with a confidence cutoff 0.9 were selected for the network analysis. The network of DEGs in OC-1 cells exhibited 727 nodes and 2623 edges (Fig. S3), and the network of DEGs in zebrafish inner ear tissues presented 141 nodes and 332 edges (Fig. S4). Next, we used MOCDE software to screen the top two sub-networks with high scores from the network of cells and tissues. In OC-1 cells, Module 1, which contained 70 nodes and 812 edges (Fig. 3A) was enriched in chemokine signalling and ubiquitin mediated proteolysis. Module 2, which contained 93 nodes and 629 edges (Fig. 3B) was enriched in
protein digestion, absorption and focal adhesion. In zebrafish inner ear tissues, Module 3, which contained 17 nodes and 136 edges (Fig. 3C) was enriched in ECM-receptor interaction and focal adhesion. Module 4, which contained 10 nodes and 45 edges (Fig. 3D) was enriched in ubiquitin mediated proteolysis. PPI analysis indicated that Osbpl2/osbpl2b was not only associated with cell adhesion, but also was associated with ubiquitination. 3.5. Verification of RNA-seq results by quantitative real-time PCR To verify the accuracy of the RNA-seq results, we conducted real-time PCR analysis of selected genes in OC-1 cells (Fig. 4A) and zebrafish inner ear tissues (Fig. 4B). The selected genes were associated with ECM-receptor interaction, focal adhesion, and ubiquitin mediated proteolysis pathways in OC-1 cells and zebrafish inner ear tissues. The results showed that the trend of the differential expression of these genes was consistent with the RNAseq data. 3.6. Osbpl2/ decreases focal adhesion formation and inhibits FAK activity To investigate the effect of Osbpl2/ on the focal adhesion and ECM-receptor interaction, the activity of FAK (the key protein of focal adhesion pathway and ECM-receptor interaction) (Fig. 4C) was measured in WT/Osbpl2/ OC-1 cells. The results showed that
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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Fig. 3. PPI analysis of DEGs in OC-1 cells and zebrafish inner ear tissues. (A) Module 1 identified by the MCODE algorithm in OC-1 cells. (B) Module 2 identified by the MCODE algorithm in OC-1 cells. (C) Module 3 identified by the MCODE algorithm in zebrafish inner ear tissues. (D) Module 4 identified by the MCODE algorithm in zebrafish inner ear tissues. (Red nodes represent the up regulated proteins, and blue nodes represent the down regulated proteins). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
knockout of Osbpl2 significantly reduced FAK phosphorylation at Tyr397 in OC-1 cells. In contrast, there was no difference in levels of total FAK in WT/Osbpl2/ OC-1 cells (Fig. 4D). The real-time PCR results for total FAK are consistent with western blot (Fig. S5). FAK localizes to sites of transmembrane integrin receptor clustering and facilitates focal adhesion formation [16]. As expected, compared with WT OC-1 cells, the rate of cell adhesion in Osbpl2/ cells was decreased significantly (Fig. 4E and F). In addition, a significant decrease in focal adhesion was observed in Osbpl2/ cells (Fig. 4G). These results demonstrated that Osbpl2/ inhibits FAK activity and decreases focal adhesion formation. Interestingly, Antoniades et al. has reported the important role of FAK in ciliogenesis [17,18]. Therefore, it is deduced that FAK may plays an important role in OSBPL2-induced hearing loss. 4. Discussion In a previous, we successfully generated Osbpl2/ OC-1 cells and osbpl2b/ zebrafish by using CRISPR/Cas9 gene editing technology (results not shown) based on Rattus norvegicus Osbpl2 and Danio rerio osbpl2b, which are highly homologous to Homo sapiens OSBPL2 (Fig. S6). Here, we performed a series of transcriptome analyses of Osbpl2/ OC-1 cells and osbpl2b/ zebrafish inner tissues and partite experiments to provide new evidence regarding
