Int J Clin Exp Med 2017;10(2):2241-2251 www.ijcem.com /ISSN:1940-5901/IJCEM0042143 Original Article Transcript expression profiles of stria vascularis in Mitf-m knockout mice Wei Chen1*, Xi Shi2*, Lili Ren1, Keshuang Wang1, Xiaojie Liang3, Lei Chen4, Wei Sun5, Weiwei Guo1, Shiming Yang1 1Department of Otolayngology, Head & Neck Surgery, Institute of Otolaryngology of PLA, Chinese PLA General Hospital, Beijing 100853, China; 2Xuzhou Medical College listening Center, Xuzhou 221006, China; 3Department of Otolaryngeal-Head Neck Surgery, The Army General Hospital of PLA, Beijing 100700, China; 4Key Laboratory of Pig Industry Sciences (Ministry of Agriculture), Chongqing Academy of Animal Science, Chongqing 402460, China; 5Department of Communicative Disorders & Sciences, Center for Hearing and Deafness, State University of New York at Buffalo, Buffalo, New York, United States. *Equal contributors. Received October 17, 2015; Accepted November 15, 2015; Epub February 15, 2017; Published February 28, 2017 Abstract: Objective: Mitf-m gene plays an important role in the development of cochlear stria vascularies. The mutation of Mitf-m gene can cause severe hearing loss, such as Wanderburg syndrome. However, the gene ex- pression change caused by Mitf-m has not been systemically studied. In this paper, we studied the transcriptome of Mitf-m knockout mouse on cochlear stria vascularies. Methods: Total RNA extracted from the stria vascularies of the Mitf-m knockout mice (Mitfmi-ΔM/mi-ΔM, MM group) and the wild type mice (Mitf-m+/+, WW group) were used for RNA-Seq analysis. The clean reads were mapped to the reference sequence and cufflinks were used to evaluate the RNA sequencing results and gene expression. The program edgeR and DESeq were applied to identify the dif- ferentially expressed genes (DEGs). The down-regulated DEGs were further analyzed. Results: A total of 42,463,888 and 39,718,542 clean reads have been mapped to the reference sequence in the MM group and the WW group, respectively. For screening DEGs, 88.7% and 87.4% of the reads were uniquely mapped to the reference sequence suggesting a confidence of the RNA-Seq result. Out of the 45 DEGs, 7 genes were found to be up-regulated and 38 genes were down-regulated. GO enrichment analysis indicated that the down-regulated genes were mainly enriched in pigmentation, the melanin biosynthetic and metabolic process. In addition, these DEGs were involved with the inward rectifier potassium channel activity (Kcnj10 and Kcnj13). KEGG enrichment analysis showed that the down- regulated DEGs mainly enriched in melanogenesis pathways. Conclusion: Our data provide a large amount of useful information about mRNAs that are related in Mitf-m related pathways. The result will help researchers to further understand the functions of Mitf-m as a master regulator in more pathways, such as cell proliferation, DNA repair, cell cycle, apoptosis, proliferation, malanosome, and Na+/K+ iron pathway. Together, our data provide a novel insight for Mitf-m network function. Keywords: Mitf-m, stria vascularis, RNA sequencing, knockout mice Introduction WS 3 are caused by loss of function mutation in the PAX3 gene, while mutation in EDNRB, EDN3 According to recent estimates of the World and SOX10 can be the cause of WS 4 [2]. Health Organization (WHO), more than 5% of Among those subtypes of WS, WS 2 is a domi- the world’s total population suffered from some nantly inherited disorder and is associated with form of disabling hearing loss (WHO, 2014). It mutation in the microphthalmia-associated was found that abnormal pigmentation and transcription factor (Mitf) gene [3]. Mitf is a tis- hearing loss were frequently combined in most sue restricted, basic helix-loop-helix leucine zip- mammals. In human, Waardenburg syndrome per (b-HLH-Zip), and dimeric transcription fac- (WS) is characterized by varying combinations tor. Mitf plays a critical role in the development of sensorineural hearing loss, heterochromia of both neural-crest-derived melanocytes and iridis and patchy abnormal depigmentation of optic cup-derived retinal pigmented epithelium the hair and skin [1]. WS is clinically and geneti- (RPE); the loss of a functional Mitf in mice cally heterogeneous and classified into four results in complete absence of all pigment types (WS 1, WS 2, WS 3 and WS 4). WS 1 and cells, which in turn induces microphthalmia and Transcript profiles of stria vascularis in Mitf-m KO mice pathway. Together, our data provide a novel insight for Mitf network function. Materials and methods Mice Mitfmi-ΔM/mi-ΔM targeted mice were generated using the recombineering technology. To gener- ate the targeting construct, the M-promoter/M- exon and its flanking regions (10,868 bp) were cloned using plasmid rescued from BACRP23- Figure 1. Schematics