Cholesteatomas are benign, but aggressive, lesions typically found in the outer and middle ears. Epidermally derived, they are more accurately termed keratomas and represent dysregulation of skin growth. Cholesteatomas may be congenital or secondarily acquired and have an incidence of approximately 6-10 per 100,000 population (Kemppainen et al., 1999, Homoe, 2001, Homoe and Rosborg, 2007). Although not malignant, cholesteatomas are locally destructive and their unregulated growth can have serious sequelae, including hearing loss, facial nerve paralysis, and intracranial infections. (Vikram et al., 2008, Friedland et al., 2008, Smith and Danner, 2006). There are no medical therapies for the treatment of cholesteatoma, only surgical resection. Because of high recurrence rates, individuals with this process typically undergo many operations.
Gene expression studies in cholesteatoma have demonstrated significant up-regulation of messenger RNAs of a number of genes involved with cell growth and proliferation. In contrast, some studies examining the same genes have found no significant difference in mRNA expression. One explanation of these conflicting data would be that the level of messenger RNA is variable but irrelevant as the phenotype would be more closely related to protein levels. However, some protein expression studies have shown conflicting results as well.
It is becoming increasingly evident that individual protein and gene expression levels are not the molecular pathology leading to disease. Rather, the relative levels among proteins in complex pathways may ultimately determine the cellular phenotype. Regulators of such pathways have recently been identified and are called microRNAs (hsa-mirs in humans). MicroRNAs are evolutionary conserved, 22-24 nucleotide long, non-coding RNA molecules that inhibit protein translation. They are significant in cellular pathways subserving differentiation, development, apoptosis, and oncogenesis. Many oncogenic microRNAs have been identified including hsa-mir-21. Hsa-mir-21 has been shown to be up-regulated in neoplasms and appears to down-regulate tumor suppressor proteins. This one molecule is able to affect many proteins downstream thereby amplifying its effect. It has been shown to be significantly up-regulated in acoustic neuroma -another benign tumor in the otology discipline.
We hypothesize that cholesteatoma growth and proliferation is regulated at the translational level by microRNAs. Based on preliminary data, this study examines the potential role of hsa-mir-21 in cholesteatoma growth and proliferation. We investigated upstream regulators of hsa-mir-21 expression and levels of downstream protein targets to develop a model of cholesteatoma formation. This model may identify novel targets for RNA-and protein-based therapeutics that can provide non-surgical or adjunctive treatments for cholesteatoma.
Objectives: Identify novel regulatory mechanisms controlling growth and proliferation of cholesteatoma. Specifically, the potential role of microRNAs and downstream proteins was studied in cholesteatoma.
Study Design: This study represents a molecular biological investigation characterizing and comparing microRNA and protein expression. Methods: Cholesteatoma and normal skin were taken from patients at the time of surgery. Tissue was processed for RNA and protein extraction. Real-time RT-PCR was used to assess levels of human microRNAs. Western blot analyses were used to assess levels of upstream and downstream regulatory proteins. Results: Several microRNAs were found to be up-regulated in cholesteatoma as compared to normal skin, especially microRNA-21 (hsa-mir-21), which has been associated with numerous other human neoplasms. Further characterization of hsa-mir-21 showed a greater than 4-fold higher expression in cholesteatoma. The downstream targets of hsa-mir-21, PTEN and PDCD4, were found to be reduced in cholesteatoma compared to normal skin. Activators of hsa-mir-21 expression were also differentially regulated between the tissues. Conclusions: Hsa-mir-21 causes down-regulation of the potent tumor suppressor gene PTEN which regulates apoptosis, proliferation, invasion and migration. It also regulates PDCD4 which has been implicated in cutaneous neoplasms. This study has identified differential regulation of these factors in cholesteatoma when compared to normal skin. The presence of microRNA regulators provides pharmacological targets for the adjunctive or primary treatment of cholesteatoma.