how OSBPL2 deficiency contributes to hearing loss. In the present study, we first performed a sequencing quality assessment. Over 99% of the sequence reads passed the QC step, and the mapping rate of the sequence reads matched with the rat and zebrafish genomes was very high. In addition, more than 60 M reads were obtained from most of the samples, which indicated that the sequencing depth was deep enough for this study. To verify the findings of the transcriptome analysis, qRT-PCR was used to quantify the expression of the five DEGs in OC-1 cells and zebrafish inner ear tissues. The above results demonstrated the reliability of the transcriptome data. In addition, GO analysis showed that Osbpl2//osbpl2b/ mainly affected lipid metabolism, cell adhesion and the extracellular matrix in the cells and zebrafish inner ear tissues, and KEGG pathway analysis indicated that ECM-receptor interaction and focal adhesion were the most significant pathways involved in both Osbpl2/ OC-1 cells and osbpl2b/ zebrafish inner ear tissues. PPI analysis also indicated that most of the hub genes in sub-networks were associated with ECM and focal adhesion. Hence, transcriptome analysis revealed that ECM-receptor interaction and focal adhesion might be involved in the mechanism of OSBPL2-induced hearing loss. OSBPL2 was previously reported to participate in lipid/cholesterol transport, triglyceride metabolism and adrenocortical steroid hormone production [5,6]. In addition, a recent study showed that
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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Fig. 4. Osbpl2/ inhibits FAK activity and focal adhesion formation. (A) mRNA expression of Itgb7, Col6a1, Pdgfc, Ubac2 and Cd9 in WT/Osbpl2/ OC-1 cells. (B) mRNA expression of itgb7, col6a1, pdgfbb, uba3 and cd8a in WT/osbpl2b/- zebrafish inner ear tissues. mRNA expression was normalized to Actin (Rattus norvegicus)/actin (Danio rerio) (mean ± SEM, n ¼ 3), *p < 0.05, **p < 0.01. (C) Schematic diagram of the “focal adhesion” pathway showing all identified DEGs. Red squares represent up-regulated genes, green squares represent down-regulated genes and blue squares represent mix-regulated genes. (D) Knockout of Osbpl2 reduced FAK Y397 phosphorylation in OC-1 cells. (E) Knockout of Osbpl2 reduced cell adhesion to Matrigel in OC-1 cells. The experiments were performed in triplicate. (F) The relative adhesion ability of cells in WT and Osbpl2/ OC1 cells, **p < 0.01. (G) Knockout of Osbpl2 reduced the formation of focal adhesion in OC-1 cells. Green: F-actin (stained with phalloidin); Red: Focal adhesion (stained with anti-paxillin); Blue: nucleus (stained with DAPI). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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OSBPL2 can regulate the cellular actin cytoskeleton [8]. Consistent with this finding, the present study first showed that ECMreceptor-interaction and focal adhesion might be significant in the development of hearing loss. Extracellular matrix (ECM) is mainly composed of collagen, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminin and other glycoproteins. It not only acts as a scaffold for tissue formation, but also interacts with cells to regulate cell adhesion, migration, proliferation, apoptosis, survival and differentiation. Excessive ECM deposition can lead to pathological fibrosis, increasing the risk of cancer. On the other hand, increasingly ECM breakdown causes tissue destruction, a process that is mainly mediated by proteinases [19,20]. Additionally, many studies have indicated that collagen family ECM components including COL2A1, COL4A6, COL11A1 and COL11A2 are associated with hearing loss [21e24]. At focal adhesion sites, integrin and proteoglycan mediate adhesion to the actin cytoskeleton, which is composed of scaffolding molecules, GTPases, and enzymes such as