MicroRNAs in programmed cell death

Programmed cell death, which includes apoptosis, autophagy, and programmed necrosis or necroptosis, is defined as regulated cell death executed by an intracellular programme.^{Reference Su, Yang, Xu, Chen and Yu24} Accumulated evidence has shown a close interaction between apoptosis, autophagy and necroptosis, and the molecular mechanism underlying the crosstalk is regulated by microRNAs. One microRNA may target more than one component of a programmed death pathway and even more than one death pathway.

Apoptosis is a process by which the body rids itself of up to 70 billion superfluous cells a day without triggering an inflammatory process. It occurs normally during development and ageing, and as a homeostatic mechanism to maintain cell populations in tissues. Inappropriate apoptosis is a factor in many human conditions, including neurodegenerative diseases, ischaemic damage, autoimmune disorders and many types of cancer. Many microRNAs have been reported as regulating the apoptotic signalling pathways, such as executioner caspases, and key regulators transcription factor p53 and death-associated protein kinase (DAPK).^{Reference Su, Yang, Xu, Chen and Yu24}

Pro-apoptotic microRNA upregulation in presbyacusis

Pro-apoptotic microRNAs, such as miR-29a/b/c, miR-34a/b/c, let-7a/b/c/e/f/g/i, miR-141, miR-146, miR-203 and miR-429, work through different direct and indirect pathways to induce apoptosis.^{Reference Zhang, Liu, McGee, Walsh, Soukup and He16} Upregulation of both pro-apoptotic miR-29a/b/c and miR-34a/b/c families in the organ of Corti likely contribute to hair cell apoptosis and tissue degeneration in age-related hearing loss. MiR-29 regulates genes upstream of p53 pathways and miR-34a regulates genes downstream of p53 pathways, suggesting that microRNAs play a role in p53-mediated apoptosis in the organ of Corti.^{Reference Tadros, D'Souza, Zhu and Frisina25} The protein p53 is a key transcription factor which regulates many genes that initiate anti-proliferative responses, such as cell-cycle arrest, DNA repair, apoptosis and cellular senescence. At an organism level, p53 activity has been implicated in tissue degeneration and ageing.

In age-related hearing loss animal models, signalling pathways miR-34a/B-cell lymphoma2 (Bcl-2)^{Reference Huang, Zheng, Ou, Xiong, Yang and Zhang18} and miR-34a/sirtuin 1 (SIRT1)/p53^{Reference Xiong, Pang, Yang, Dai, Liu and Ou21} were demonstrated to be associated with cochlear hair cell apoptosis. Genes Bcl-2 and SIRT1 are direct targets of miR-34a.^{Reference Huang, Zheng, Ou, Xiong, Yang and Zhang18,Reference Xiong, Pang, Yang, Dai, Liu and Ou21} The Bcl-2 family contains both anti-apoptotic and pro-apoptotic members, and their interactions determine the likelihood of the cell undergoing apoptosis.^{Reference Gross, McDonnell and Korsmeyer26} Anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-2l1, Bcl-2l2, Mcl-1 and Bcl-2a1) are thought to exert their effects by stabilising the mitochondrial membrane potential and preventing the release of cytochrome C and apoptosis-inducing factors. The binding of miR-34a to the 3’-untranslated region of the Bcl-2 gene halts its transcription, thus cancelling the anti-apoptotic effect of Bcl-2. In aged mice, higher expression of Bcl-2 and Bcl-211 genes was demonstrated to exert a protective effect during cochlear ageing.^{Reference Tadros, D'Souza, Zhu and Frisina25}

Similarly, miR34a overexpression suppressed SIRT1 gene messenger RNA and induced cochlear hair cell apoptosis via a p53-mediated pathway.^{Reference Xiong, Pang, Yang, Dai, Liu and Ou21} The SIRT1 protein is a nicotinamide adenine dinucleotide dependent deacetylase that regulates apoptosis in response to oxidative and genotoxic stress by deacetylating its substrates, including p53.^{Reference Yamakuchi and Lowenstein27} Thus, miR-34a blockage of SIRT1 transcription induces cochlear hair cell apoptosis via a p53-mediated pathway.