show the process of Mitf-m targeting. Top: The region of the Mitf gene contain- 9A13. A 1,743 bp floxed neomycin resistance ing exon m, exon 2, exon 3, and exon 4; Middle: The expression cassette, which was flanked by 160 targeting construct with a floxed neomycin cassette bp of sequence flanking the M-promoter/M-ex- in the M promoter/Mexon region; Bottom: Mitf gene on, was used to replace 790 bp of the M-pro- portion after targeting. moter/M-exon from the above plasmid (Figure 1). The targeting construct was used for stan- inner ear deafness [4]. Mitf is also a melano- dard targeting of CRIB 6.1 ES cells (genotype cyte master regulator gene and a melanoma [C57Bl6J/6Nx129S6] F1), giving 6 correctly oncogene [5]. The Mitf gene contains multiple targeted colonies out of 40 colonies tested. promoters and consecutive first exons, fol- Positive ES cell clones were micro injected into lowed by common downstream exons, thereby 8-cell embryos to obtain chimeric mice. The generating distinct isoforms that differ in Chimeric mice that transmitted the modified Mitf M-exon allele to their progeny were crossed theamino-termini, such as Mitf-M, Mitf-A, Mitf-D mi-ΔM/+ and Mitf-H [6]. with Kunming mice to generate Mitf mice. Mitfmi-ΔM/mi-ΔM targeted mice were obtained by mi-ΔM/+ mi-ΔM/+ In 1957, Kreinter reported a recessive white Mitf ×Mitf mating. Genotyping of spotting mutation that was subsequently mice was performed by PCR using primers termed black-eyed white (Mitfmi-bw) [7]. The which flanking M-exon of Mitf shown in mouse homozygous for the Mitf mi-bw allele Supplementary Table 1. The use of mice has shows a complete black-eyed white phenotype been approved by the Institutional Animal Care with severe hearing loss but without apparent and Use Committee of the General Hospital of ocular abnormalities [4, 8]. It was found that PLA in Beijing. the 7.2 Kb L1 element was inserted into intron Dissection of mouse stria vascularis 3 that specifically repress the transcription from the melanocyte-specific promoter and Heads of anesthetized animals were cut down lead to the decreased expression of Mitf-m after the auricle with a guillotine. The heads mRNA [4]. Although Mitf mi-bw provided a good were split along the middle line to remove the model to study the function of Mift-m, the 7.2 brain tissues. The bilateral otic vesicle was dis- kb L1 element insertion within intron 3 is spon- sociated carefully using the rongeur. The bone taneous and there is possible affect on the shell of the otic vesicle was opened to expose transcription of other type of Mift. the cochlear structures. The dissected cochlea was put into the glass dish that filled with RNA In the present study, we investigated the differ- later solution (Life Technologies, Carlsbad, CA). ent expression of mRNAs in transgenic Mitf- The cochlea volute was quickly removed under gene using RNA-seq technology. We used tran- the dissection microscope and the stria vascu- scriptome sequencing technology to analyses lar was removed from the outer wall of the and identify the full repertoire of mRNAs cochlea by the gossamer tweezers. The expressed in the Mitf pathways. The data pro- Reissner’s membrane was to from the edge of vide a large amount of useful information about the stria vascular to get the complete stria mRNAs that are associated to Mitf related vasculars. pathways. The result will help researchers to further understand the functions of Mitf as a RNA extraction master regulator in more pathways, such as cell proliferation, DNA repair, cell cycle, apopto- Total RNA of stria vascularis was isolated sis, proliferation, malanosome, and Na+/K+ iron by a TRIzol Plus RNA Purification Kit (Life 2242 Int J Clin Exp Med 2017;10(2):2241-2251 Transcript profiles of stria vascularis in Mitf-m KO mice Figure 2. The phenotype of Mitf m knockout mice. A. Mitf+/+ shows black coat color, and Mitf mi­ΔM/mi­ΔM shows white coat color and black eyes. B. Mitf-m was not detected in Mitf mi­ΔM/mi­ΔM cochlea, but expressed in Mitf+/+ cochlea. C. The ABR threshold are 20~30 dB SPL in Mitf+/+ mice, and 100~110 dB SPL in Mitf mi­ΔM/mi­ΔM mice. D. The stria vas- cularis (SV) of Mitf mi­ΔM/mi­ΔM cochlea are significantly thinner and shorter than that of Mitf+/+ as indicated by stars. E. The image of transmission electron microscope (TEM) showing most of the melanocytes were abnormal or missing in the cochlea of Mitf mi­ΔM/mi­ΔM. Figure 3. Dissection of mouse stria vascularis (SV) for RNA-seq. The schematic cross-section (A) and photograph (B) show the dissection protocol. Firstly, the organ of Corti and lateral wall were peeled away from the spiral ganglion, the lateral wall was peeled away from the organ of Corti. The fraction of lateral wall contains the spiral ligament, outer sulcus, and stria vascularis.