kinases, phosphatases, proteases, and lipases. Focal adhesion maturation insufficiency is implicated in glomerular pathologies and schizophrenia [25,26]. Furthermore, there is a strong correlation between focal adhesion activation status and neurolathyrism [27]. In addition to the above diseases, focal adhesion is related to hearing loss. Cadherins are adhesion molecules and are often involved in calcium-dependent cell adhesion. Under mechanical stimulation, cadherin 23 (CDH23) and protocadherin 15 (PCDH15) can gap hair-cell mechanosensitive channels to initiate sensory perception [28]. The central mediator of ECM-receptor interaction and focal adhesion is the non-receptor-bound tyrosine kinase FAK, and the phosphorylation of FAK at Tyr397 has been demonstrated to be a key step in the assembly of focal adhesion complexes (Fig. 4C). In the present study, we observed that knockout of OSBPL2 significantly reduced FAK phosphorylation at Tyr397 in OC-1 cells (Fig. 4D). Additionally, the number of focal adhesions was significantly decreased in OC-1 cells (Fig. 4G). Bioinformatics and experimental results revealed that the OSBPL2 participates in ECMreceptor interaction and focal adhesion. Interestingly, previous studies had suggested that FAK activity is closely related to hearing loss: FAK activity is critical for ciliogenesis and ciliary function by disturbing the association between basal bodies and the actin cytoskeleton [17,18]. Considering our results and reported findings, we infer that knocking out of OSBPL2 disrupts the ECM-receptor interactions and focal adhesion pathways, thus reducing FAK activity, which may affect the formation of cilia, eventually inducing hearing loss. In summary, we identified potential pathway and gene expression changes common to tissue and cell models after OSBPL2 orthologues knockout, which provides a direction forward in the attempt to understand the mechanism of hearing loss induced by OSBPL2 deficiency.
Declaration of competing interest The authors declare no conflict of interest exists.
Acknowledgements We gratefully acknowledge generous funding from the National Natural Science Foundation of China (31571302 and 81771000), the Key Research and Development Program of Jiangsu Province (Social Development, BE2016762) and the Key Project of Science and Technology Innovation of Nanjing Medical University (2017NJMUCX001) to Xin Cao.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.061. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.10.061. References [1] N. Hilgert, R.J. Smith, G. Van Camp, Function and expression pattern of nonsyndromic deafness genes, Curr. Mol. Med. 9 (2009) 546e564. [2] M.L. Marazita, L.M. Ploughman, B. Rawlings, et al., Genetic epidemiological studies of early-onset deafness in the U.S. school-age population, Am. J. Med. Genet. 46 (1993) 486e491. [3] V.M. Olkkonen, A. Koponen, A. Arora, OSBP-related protein 2 (ORP2): unraveling its functions in cellular lipid/carbohydrate metabolism, signaling and Factin regulation, J. Steroid Biochem. Mol. Biol. 192 (2019) 105298. [4] A. Koponen, A. Arora, K. Takahashi, et al., ORP2 interacts with phosphoinositides and controls the subcellular distribution of cholesterol, Biochimie 158 (2019) 90e101. [5] R. Hynynen, S. Laitinen, R. Kakela, et al., Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis, Biochem. J. 390 (2005) 273e283. [6] S. Laitinen, M. Lehto, S. Lehtonen, et al., ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism, J. Lipid Res. 43 (2002) 245e255. [7] H. Kentala, S.G. Pfisterer, V.M. Olkkonen, et al., Sterol liganding of OSBPrelated proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure, Steroids 99 (2015) 248e258. [8] H. Kentala, A. Koponen, A.M. Kivela, et al., Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation, FASEB J. 32 (2018) 1281e1295. [9] G. Xing, J. Yao, B. Wu, et al., Identification of OSBPL2 as a novel candidate gene for progressive nonsyndromic hearing loss by whole-exome sequencing, Genet. Med. 17 (2015) 210e218. [10] M. Thoenes, U. Zimmermann, I. Ebermann, et al., OSBPL2 encodes a protein of inner and outer hair cell stereocilia and is mutated in autosomal dominant hearing loss (DFNA67), Orphanet J. Rare Dis. 10 (2015) 15. [11] N. Wu, H. Husile, L. Yang, et al., A novel pathogenic variant in OSBPL2 linked to hereditary late-onset deafness in a Mongolian family, BMC Med. Genet. 