The E2F3 gene (E2 transcription factor 3), which regulates cell-cycle progression, might be another direct target of miR-34a.^{Reference Welch, Chen and Stallings28} Pang and colleagues’ (2016) study demonstrated a simultaneous increase of miR-34a expression in the auditory pathways and plasma in aged mice.^{Reference Pang, Xiong, Yang, Ou, Xu and Huang13} The messenger RNA levels of genes SIRT1, Bcl-2 and E2F3 were correspondingly decreased within the auditory pathway and plasma with ageing. However, simultaneous increases of miR-34a expression in non-hearing organs such as the heart and liver suggest that miR-34a exerts a non-tissue-specific ageing effect in mice.

Human studies on the relationship between circulating miR-34a expression level and auditory function are inconclusive. MiR-34 expression level was significantly upregulated in age-related hearing loss patients, compared to normal hearing age-matched and younger controls.^{Reference Pang, Xiong, Yang, Ou, Xu and Huang13} However, the messenger RNA levels of miR-34a target genes SIRT1, Bcl-2 and E2F3 were similar in age-related hearing loss patients and controls, which questions the validity of a miR-34a/Bcl-2 or miR-34a/SIRT1/p53 mediated pathway in the pathogenesis of human age-related hearing loss. In addition, the similar circulating miR-34a expression levels in healthy human controls of different age groups indicates that ageing in humans is not associated with a non-tissue-specific increase in miR-34a level as demonstrated in animal models.^{Reference Pang, Xiong, Yang, Ou, Xu and Huang13}

MiR-29b overexpression was shown to induce mitochondrial dysfunction and hair cell loss via downregulation of the SIRT1/PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator 1α) in aged mice.^{Reference Xue, Wei, Zha, Qiu, Chen and Qiao20} SIRT1 is a direct target of miR-29b. Oxidative stress is believed to cause age-related hearing loss by apoptosis of auditory system cells.^{Reference Someya, Yu, Hallows, Xu, Vann and Leeuwenburgh29} SIRT1 regulates intracellular oxidative stress by the deacetylation of its substrates including PGC-1α, a transcriptional co-regulator that binds to numerous transcription factors to promote mitochondrial biogenesis and oxidative metabolism.^{Reference Tan, Tang, Zhang, Cheng, Cai and Chen30} Previous studies, as cited by Xue et al.,^{Reference Xue, Wei, Zha, Qiu, Chen and Qiao20} have shown that the upregulation of miR-29 microRNAs promotes p53-dependent apoptosis by suppressing several key regulators of cell survival, such as genes p85α and CDC42 (cell division cycle 42). There is evidence from animal studies that miR-29 contributes to age-related hearing loss, but supporting human studies are lacking.

Anti-apoptotic microRNA downregulation in presbyacusis

With ageing, more microRNA expression levels are downregulated than upregulated.^{Reference Zhang, Liu, McGee, Walsh, Soukup and He16,Reference Sekine, Matsumura, Takizawa, Kimura, Saito and Shiiba23} Amongst them are miR-181 and miR-183 family members, which are known mediators of cell proliferation and differentiation pathways. The miR-181 family consists of four members: miR-181a, miR-181b, miR-181c and miR-181d. All of these, with the exception of miR-181c, are associated with organ of Corti degeneration in aged mice.^{Reference Zhang, Liu, McGee, Walsh, Soukup and He16} Upregulation of miR-181a correlated with inner-ear sensory epithelium regeneration in chickens, by suppressing protein p27, a cell-cycle inhibitor and differentiator in the auditory sensory epithelium.^{Reference Frucht, Santos-Sacchi and Navaratnam31}

The miR-183 family is a triad consisting of miR-96, miR-182 and miR-183; these originate from a common primary transcript that begins to be expressed as early as embryonic day 9.5 within the otic vesicle.^{Reference Sacheli, Nguyen, Borgs, Vandenbosch, Bodson and Lefebvre32} Its unique expression pattern within the inner ear correlates with inner-ear development and differentiation.^{Reference Sacheli, Nguyen, Borgs, Vandenbosch, Bodson and Lefebvre32}

MiR-182 is expressed during the differentiation of inner-ear stem/progenitor cells into a hair-cell-like fate. Its function may be associated with its putative target protein Tbx1 (T-box transcription factor 1), a transcription factor that has been implicated in inner-ear development and hair cell fate.^{Reference Wang, Zhang, Du and Jiang33} MiR-182 overexpression induced ectopic hair cells in developing zebra fish, while knockdown of the miR-183 family led to reduced numbers of sensory hair cells and defects in the semi-circular canals.^{Reference Li, Kloosterman and Fekete34}

