CircRNA is a special endogenous non-coding RNA with a unique ring structure, which regulates gene expression at the post-transcriptional level and plays an important role in a series of life processes, such as cell proliferation, differentiation, apoptosis, migration and metabolism. However, there are few studies on circRNAs in middle ear cholesteatoma, and the role of circRNAs in the etiopathogenesis of middle ear cholesteatoma is rarely investigated so far. We totally identified 93 up-regulated and 85 down-regulated circRNAs in cholesteatoma with statistical significance compared with normal skin. Moreover, qRT-PCR was performed to verify the microarray discoveries by detecting the expression of 8 selected circRNAs, including 5 up-regulated circRNAs (hsa_circRNA_103670, hsa_circRNA_048764, hsa_circRNA_404864, hsa_circRNA_104327, hsa_circRNA_404655), and 3 down-regulated circRNAs (hsa_circRNA_101965, hsa_circRNA_000319, hsa_circRNA_100927). Expressions of hsa_circRNA_104327, hsa_circRNA_404655 and hsa_circRNA_00031) were in line with the microarray results, suggesting that these three circRNAs may play a pivotal role in the pathogenesis of middle ear cholesteatoma. The difference between microarray results and qRT-PCR validation minght be casue by the false positive in microarray analysis. Furthermore, a large sample size will be enrolled in our following research.
CircRNA is closely related to the pathogenesis of a variety of proliferative diseases, such as excessive proliferation of psoriatic keratinocytes [9], hypoxia-induced vascular endothelial cell proliferation [10], skeletal myoblast proliferation[11] and so on. In the process of these proliferative diseases, circRNA regulates the expression of related target genes through miRNA "sponge" and participates in the etiopathogenesis of these diseases. Wang et al [11]. found that circTitin activated insulin-like growth factor 2 (IGF2)/phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway through adsorption of miRNA-432, which lead to the proliferation and differentiation of skeletal myoblasts. In addition, circRNA is also closely related to tumorigenesis. Studies have found that the expression of circRNA was abnormal in many kinds of tumor tissues, including breast cancer [16], liver cancer [17] and esophageal cancer [18], which was closely related to tumor cell hyperproliferation, invasion and metastasis. A study found that the expression of circSMARCA5 was significantly down-regulated in hepatocellular carcinoma, and was closely related to the hyperproliferation, invasion and metastasis of hepatocellular carcinoma cells. Transfection of circSMARCA5 into the hepatocellular carcinoma cells can up-regulate the expression of target gene tissue inhibitor of matrix metalloproteinase 3 (TIMP3) through adsorption of miRNA-17-3p and miRNA-181b-5p, and ultimately inhibit the proliferation, invasion and metastasis of hepatocellular carcinoma cells [17]. Respecting to middle ear cholesteatoma, recent studies have found that hsa_circRNA_102747 acted as the endogenous sponge of miRNA-21-3p, a miRNA belonging to the miRNA-21 family, which has been demonstrated to result in excessive proliferation of cholesteatoma keratinocytes [19]. Meanwhile, hsa_circRNA-101458 was able to interact with miRNA-let-7a-3p, a miRNA belonging to the miRNA-let-7a family, the upregulation of which has been confirmed to suppress proliferation of keratinocytes in cholesteatoma [19]. Consequently, circRNAs might play an important role in the pathogenesis of middle ear cholesteatoma.
