Hsa_circRNA_000166 Promotes Cell Proliferation, Migration and Invasion by Regulating miR-330-5p/ELK1 in Colon Cancer

Abstract

Introduction: Circular RNAs (circRNAs), a novel class of non-coding RNAs, which are widely expressed in human cells, have essential roles in the development and progression of cancers. The aim of this study is to figure out the role of circ_000166 in colon cancer (CC) development and the signaling pathway involved.

Materials and methods: HT29 and HCT116 cells were transfected with siRNA of circRNA, miRNA mimics and inhibitors. Cell proliferation, migration and invasion were examined using CCK-8 assay and transwell assay, respectively. Luciferase reporter assay was used to validate the targets of circRNA and miRNA. CC cells were implanted into nude mice subcutaneously to detect tumor growth.

Results: hsa_circRNA_000166 was significantly upregulated in the human CC tissue and in the CC cell lines. Knockdown of hsa_circRNA_000166 reduced cell viability, colony formation, migration and invasion in vitro and decreased tumor size and weight in vivo. Luciferase reporter assay revealed that miR-330-5p was the target of circRNA_000166. miR-330-5p could bind to 3' untranslated region (3'UTR) of ELK1 to downregulate both mRNA and protein expression of ELK1. Dual inhibition of circRNA_000166 and miR-330-5p inhibited the suppression of cell proliferation, migration and invasion induced by si-circRNA_000166.

Conclusion: The data of this study demonstrated that the hsa_circRNA_000166 could upregulated the expression of gene ELK1 by sponging miR-330-5p, which may contribute to a better understanding of the regulatory circRNA/miRNA/mRNA network and CC pathogenesis.

Keywords: ELK1; circRNA_000166; colon cancer; miRNA-330-5p.

Figure 1

circRNA_000166 expression was increased in colon cancer. (A) Hierarchical clustering analysis of the top 15 upregulated and downregulated circRNAs in CC; (B) Relative circRNA_000166 expression in CC tissue and their adjacent normal tissue using qRT-PCR assay; (C) Relative circRNA_000166 expression in different CC cell lines (HT29, HCT116, HCT8, LOVO, SW420 and SW620 cells); (D) Relative circRNA_000166 expression and GAPDH in HT29 and HCT116 cells treated with RNase R; (E) Localization of circRNA_000166 in HT 29 and HCT116 cells. **p < 0.01, ****p < 0.0001; ns: no significance. Abbreviation: CC, colon cancer.

Figure 2

circRNA_000166 knockdown inhibited colon cancer proliferation, migration and invasion. (A) qRT-PCR analysis of knockdown efficiency of hsa_circ_000166 in HT29 and HCT116 cells transfected with si-NC and si-circRNA_000166. (B) CCK-8 assay results of cell viability in HT29 and HCT116 cells transfected with si-NC and si-circRNA_000166; (C) Colony formation in HT29 and HCT116 cells transfected with si-NC and si-circRNA_000166; (D) Tumor size and weight in nude mice implanted subcutaneously with 1 × 10⁶ of HT29 and HCT116 cells transfected with si-NC and si-circRNA_000166 (E) HT29 and HCT116 cell migration using transwell assay; (F) HT29 and HCT116 cell invasion using transwell assay. *p < 0.05; **p < 0.01. Abbreviations: si-NC, Scramble siRNA; si-circRNA_000166, siRNA of circRNA_000166.

Figure 3

circRNA_000166 sponged miR-330-5p. (A) The putative binding sites between circRNA_000166 and miR-330-5p. Relative miR-330-5p expression in cells co-transfected with wt or mut circRNA_000166 and miR-330-5p using luciferase reporter assay; (B) Enrichment of circRNA_000166 using RNA pull-down experiments; (C) Relative miR-330-5p expression in circRNA_000166 knockdown cells; (D) Relative miR-330-5p expression in CC tissue and their adjacent normal tissue; (E) Relative miR-330-5p expression from TCGA database; (F) Spearman’s rank-order correlation between miR-330-5p and circRNA_000166. **p < 0.01. Abbreviations: ns, no significance. miR-NC, negative control of miR-330-5p; wt, wild type; mut, mutant.

Figure 4

circRNA_000166 accelerated colon cancer progression by targeting miR-330-5p. (A) qRT-PCR analysis of relative miR-330-5p expression in HT29 and HCT116 cells transfected with si-NC, si-circRNA_000166 and si-circRNA_000166 + miR-330-5p inhibitor; (B) CCK-8 assay of cell viability in HT29 and HCT116 cells transfected with si-circRNA_000166 + miR-330-5p inhibitor; (C) Colony formation in HT29 and HCT116 cells transfected with si-circRNA_000166 + miR-330-5p inhibitor; (D) HT29 and HCT116 cell migration using transwell assay; (E) HT29 and HCT116 cell invasion using transwell assay. **p < 0.01 versus (vs) si-NC; ^## p < 0.01 vs si-circ_000166.

Figure 5

circRNA_000166 promoted ELK1 expression through sponging miR-330-5p. (A) The putative binding sites between ELK1 and miR-330-5p. Relative ELK1 expression in cells transfected with wt and mut circRNA_000166 using luciferase reporter assay; (B) Relative ELK mRNA and protein expression in HT29 and HCT116 cells transfected with miR-NC, miR-330-5p mimics and inhibitors; (C) Relative ELK mRNA and protein expression in HT29 and HCT116 cells transfected with vector, circRNA_000166 and circRNA_000166 + miR-330-5p mimics; (D) Relative ELK1 expression in 30 pairs of CC tissue and their adjacent normal tissue. Spearman’s rank-order correlation between miR-330-5p and ELK1, and between circRNA_000166 and ELK1. **p < 0.01. Abbreviations: ns, no significance. miR-NC, miRNA negative control.