YY1 and eIF4A3 are mediators of the cell proliferation, migration and invasion in cholangiocarcinoma promoted by circ-ZNF609 by targeting miR-432-5p to regulate LRRC1

Abstract

Cholangiocarcinoma is a highly aggressive malignant tumor, and its incidence is increasing all over the world. More and more evidences show that the aberrant expression of circular RNAs play important roles in tumorigenesis and progression. Current studies on the expression and function of circRNAs in cholangiocarcinoma are scarce. In this study, circ-ZNF609 was discovered as a novel circRNA highly expressed in cholangiocarcinoma for the first time. The circ-ZNF609 expression is connected with the advanced TNM stage, lymphatic invasion and survival time in cholangiocarcinoma patients, and can be used as an independent prognostic factor for the patients. Circ-ZNF609 can promote the cholangiocarcinoma cells proliferation, migration and invasion in vitro, it can also catalyze the xenograft growth in vivo. The promoting effect of circ-ZNF609 on cholangiocarcinoma is achieved via oncogene LRRC1 up-regulation through targeting miR-432-5p by endogenous competitive RNA mechanism. In addition, transcription factor YY1 can bind to the promoter of ZNF609 to further facilitate the transcription of circ-ZNF609. RNA binding protein eIF4A3 can bind to the pre-mRNA of circ-ZNF609 which promotes the circ-ZNF609 circular formation. Overall, YY1/eIF4A3/circ-ZNF609/miR-432-5p/LRRC1 have a significant role in progression of cholangiocarcinoma, and circ-ZNF609 is expected to become a novel biomarker for targeted therapy and prognosis evaluation of cholangiocarcinoma.

Keywords: LRRC1; YY1/eIF4A3; cholangiocarcinoma; circ-ZNF609; miR-432-5p.

Figure 1

Circ-ZNF609 was significantly high expression in CCA and associated with clinicopathological characteristics. (A) Schematic representation of circ-ZNF609 genomic structure. (B) The circ-ZNF609 and ZNF609 expression were detected through using the qRT-PCR in CCA cells treated with RNase R. (C) qRT-PCR was used to detect the relative expression of circ-ZNF609 in CCA tumor tissues and adjacent normal tissues. (D) Circ-ZNF609 expression of patients with different TNM stages. (E) Circ-ZNF609 expression of patients with or without lymph node metastasis. (F) The overall survival of high and low circ-ZNF609 expression group patients. (G) The correlation between circ-ZNF609 expression and survival time of CCA patients. (H) The ROC curve of circ-ZNF609 detection as a prognostic biomarker. (I) The ROC curve of CA19-9 detection as a prognostic biomarker. (J) The ROC curve of circ-ZNF609+CA19-9 detection as a prognostic biomarker. **P< 0.01; ***P< 0.001.

Figure 2

Silencing circ-ZNF609 inhibited the proliferation of cholangiocarcinoma cells. (A) The relative circ-ZNF609 expression in CCLP-1, QBC939, TFK-1, RBE cells and normal HIBEC. (B) The two siRNAs interference efficiency detection of circ-ZNF609. (C–E) CCK-8 assay, EdU and colony formation assay were used to detect the proliferation ability of CCA cells after circ-ZNF609 knockdown. (F) Tumor xenograft assay was used to measure tumor growth after silencing circ-ZNF609 in vivo. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 3

Silencing circ-ZNF609 inhibited cholangiocarcinoma cells migration and invasion. (A) Wound healing assay was used to detect the migration ability of CCA cells after circ-ZNF609 knockdown. (B) The migration and invasive abilities of CCLP-1 and QBC939 were confirmed by transwell assay after silencing circ-ZNF609. (C) Western blot was used to detect EMT-related proteins including E-cadherin, N-cadherin and vimentin. **P< 0.01; ***P< 0.001.