By analyzing the differentially expressed circRNA between cholesteatoma and normal skin, expressions of hsa_circRNA_104327 and hsa_circRNA_404655 in cholesteatoma were significantly increased, while the expression of hsa_circRNA_000319 was significantly decreased. Moreover, our results revealed that hsa_circRNA_000319 interacted with miRNA-4436b-5p, hsa_circRNA_104327 interacted with miRNA-152-5p, and hsa_circRNA_404655 interacted with miRNA-3664-3p. Some studies have found that the decreased expression of miRNA-3664-3p in spinal cord glioblastoma (SC-GBM) may lead to an increase in the expression of its target gene growth diferentiation factor 15 (GDF15), which in turn increases the expression of p53 and promotes tumor progression [20]. However, so far, the role of miRNA-3664-3p in cholesteatoma disease is not clear. It is well known that although cholesteatoma is pathologically benign, it is locally invasive and leads to bone resorption. We speculate that hsa_circRNA_404655, as a sponge of miRNA-3664-3p, may play a potential regulatory role in the proliferation and local invasion of cholesteatoma keratinocytes and participate in the formation of cholesteatoma. Moreover, we also discovered several targeted mRNAs for hsa_circRNA_404655 and miRNA-3664-3p, including IQGAP3, ROR2, EPHB1, NPM1, RCC2, NCOA5, and so on. Although these targeted mRNAs have not been investigated in middle ear cholesteatoma, their biological functions were explored in other proliferative and invasive diseases. For instance, IQGAP3, EPHB1, ROR2 and RCC2 were demonstrated to markedly enhance cell migration, invasion, and epithelial-to-mesenchymal transition in hepatocellular carcinoma, medulloblastoma and breast carcinoma, respectively [21–24]. Meanwhile, epithelial-to-mesenchymal transition was also observed in middle ear cholesteatoma [25]. Moreover, NPM1 contributed to enhanced cell proliferation with phosphatase and tensin homologue deleted on chromosome ten (PTEN) inactivation [26], while NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/Akt pathway [27]. Similarly, PTEN inactivation and PI3K/Akt pathway activation were also noticed in middle ear cholesteatoma [28]. These results provide new insights into the pathogenesis of cholesteatoma and suggest that circRNAs may be a potential target for the treatment of cholesteatoma. The specific mechanism and regulatory pathway need to be further studied. In addition, our study also found that the expression of hsa_circRNA_000319 was significantly decreased in cholesteatoma tissues, and caused the up-regulation of miR-4436b-5p expression in cholesteatoma tissues. However, its potential role needs to be further explored.
Through KEGG pathway analysis, autophagy and hypoxia inducible factor-1 (HIF-1) signaling pathway were indentified. Li et al [29]. proposed that enhanced autophagy might contribute to the pathogenesis of middle ear cholesteatoma through PI3K/Akt pathway activation. However, Ho ea al [30]. put forward that autophagy is significantly suppressed in cholesteatoma. Consequently, autophagy is involved in the pathogenesis of middle ear cholesteatoma with unclear function. Moreover, activated HIF-1 pathway was detected in middle ear cholesteatoma, indicating that cholesteatoma might be hypoxic [31]. Furthermore, KEGG pathway analysis exhibited several potential signaling pathway, which broaden our horizons on the pathogenesis of middle ear cholesteatoma.
More and more evidence shows that m6A modification can regulate the function of circRNAs. It is reported that m6A modification can also initiate circRNAs translation, and this m6A-driven translation requires the initiation factor eukaryotic translation initiation factor 4 gamma 2 (eIF4G2) and m6A reader YTH domain family 3 (YTHDF3), which is enhanced by methyltransferase like 3 (METTL3) or METTL14, inhibited by demethylase fat mass and obesity associated protein (FTO), and upregulated upon heat shock [32]. Park et al [33]. demonstrated that m6A modification could reduce the stability of circRNA. In addition, m6A modification can also regulate cytoplasmic output. It has been found that YTH domain-containing protein 1 (YTHDC1) mediates the nuclear output of circNSUN2 in a m6A methylation-dependent manner, which increases the cytoplasmic expression of CircNSUN2 and promotes liver metastasis of colorectal cancer [34]. Some studies have found that unmodified circRNA is an effective adjuvant to induce specific T and B cell responses, and can activate Retinoic acid-inducible gene I (RIG-I) and innate immune signals with K63-polyubiquitin, while m6A RNA modification marks its own circRNA, and makes circRNA immune ineffective [35]. However, these potentially important functions of circRNA have not been fully studied. Therefore, the prediction and analysis of m6A modification of circRNA need more in-depth research.