CRISPR/Cas9‐mediated knockout of Lim‐domain only four retards organ of Corti cell growth

Abstract

Lim-domain only 4 (LMO4) plays a critical role in mediating the ototoxic side-effects of cisplatin, a highly effective anti-cancer drug. However, the signaling mechanism by which cochlear LMO4 mediates otopathology is yet to be fully understood. Knockout cell culture models are useful tools for investigating the functional roles of novel genes and delineating associated signaling pathways. Therefore, LMO4 knockout organ of Corti cells were generated by using the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system. Successful knockout of LMO4 in UB/OC1 cells was verified by the absence of LMO4 protein bands in immunoblots. Though the Knockout of LMO4 retarded the growth rate and the migratory potential of the cells it did not inhibit their long-term viability as the LMO4 knockout UB/OC1 cells were able to survive, proliferate, and form colonies. In addition, the knockout of LMO4 did not alter the expression of myosin VIIa, a biomarker of hair cells, suggesting that the knockout cells retain important characteristic features of cochlear sensory receptor cells. Thus, the findings of this study indicate that CRISPR/Cas9 system is a simple and versatile method for knocking out genes of interest in organ of Corti cells and that LMO4 knockout UB/OC1 cells are viable experimental models for studying the functional role of LMO4 in ototoxicity.

Advanced Omics Techniques for Understanding Cochlear Genome, Epigenome, and Transcriptome in Health and Disease

“…In particular, CRISPR/Cas9 has been successfully applied to the creation of new in vitro and in vivo models of cochlear diseases [31]. For instance, this technology has allowed the study of genes associated with ototoxicity via the knock-out of Lim-domain only 4 (LMO4), for cisplatin, and HtrA Serine Peptidase 2 (htra2), for aminoglycosides, in vitro and in vivo models, respectively [32,33]. It has also allowed the study of inherited hearing loss genes, such as MYO7A, CIB2, and CDH23, for Usher syndrome [31].…”

lmo4a Contributes to Zebrafish Inner Ear and Vestibular Development via Regulation of the Bmp Pathway

“…In humans, LMO4 is known for its association with breast neoplasia and other types of carcinoma [9,[35][36][37]. Apart from a series of studies by Jamesdaniel and colleagues, which demonstrated that LMO4 and its downstream targets mediate the ototoxic effects of the commonly used anti-neoplastic agent cisplatin [13,14,16,17,40], its role in overall embryonic and inner ear development has not been a particular focus of clinical attention However, the possibility that Lmo4 serves as a regulator of Bmps (Figure 6B) or that Lmo4 and Bmps are involved in two parallel signaling pathways with mutual regulatory interactions (Figure 6C) cannot be ruled out.…”

“…Targeted disruption of Lmo4 in the mouse model led to dysmorphogenesis of the vestibule and negative regulation of sensory organ formation in the mammalian cochlea [11,12]. In addition to its role in inner ear development, recent research has revealed that Lmo4 and its downstream proteins play vital roles in mediating the ototoxicity of cisplatin, including cochlear apoptosis and hearing loss [13][14][15][16][17][18]. Lmo4 is proposed to act under the regulation of Bmp4 [19], with more recent evidence showing that Bmp2 regulates Lmo4 via an intermediate gene, Ntn1 [20].…”

Advanced Omics Techniques for Understanding Cochlear Genome, Epigenome and Transcriptome in Health and Disease

“…In particular, CRISPR/Cas9 has been successfully applied for creating new in vitro and in vivo models of cochlear diseases [30]. For instance, this technology has allowed to study genes associated with ototoxicity, via the knock-out of Lim-domain only 4 (LMO4), for cisplatin, and HtrA Serine Peptidase 2 (htra2), for aminoglycosides, in in vitro and in vivo models respectively [31,32]. It also allowed the study of inherited hearing loss genes, such as MYO7A, CIB2 and CDH23, for the Usher syndrome [30].…”