Figure 4

Circ-ZNF609 was involved in CCA progression via sponging miR-432-5p. (A) Detection of relative expression of circ-ZNF609 in cytoplasm and nucleus of CCA cells by qRT-PCR. (B) Targetscan, circinteractome and mirMap were used to identify potential miRNA target genes for circ-ZNF609. (C) Relative expression of potential target genes in CCA cells transfected with si-circ-1 and si-circ-2. (D) qRT-PCR was used to detect the relative expression of c miR-432-5p in CCA tumor tissues and adjacent normal tissues. (E) qRT-PCR was used to detect the correlation between the expression of circ-ZNF609 and miR-432-5p in CCA specimens. (F) The relative expression of miR-432-5p in CCA cells detected by qRT-PCR. (G) The efficiency detection of miR-432-5p mimics and inhibitor transfection confirmed by qRT-PCR. (H) Diagrammatic sketch of the binding sites between miR-432-5p and circ-ZNF609. (I) Luciferase activity in CCA cells after transfection of circ-ZNF609 WT or circ-ZNF609 MUT, miR-432-5p mimics or NC mimics. (J) RIP assay was utilized to further demonstrate the direct binding between miR-432-5p and circ-ZNF609. (K) Colony formation assay was used to detect the proliferation ability after miR-432-5p down-regulation and up-regulation. (L, M) The migration and invasion changes of tumor cells after miR-432-5p down-regulation and up-regulation detected by transwell. **P< 0.01; ***P< 0.001.

Figure 5

LRRC1 was a direct target of miR-432-5p in CCA. (A) Targetscan, circinteractome and mirMap were used to identify potential mRNA target genes for miR-432-5p. (B) Relative expression of LRRC1 mRNA in CCA tissues and adjacent normal tissues. (C) qRT-PCR was used to detect the correlation between miR-432-5p and LRRC1 expression in CCA specimens. (D, E) The expression of LRRC1 mRNA and protein in CCLP-1 and QBC939. (F, G) The LRRC1 expression was detected in CCA cells with miR-432-5p up-regulation and down-regulation by qRT-PCR and western blot. (H) Diagrammatic sketch of the binding sites between miR-432-5p and LRRC1. (I) Luciferase reporter assay in CCA cells transfected with LRRC1 WT or LRRC1 MUT, along with miR-432-5p mimics or NC mimics. (J) RIP assay further verified the direct interaction between miR-432-5p and LRRC1. (K) Colony formation assay was used to detect the proliferation ability change after LRRC1 down-regulation and up-regulation. (L, M) The migration and invasion changes of tumor cells after LRRC1 down-regulation and up-regulation detected by transwell. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 6

Circ-ZNF609 up-regulated LRRC1 expression by competitively binding miR-432-5p to induce cholangiocarcinoma development. (A) After co-transfection of circ-ZNF609 siRNA and miR-432-5p inhibitor, the LRRC1 expression was detected by qRT-PCR and western blot. (B–E) CCK-8, EdU and transwell assays confirmed that the effects of proliferation, migration and invasion induced by miR-432-5p inhibitor were partly reversed by silencing LRRC1. (F–I) Silencing circ-ZNF609 could partially reverse the proliferation and invasion induced by LRRC1 overexpression in CCK-8, EdU and transwell assays. *P< 0.05; **P< 0.01; ***P< 0.001.

Figure 7

YY1 and eIF4A3 promoted circ-ZNF609 expression via linking to the circ-ZNF609 promoter and pre-mRNA, respectively. (A) YY1 sequence and binding sites (E1, E2 and E3) to circ-ZNF609 promoter region were predicted by using JASPAR database. (B) The efficiency detection of YY1 overexpression and siRNA transfection confirmed by qRT-PCR. (C) The circ-ZNF609 expression was detected in CCA with YY1 up-regulation and down-regulation by qRT-PCR. (D) ChIP assay was conducted to affirm the direct binding of YY1 to circ-ZNF609 promoter in QBC939 and CCLP-1 cells. (E) Schematic diagram of plasmids construction. (F) The luciferase activity of E3 wild type was markedly promoted by oe-YY1 co-transfection compared with controls in CCA cells. (G) The prediction results of circinteractome database indicated that eIF4A3 had binding sites in the upstream and downstream regions of circ-ZNF609 pre-mRNA. (H) The results of RIP demonstrated that eIF4A3 could directly bind to the circ-ZNF609 pre-mRNA in CCA cells. (I) Knockdown efficiency and amplification efficiency of eIF4A3 confirmed by qRT-PCR. (J) The expression of circ-ZNF609 in CCLP-1 and QBC939 transfected with high expression eIF4A3 and low expression eIF4A3 was detected by qRT-PCR. (K) Schematic diagram of circ-ZNF609 tumorigenesis mechanisms in cholangiocarcinoma. **P< 0.01; ***P< 0.001.