ESCC is one of the most lethal malignant tumors in the world, and its treatment is limited [3]. In addition, the biological mechanism of the occurrence and development of ESCC is still largely unknown. Therefore, it is urgent to explore the biological mechanisms of ESCC to identify molecular biomarkers for early diagnosis and prognosis. CircRNA is a long noncoding RNA (ncRNA), whose transcript length is usually more than 200 and contains no coding region [15]. Emerging evidence has revealed that circRNA plays a critical role in different types of human cancers[16]. As a miRNA sponge, circRNA regulates many biological processes by regulating the function of miRNA, affecting RNA splicing, chromatin structure and mRNA stability [17, 18].
Current studies have found that circHIPK3 plays a dual role in a variety of human cancers. The overexpression of circHIPK3 can effectively reverse the miR-7-induced decrease in the progression of colorectal cancer cells by up-regulating the expression of several key miR-7 target genes (including EGFR, IGF1R, FAK and YY1) [8]. In gallbladder cancer cells, knockdown the expression of circHIPK3 could increase the expression of miR-124, thereby reducing tumor cell proliferation and survival [19]. Overexpression of circHIPK3 sponges miR-124, inhibits its activity and increases the expression of its target genes, such as IL6R and DLX2, thereby causing tumor cell growth. However, in some kinds of cancer, circHIPK3 also displays the function of inhibiting tumor growth. Overexpression of circHIPK3 significantly inhibited the growth of bladder cancer in vivo [20], while knockdown the expression of circHIPK3 promoted the proliferation of ovarian cancer cells (A2780 and SKOV3) and normal ovarian epithelial cells [21]. These studies proved that circHIPK3 participated in complex regulatory networks, and confers cell-type-specific regulation of cell function in different cancers.
In this study, we investigated the expression of circHIPK3 in ESCC tumor tissues and ESCC cell lines. We further identified that circHIPK3 negatively regulated the expression of miR-124 in ESCC
cell lines. Our in vitro experiments showed that miR-124 suppressed cell proliferation and migration in ESCC cells. Moreover, the expression of miR-124 was remarkably down-regulated in ESCC tumor tissues. Therefore, we suggested that miR-124 functioned as a tumor suppressor gene that was dependent on circHIPK3 in ESCC.
Moreover, our data for the first time proved that AKT3 was suggested to be a target gene of miR-124. AKT3 encodes a serine/threonine protein kinase that belongs to the AGC kinase family [22]. AKT-kinases play roles in signaling pathways involved in cell proliferation, oncogenic transformation, cell survival, cell migration, and intracellular protein trafficking [23, 24]. AKT3 alterations have been related with tumor growth and migration. Previously, gain of function mutations in AKT3 were found in several cancer types, such as breast cancer and endometrium cancer, which were relied on the mediation of PI3K signaling pathways [25, 26]. In this study, using luciferase and western blot analysis, we found that miR-124 negatively modulated AKT3 expression. In ESCC tumor tissues, AKT3 expression was highly increased, which was in line with the TCGA database. Intriguingly, promoting the expression of circHIPK3 enhanced AKT3 expression in ESCC cells, while the opposite result was observed in circHIPK3-knockdown cells. Moreover, enhancing AKT3 expression significantly enhanced ESCC cell proliferation and migration, which were in line with those promoting tumorigenesis in various cancers[25].