Long Non-Coding RNA ZEB2-AS1 Promotes Hepatocellular Carcinoma Progression by Regulating The miR-582-5p/FOXC1 Axis

Abstract

Objective: Long non-coding RNAs (lncRNAs) feature prominently in tumors. Reportedly, lncRNA zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) is aberrantly expressed in a variety of tumors. The present study was aimed to explore ZEB2-AS1 functions and determine mechanism in hepatocellular carcinoma (HCC) progression.

Materials and methods: In this experimental study, expressions of ZEB2-AS1, microRNA (miR)-582-5p and forkhead box C1 (FOXC1) mRNA in HCC tissues and cell lines were detected via quantitative reveres transcription polymerase chain reaction (qRT-PCR). After establishing gain- and loss-of-functions models, cell counting kit-8, 5-bromo-2'-deoxyuridine (BrdU), Transwell assays and flow cytometry analysis were conducted to examine HCC cell multiplication, migration, invasion and apoptosis, respectively. The targeted relationship between miR-582- 5p and ZEB2-AS1 was verified via dual-luciferase reporter gene assay. Western blot was utilized for detecting FOXC1 expression in HCC cells after selectively regulating ZEB2-AS1 and miR-582-5p.

Results: In HCC tissues and cells, ZEB2-AS1 expression was increased. High ZEB2-AS1 expression was related to relatively large tumor volume, increased tumor-node-metastasis (TNM) stage and positive lymph node metastasis of the patients. ZEB2-AS1 overexpression facilitated HCC cell multiplication, migration, invasion and suppressed apoptosis, while ZEB2-AS1 knock-down caused the opposite effects. It was also confirmed that ZEB2-AS1 could competitively bind with miR-582-5p to repress its expression, and indirectly up-regulate FOXC1 expression level in HCC cells.

Conclusion: The current study revealed that ZEB2-AS1 was over-expressed in HCC tissues and cells. It also upregulated (FOXC1), through sponging miR-582-5p, to promote HCC progression. This provides new perspectives for elucidating the pathogenesis of HCC.

Keywords: Forkhead Box C1; Hepatocellular Carcinoma; Long Non-Coding RNA; miR-582-5p.

Fig 1

ZEB2-AS1 is over-expressed in HCC tissues and cells. A. Detection by qRT-PCR of ZEB2-AS1 expression in HCC tissues (n=50), adjacent normal tissues (n=50) and breast cancer tissues (n=20). B. Detection by qRT-PCR of ZEB2-AS1 expression in normal human liver cell line (MIHA cells), HCC cell lines (BEL7402, SMMC-7721, HCCLM3, and Huh7 cells) and human breast cancer cells MCF-7. C. GEPIA database was employed for analyzing the relationship between ZEB2-AS1 expression and HCC patient prognosis. All experiments were repeated 3 times, each in triplicate. **; P<0.01, ***; P<0.001, HCC; Hepatocellular carcinoma, qRT-PCR; Quantitative reverse transciption polymerase chain reaction, and GEPIA; Gene expression profiling interactive analysis.

Fig 2

Effects of ZEB2-AS1 on HCC cell proliferation, migration, invasion and apoptosis. A. Detection by qRT-PCR of ZEB2-AS1 expression in HCCLM3 cells transfected with ZEB2-AS1 overexpression plasmids and BEL7402 cells transfected with ZEB2-AS1 siRNAs. B-D. CCK-8 and BrdU assays (scale bars: 75 μm) were conducted for detecting HCC cell proliferation after ZEB2-AS1 overexpression or knock-down. E-H. Transwell assay was used to detect HCC cell migration and invasion (scale bars: 250 μm). I, J. Flow cytometry was conducted to evaluate apoptosis rate of HCCLM3 and BEL7402 cells after overexpression or knock-down of ZEB2-AS1. All experiments were repeated 3 times, each in triplicate. **; P<0.01, ***; P<0.001, HCC; Hepatocellular carcinoma, qRT-PCR; Quantitative reverse transciption PCR, siRNA; Small interfering RNA, CCK-8; Cell counting kit-8, and BrdU; 5-Bromo-2-deoxyUridine.

Fig 3

miR-582-5p is the target of ZEB2-AS1 in HCC cells. A. Bioinformatics was adopted for predicting binding site between ZEB2-AS1 and miR-582- 5p. B. Binding relationship between miR-582-5p and ZEB2-AS1 in HCC cells was detected by dual-luciferase reporter gene assay. C. Detection via qRTPCR of miR-582-5p expression in HCC cells with overexpression or knockdown of ZEB2-AS1. D. Detection via qRT-PCR of miR-582-5p expression in 50 cases of HCC tissues and adjacent normal tissues. E. Detection of the correlation between ZEB2-AS1 and miR-582-5p expressions in HCC tissues via qRT-PCR. All experiments were repeated 3 times, each in triplicate. **; P<0.01, ***; P<0.001, HCC; Hepatocellular carcinoma, and qRT-PCR; Quantitative reverse transciption polymerase chain reaction.

Fig 4

miR-582-5p reverses effects of ZEB2-AS1 on HCC cell proliferation, migration, invasion and apoptosis. ZEB2-AS1 overexpression plasmid, miR-582-5p mimic, ZEB2-AS1 overexpression plasmid+miR-582-5p were transfected into HCCLM3 cells, respectively, and si-ZEB2-AS1-1, miR-582- 5p inhibitors, si-ZEB2-AS1-1+miR-582-5p inhibitor were transfected into BEL7402 cells, respectively. A, B. CCK-8 and BrdU assays were utilized for examining HCCLM3 and BEL7402 cell proliferation. C, D. HCCLM3 and BEL7402 cell migration and invasion were detected through Transwell assays. E. Flow cytometry analysis was utilized to detect HCCLM3 and BEL7402 cell apoptosis. All experiments were repeated 3 times, each in triplicate. *; P<0.05, **; P<0.01, ***; P<0.001, HCC; Hepatocellular carcinoma, qRT-PCR; Quantitative reverse transciption polymerase chain reaction, CCK-8; Cell counting kit-8, and BrdU; 5-Bromo-2-deoxyUridine.

Fig 5

Regulation of FOXC1 expression by the ZEB2-AS1/miR-582-5p axis. A. Bioinformatics was used for predicting binding site between miR-582- 5p and FOXC1. B, C. Binding relationship between miR-582-5p and FOXC1 in HCC cells was detected via dual-luciferase reporter gene assay. D, E. Detection of FOXC1 expression by qRT-PCR in 50 cases of the HCC tissues and adjacent normal tissues, as well as normal human liver cell line (MIHA cells) and HCC cell lines (BEL7402, SMMC-7721, HCCLM3, and Huh7 cells). F-I. qRT-PCR and Western blot were used to detect regulatory effect of miR582-5p or ZEB2-AS1 on FOXC1 protein expression. Original blots are shown in Supplementary material. J, K. qRT-PCR was employed for analyzing the correlation between FOXC1 mRNA and miR-582-5p as well as ZEB2-AS1 expression in HCC tissues. All experiments were repeated 3 times, each in triplicate. *; P<0.05, **; P<0.01, ***; P<0.001, HCC; Hepatocellular carcinoma, and qRT-PCR; Quantitative reverse transcription polymerase chain reaction.