MiR-96 overexpression demonstrated the same effect as miR-182 in developing zebra fish.^{Reference Li, Kloosterman and Fekete34} Mutation in the miR-96 seed region (2–7 nucleotides in length), which confers binding specificity,^{Reference Lewis, Burge and Bartel35} results in autosomal dominant, progressive hearing loss in humans^{Reference Mencia, Modamio-Hoybjor, Redshaw, Morin, Mayo-Merino and Olavarrieta36} and in mice.^{Reference Lewis, Quint, Glazier, Fuchs, De Angelis and Langford37} In addition, a mutation located in the non-seed region of the miR-96 identified in non-syndromic hearing loss in humans was shown to interfere with miR-96 pre-microRNA secondary structure.^{Reference Solda, Robusto, Primignani, Castorina, Benzoni and Cesarani38} MiR-96 mutation in mice alters the function and gene expression profile of the organ of Corti.^{Reference Lewis, Quint, Glazier, Fuchs, De Angelis and Langford37} Its downstream effect on Oncomodulin, Pitpnm1, Slc26a5 (prestin), Ptprq and Gfi1 genes results in impaired hair cell function and hair cell degeneration, and causes deafness.^{Reference Solda, Robusto, Primignani, Castorina, Benzoni and Cesarani38} Interestingly, Zhang and colleagues’ (2013) microarray analysis detected miR-96 downregulation only in C57BL/6J mice, and not in CBA/J mice, suggesting that the different genetic backgrounds of these two mouse strains may result in differences in the microRNAs involved in the pathogenesis of age-related hearing loss.^{Reference Zhang, Liu, McGee, Walsh, Soukup and He16}

Anti-autophagy microRNA upregulation in presbyacusis

In a recent study, miR-34a was shown to induce cochlear hair cell death via the suppression of autophagy.^{Reference Pang, Xiong, Lin, Lai, Yang and Liu19} Autophagy is a regulated intracellular programme that seeks to clear accumulated intracellular toxins; if unsuccessful, it results in cell death. It is triggered by growth signal deficiency, nutrient deprivation, genotoxic stress, hypoxic stress, endoplasmic reticulum stress, and/or reactive oxygen species accumulation.

In Pang and colleagues’ (2017) study, miR-34a overexpression was associated with a reduction in autophagy in aged mice and HEI-OC1 (House Ear Institute-organ of Corti 1) cells.^{Reference Pang, Xiong, Lin, Lai, Yang and Liu19} In HEI-OC1 cells, miR-34a overexpression suppressed protein ATG9A (autophagy-related protein 9A) level and impaired autophagy via p62 elevation.^{Reference Tai, Wang, Gong, Han, Zhou and Wang39} ATG9A is one target gene of miR-34a, and its corresponding protein AT9A is necessary for optimal autophagy.^{Reference Huang, Sun, Huang, Ye, Pan and Zhong40}

Further research is required to demonstrate the role of miR-34a mediated autophagy in human age-related hearing loss.

MicroRNAs in stria vascularis

A number of differentially expressed microRNAs have been reported in the ageing lateral wall of the cochlear duct or stria vascularis.^{Reference Zhang, Liu, Soukup and He17} MiR-203, a pro-apoptotic microRNA, was upregulated in the lateral wall of aged mice. Genotoxic stress agents (e.g. camptothecin) were demonstrated to induce miR-203 expression by p53 acetylation.^{Reference Chang, Davis-Dusenbery, Kashima, Jiang, Marathe and Sessa41} MiR-203 overexpression downregulates anti-apoptotic genes Bclw or Bcl-2l2 (a member of the Bcl-2 family), and promotes cell death in a p53-dependent manner. Other upregulated microRNAs (miR-762 and miR-1224) and downregulated microRNAs (miR-107, miR-145, miR-342 and miR-455) are also suggested to play a crucial role in the function and maintenance of the stria vascularis.

