circFAT1(e2) Promotes Papillary Thyroid Cancer Proliferation, Migration, and Invasion via the miRNA-873/ZEB1 Axis

Abstract

Circular RNAs (circRNAs) play an extremely important regulatory role in the occurrence and development of various malignant tumors including papillary thyroid cancer (PTC). circFAT1(e2) is a new type of circRNA derived from exon 2 of the FAT1 gene, which is distributed in the cytoplasm and nucleus of PTC cells. However, so far, the role of circFAT1(e2) in PTC is still unclear. In this study, circFAT1(e2) was found to be highly expressed in PTC cell lines and tissues. circFAT1(e2) knockdown suppressed PTC cell growth, migration, and invasion. Also, circFAT1(e2) acted as a sponge for potential microRNAs (miRNAs) to modulate cancer progression. A potential miRNA target was discovered to be miR-873 which was targeted by circFAT1(e2) in PTC. The dual-luciferase assay conducted later also confirmed that there was indeed a direct interaction between circFAT1(e2) and miR-873. This study also confirmed that circFAT1(e2) inhibited the miR-873 expression and thus promoted the ZEB1 expression, thus affecting the proliferation, metastasis, and invasion of PTC cells. In conclusion, the results of this study indicated that circFAT1(e2) played a carcinogenic role by targeting the miR-873/ZEB1 axis to promote PTC invasion and metastasis, which might become a potential novel target for therapy of PTC.

Figure 1

circFAT1(e2) increased abnormally in PTC samples and cells. (a) The circFAT1(e2) was more expressed in PTC cells. (b) PTC tissues had higher expression level of circFAT1(e2). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 2

Silencing circFAT1(e2) suppressed PTC cell proliferation. (a–c) circFAT1(e2) mainly located in the cytoplasm of TC cells. The efficiency of circFAT1(e2) knockdown was detected by qRT-PCR in CAL-62 (d) and TPC-1 (f) cells. CCK-8 experiments showed that circFAT1(e2) knockdown attenuated the proliferation capacity of CAL-62 (e) and TPC-1 (g) cells. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

circFAT1(e2) knockdown remarkably weakened the migratory capability of CAL-62 (a, b) and TPC-1 (c, d) cells. (a) Representative images of transwell migration assays in CAL-62 cells (scale bar, 45 μm). (b) The quantification of transwell migration assays in CAL-62 cell. (c) Representative images of transwell migration assays in TPC-1 cells (scale bar, 45 μm). (d) The quantification of transwell migration assays in TPC-1 cell. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 4

circFAT1(e2) silenced inhibited invasive capability of CAL-62 (a, b) and TPC-1 (c, d) cells. (a) Representative images of transwell invasion assays in CAL-62 cells (scale bar, 45 μm). (b) The quantification of transwell invasion assays in CAL-62 cell. (c) Representative images of transwell invasion assays in TPC-1 cells (scale bar, 45 μm). (d) The quantification of transwell invasion assays in TPC-1 cell. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

circFAT1(e2) served as a sponge of miR-873 to augment ZEB1. ZEB1 expression levels were lower in TPC-1 (a) and CAL-62 (b) cells with circFAT1(e2) knockdown by qRT-PCR. TPC-1 (c, e) and CAL-62 (d, f) cells transfected with miR-873 had less expression of circFAT1(e2), ZEB1. The luciferase activities of circFAT1(e2) and ZEB1 were measured in TPC-1 (g, i) and CAL-62 (h, j) cells transfected miR-873 mimics or miR-NC with dual-luciferase reporter assay. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.