Circular RNA Hsa_circ_0004018 Inhibits Wnt/β-Catenin Signaling Pathway by Targeting microRNA-626/DKK3 in Hepatocellular Carcinoma

Background and aim: Dysexpression of circular RNAs has been identified in multiple types of cancer. Hsa_circ_0004018 was reported to be significantly downregulated in hepatocellular carcinoma (HCC) and to display HCC-stage-specific expression features. However, the role of hsa_circ_0004018 in HCC progression remains unclear.

Methods: The expression of hsa_circ_0004018 or microRNA-626 (miR-626) was detected in tumor tissues and paired non-tumor tissues from HCC patients, as well as in one normal human liver cell line and 5 HCC cell lines by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, dye exclusion assay, clonogenic assay, scratch migration assay and transwell assay were used to measure cell proliferation and migration capacity, respectively. Luciferase report assay and RNA pull down assay were performed to explore the regulatory effect of certain molecules on the expression of target genes.

Results: We found that the expression of hsa_circ_0004018 was lower in tumor tissues than in their paired non-tumor tissues from 28 out of 41 HCC patients. The difference in the expression between tumor tissues and non-tumor tissues was statistically significant (p<0.001). Further analysis revealed that such lower expression in tumor tissues was much more common in bigger tumor size group (≥5cm) compared with the smaller tumor size group (<5cm) (85% vs 42%, p=0.0007). Similarly, hsa_circ_0004018 was downregulated in HCC cell lines. Additionally, a negative correlation between hsa_circ_0004018 and miR-626 expression was noticed in HCC tissues. Moreover, we observed that hsa_circ_0004018 interacted with miR-626/DKK3 and contributed to HCC cell proliferation and migration through inhibiting Wnt/β-catenin signaling pathway in vitro. Furthermore, hsa_circ_0004018 blocked xenograft tumor growth in vivo through inhibiting Wnt/β-catenin signaling pathway by targeting miR-626/DKK3.

Conclusion: We revealed that hsa_circ_0004018/miR-626/DKK3 regulatory axis may be a possible novel therapeutic target for HCC.