20 (2019) 43. [12] S.Q. Shen, X.W. Yan, P.T. Li, et al., Analysis of differential gene expression by RNA-seq data in ABCG1 knockout mice, Gene 689 (2019) 24e33. [13] S. Yang, Q. Cai, J. Bard, et al., Variation analysis of transcriptome changes reveals cochlear genes and their associated functions in cochlear susceptibility to acoustic overstimulation, Hear. Res. 330 (2015) 78e89. [14] J.C. Maass, R. Gu, T. Cai, et al., Transcriptomic analysis of mouse cochlear supporting cell maturation reveals large-scale changes in notch responsiveness prior to the onset of hearing, PLoS One 11 (2016) e0167286. [15] Y. Fan, G. Abrahamsen, R. Mills, et al., Focal adhesion dynamics are altered in schizophrenia, Biol. Psychiatry 74 (2013) 418e426. [16] D.J. Sieg, C.R. Hauck, D.D. Schlaepfer, Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration, J. Cell Sci. 112 (Pt 16) (1999) 2677e2691. [17] I. Antoniades, P. Stylianou, N. Christodoulou, et al., Addressing the functional determinants of FAK during ciliogenesis in multiciliated cells, J. Biol. Chem. 292 (2017) 488e504. [18] I. Antoniades, P. Stylianou, P.A. Skourides, Making the connection: ciliary adhesion complexes anchor basal bodies to the actin cytoskeleton, Dev. Cell 28 (2014) 70e80. [19] H. Bao, T.K. Sin, G. Zhang, Activin A induces leiomyoma cell proliferation, extracellular matrix (ECM) accumulation and myofibroblastic transformation of myometrial cells via p38 MAPK, Biochem. Biophys. Res. Commun. 504 (2018) 447e453. [20] C. Bonnans, J. Chou, Z. Werb, Remodelling the extracellular matrix in development and disease, Nat. Rev. Mol. Cell Biol. 15 (2014) 786e801. [21] T. Lopponen, J. Korkko, T. Lundan, et al., Childhood-onset osteoarthritis, tall stature, and sensorineural hearing loss associated with Arg75-Cys mutation in procollagen type II gene (COL2A1), Arthritis Rheum. 51 (2004) 925e932. [22] S. Rost, E. Bach, C. Neuner, et al., Novel form of X-linked nonsyndromic hearing loss with cochlear malformation caused by a mutation in the type IV collagen gene COL4A6, Eur. J. Hum. Genet. 22 (2014) 208e215. [23] K.T. Booth, J.W. Askew, Z. Talebizadeh, et al., Splice-altering variant in COL11A1 as a cause of nonsyndromic hearing loss DFNA37, Genet. Med. 21 (2018) 948e954. [24] W.T. McGuirt, S.D. Prasad, A.J. Griffith, et al., Mutations in COL11A2 cause nonsyndromic hearing loss (DFNA13), Nat. Genet. 23 (1999) 413e419.
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061
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[25] C. Schell, M. Rogg, M. Suhm, et al., The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E4621eE4630. [26] M.A. Wozniak, K. Modzelewska, L. Kwong, et al., Focal adhesion regulation of cell behavior, Biochim. Biophys. Acta 1692 (2004) 103e119. [27] R.Y. Tan, G.Y. Xing, G.M. Zhou, et al., Plant toxin beta-ODAP activates integrin
beta 1 and focal adhesion: a critical pathway to cause neurolathyrism, Sci. Rep. 7 (2017) 40677. [28] A. Jaiganesh, Y. Narui, R. Araya-Secchi, et al., Beyond cell-cell adhesion: sensational cadherins for hearing and balance, Cold Spring Harb Perspect Biol 10 (2018).
Please cite this article as: H. Shi et al., Comparative transcriptome analysis of auditory OC-1 cells and zebrafish inner ear tissues in the absence of human OSBPL2 orthologues, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.061