Potential microRNA–messenger RNA networks in age-related hearing loss

Zhang et al. (2014) proposed networks of microRNA–messenger RNA interactions in age-related hearing loss based on studies of ageing mice.^{Reference Zhang, Liu, Soukup and He17} Differentially expressed microRNAs and their predicted target messenger RNAs associated with apoptosis were used. The networks were complex, with examples of downregulated and upregulated microRNAs targeting upregulated and downregulated pro- and anti-apoptotic genes. For example, Tnfsf10, a critical pro-apoptotic gene was predicted to be upregulated by the downregulation of anti-apoptotic miR-181, amongst others.

Lewis et al. (2016) also explored the potential networks controlled by miR-96 in miR-96 mutant mice (diminuendo mice).^{Reference Lewis, Buniello, Hilton, Zhu, Zhang and Evans42} Three methods of network analysis were used: a manually created regulatory network, protein–protein interactome analysis and gene set enrichment analysis. Myc, Gfi1 and Fos genes were suggested as important targets of miR-96 mediated regulatory networks involved in hearing and deafness. Fos was consistently identified by more than one network analytical method. It regulates genes Slc26a5 (prestin) and Ocm (oncomodulin), both of which are associated with hearing and deafness.^{Reference Tong, Hornak, Maison, Ohlemiller, Liberman and Simmons43} Gfi1 controls Fos via Myc.^{Reference Lewis, Buniello, Hilton, Zhu, Zhang and Evans42} However, more work is required in this area.

MicroRNAs in sudden sensorineural hearing loss pathogenesis

Li et al. predicted that the microRNAs hsa-miR-34a/548n/15a/143/23a/210/18b regulated target genes which may have a critical role in sudden SNHL, based on a small sample of nine sudden SNHL patients and three controls.^{Reference Li, Peng, Huang, Li, Wang and Wang22} Experimental studies not specifically focused on sudden SNHL corroborate a potential role for miR-34a,^{Reference Pang, Xiong, Yang, Ou, Xu and Huang13} miR-210,^{Reference Tra, Frisina and D'Souza44} miR18B,^{Reference Niceta, Stellacci, Gripp, Zampino, Kousi and Anselmi45} miR-23A^{Reference Ohlemiller, Rosen and Gagnon46} and miR15a-5p^{Reference Hedlund, Tangvoranuntakul, Takematsu, Long, Housley and Kozutsumi47} in sudden SNHL. Further studies of larger patient samples are required to validate a role for microRNAs in human sudden SNHL.

Two human-subject studies investigated microRNA biogenesis associated components in sudden SNHL, though neither sought direct evidence of altered microRNA levels.^{Reference Han, Kim, Shin, Cho and Nam48,Reference Kim, Lee and Nam49} The microRNA biogenesis pathway proteins DiGeorge syndrome critical region gene 8 (DGCR8) and argonaute 2 (AGO2) directly influence the biosynthesis of all microRNAs.^{Reference Han, Kim, Shin, Cho and Nam48} AGO2 is a component of the RNA-induced silencing complex (RISC) that silences microRNA activity.^{Reference Kobayashi and Tomari50} DGCR8 is a unit of the microprocessor complex, which mediates microRNA biogenesis via the generation of a precursor microRNA (pre-microRNA).^{Reference Lee, Ahn, Han, Choi, Kim and Yim51}

Han and colleagues’ study identified elevated AGO2 gene expression in sudden SNHL patients’ peripheral blood samples, and it was positively correlated with DGCR8 gene expression in both sudden SNHL patients and healthy controls.^{Reference Han, Kim, Shin, Cho and Nam48}

Kim and colleagues’ study on circulating messenger RNA levels of RNAse III endonucleases Dicer and Drosha identified dysregulation of Dicer, another RISC component that was reduced in sudden SNHL patients compared to controls.^{Reference Kim, Lee and Nam49} Drosha expression was not altered, suggesting that the processing of microRNAs at the nuclear level was not affected. Dicer is essential for the processing of mature microRNAs from their pre-microRNA form.^{Reference Bernstein, Kim, Carmell, Murchison, Alcorn and Li52} Normal development of the functioning inner ear is strongly dependent on normal microRNA maturation, and interrupting the microRNA maturation process results in profound inner-ear malformation.^{Reference Friedman and Avraham53}

The expression of individual microRNA biogenesis related components has been found to be changed in various human diseases.^{Reference Sand, Skrygan, Georgas, Arenz, Gambichler and Sand54} Therefore, the impact of alterations in AGO2 and Dicer expressions on the differential expression of microRNAs in sudden SNHL patients is worthy of further study.