Circular RNA circ-LRP6 facilitates Myc-driven tumorigenesis in esophageal squamous cell cancer

Abstract

Circular RNA (circRNA) circ-LRP6 was recently proven to be a pivotal player in various human diseases. Nevertheless, its role in esophageal squamous cell cancer (ESCC) remains unknown. In the current study, we investigated the expression level, biological function and mechanism of circ-LRP6 in ESCC. Circ-LRP6 was significantly upregulated in ESCC tissues and correlated with malignant clinicopathological characteristics and poor prognosis. Knockdown of circ-LRP6 evidently reduced ESCC cell viability, colony formation and invasion. Circ-LRP6 was mainly located in the cytoplasm and could sponge miR-182 to increase the expression of Myc, a well-documented proto-oncogene. Importantly, circ-LRP6 depletion significantly retarded tumor growth in vivo. And silencing of miR-182 or overexpression of Myc effectively rescued the attenuated aggressive phenotype of ESCC cells caused by circ-LRP6 knockdown. Therefore, our data indicate that circ-LRP6 is a novel oncogenic circRNA in ESCC, targeting the circ-LRP6/miR-182/Myc signaling may be a promising therapeutic approach for ESCC patients.

Keywords: Esophageal squamous cell cancer; Myc; circular RNA; miRNA; prognosis.

Graphical abstract

Figure 1.

Circ-LRP6 is overexpressed in ESCC tissues. (a) qRT-PCR analysis of circ-LRP6 level in ESCC and normal tissues. (b) The survival curve showing that high circ-LRP6 level predicted poor outcome. (c) qRT-PCR analysis of circ-LRP6 and LRP6 expression at the indicated time after treatment with Actinomycin D. (d) qRT-PCR analysis of circ-LRP6 and LRP6 expression after treatment with RNase R. (e, f) qRT-PCR and FISH assays detecting the location of circ-LRP6. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Circ-LRP6 promotes ESCC cell proliferation and invasion. (a) qRT-PCR testing the knockdown efficiency of circ-LRP6 in TE-1 and EC109 cells. (b, c) CCK-8 and colony formation assays detecting the viability and clonal ability of ESCC cells after circ-LRP6 depletion. (d) Transwell assay detecting ESCC cell invasive ability after circ-LRP6 depletion. **p < 0.01.

Figure 3.

Circ-LRP6 sponges miR-182. (a, b) RNA pull-down assay in two ESCC cells using biotin-labeled circ-LRP6 probe, followed by qRT-PCR analysis. (c) The binding site of miR-182 on circ-LRP6. (d) Luciferase reporter assay in two ESCC cells using wild-type or mutant circ-LRP6 vector. (e) qRT-PCR analysis of miR-182 expression after circ-LRP6 knockdown. (f, g) CCK-8 and Transwell assays testing cell viability and invasion in circ-LRP6-silenced ESCC cells transfected with miR-182 inhibitors. *p < 0.05, **p < 0.01.

Figure 4.

Circ-LRP6 regulates the miR-182/Myc axis. (a) The binding site of miR-182 on Myc 3`-UTR. (b) qRT-PCR analysis of Myc expression after overexpression or silencing of miR-182. (c) Luciferase reporter assay in two ESCC cells using wild-type or mutant Myc 3`-UTR vector. (d, e) Western blot analyzing Myc protein level in circ-LRP6-silenced ESCC cells transfected with miR-182 inhibitors. (f, g) CCK-8 and Transwell assays testing cell viability and invasion in circ-LRP6-silenced ESCC cells transfected with Myc expression vector. *p < 0.05, **p < 0.01.

Figure 5.

Knockdown of circ-LRP6 inhibits tumor growth. (a) The image showing the tumors of control and circ-LRP6-silenced groups. (b) Tumor volume and weight of control and circ-LRP6-silenced groups. (c) qRT-PCR analysis of circ-LRP6, miR-182, and Myc expression in control and circ-LRP6-silenced groups. (d) Western blot analyzing Myc protein level in control and circ-LRP6-silenced groups. **p < 0.01.