Yonsei Med J.  2019 Oct;60(10):905-913. 10.3349/ymj.2019.60.10.905.

The Long Non-Coding RNA CASC2 Suppresses Cell Viability, Migration, and Invasion in Hepatocellular Carcinoma Cells by Directly Downregulating miR-183

• Sun J
• Liu L
• Zou H
• Yu W

• KMID: 2459143
• DOI: http://doi.org/10.3349/ymj.2019.60.10.905

Abstract

PURPOSE
Hepatocellular carcinoma (HCC) is the most common malignant tumor of liver cells. Researchers have reported that cancer susceptibility candidate 2 (CASC2), a long non-coding RNA, is down-regulated in various cancers, including HCC. Our study aimed to investigate the molecular mechanism(s) of CASC2 in HCC.
MATERIALS AND METHODS
Real-time quantitative PCR (RT-qPCR) was used to analyze the expression of CASC2 and miR-183 in HCC tissues and cells. The viability of HCC SMMC-7721 and Huh-7 cells was detected through MTT assay. Colony formation assay was performed to assess the colony formation ability of HCC cells. The migration and invasion abilities of HCC cells were evaluated by Transwell assay. Western blot was conducted to examine levels of key Wnt/β-catenin signaling pathway factors, C-myc, cyclinD, survivin, and β-catenin. The interaction between CASC2 and miR-183 was affirmed by bioinformatics analysis and luciferase reporter assay.
RESULTS
CASC2 was down-regulated in HCC tissues and cell lines, while miR-183 was up-regulated. The expression of miR-183 was negatively correlated with CASC2 expression in HCC tissues. Overexpression of CASC2 inhibited cell viability, colony formation, migration, and invasion in HCC cells, as well as Wnt/β-catenin signaling pathway activity. miR-183 was a downstream target of CASC2 and negatively regulated by CASC2. Introduction of miR-183 rescued CASC2-induced suppressive effects on HCC cell viability, colony formation, migration, and invasion and Wnt/β-catenin signaling.
CONCLUSION
CASC2 inhibited cell viability and the colony formation, migration, and invasion abilities of HCC cells by directly downregulating miR-183 through inactivation of the Wnt/β-catenin signaling pathway.

Keyword

CASC2; hepatocellular carcinoma; miR-183; Wnt/β-catenin signaling pathway

MeSH Terms

Blotting, Western
Carcinoma, Hepatocellular*
Cell Line
Cell Survival*
Computational Biology
Liver
Luciferases
Polymerase Chain Reaction
RNA, Long Noncoding*
Luciferases
RNA, Long Noncoding

Figure

• 

  Fig. 1 Cancer susceptibility candidate 2 (CASC2) significantly downregulated in hepatocellular carcinoma (HCC) tissues and cells. (A) The expression levels of CASC2 in 30 pairs of HCC tumor and matched neighboring non-tumor tissues (NTTs) were detected by real-time quantitative PCR (RT-qPCR). (B) Survival analysis in 30 HCC patients with high and low expression of CASC2. (C) CASC2 expression in human LO2 and five HCC cell lines (HepG2, Hep3B, QSG-7701, SMMC-7721 and Huh-7) was also analyzed by RT-qPCR. *p<0.05 compared to NTT; †p<0.05 compared to LO2 cells.

• 

  Fig. 2 Upregulation of cancer susceptibility candidate 2 (CASC2) inhibits cell viability, colony formation, migration, and invasion in hepatocellular carcinoma (HCC) cells in vitro. SMMC-7721 and Huh-7 cells were transfected with pcDNA3.1-CASC2 or pcDNA3.1 vector. The image magnification of transwell assay is ×100. The cells in colony formation assay and transwell assay were stained using crystal violet dye. (A and B) Real-time quantitative PCR was performed to analyze CASC2 expression in SMMC-7721 and Huh-7 cells after transfection. (C and D) MTT assay in SMMC-7721 and Huh-7 cells after transfection. (E and F) Colony formation assay in SMMC-7721 and Huh-7 cells after transfection. (G and H) Transwell assay detected migratory abilities of SMMC-7721 and Huh-7 cells after transfection. (I and J) Transwell assay detected the invasive abilities of SMMC-7721 and Huh-7 cells after transfection. *p<0.05 compared to cells transfected with pcDNA3.1 vector. OD, optical density.

• 

  Fig. 3 miR-183 is upregulated in hepatocellular carcinoma (HCC) tissues and cells. (A) Real-time quantitative PCR (RT-qPCR) was performed to analyze miR-183 expression in 30 pairs of HCC tumor and neighboring non-tumor tissues (NTTs). (B) RT-qPCR was conducted to analyze miR-183 expression in SMMC-7721 and Huh-7 cells. *p<0.05 compared to NTT; †p<0.05 compared to LO2 cells.

• 

  Fig. 4 miR-183 is directly targeted by cancer susceptibility candidate 2 (CASC2). (A) The sequence alignment of miR-183 with the 3′UTR of CASC2 is shown. (B and C) Luciferase assays in SMMC-7721 and Huh-7 cells transfected with wt CASC2 or mut CASC2 luciferase reporter plasmid, with miR-183 mimic or negative control (NC) mimic. (D and E) The expression of miR-183 in SMMC-7721 and Huh-7 cells transfected with pcDNA3.1-CASC2 or si-CASC2. (F) Correlation analysis between CASC2 and miR-183 in hepatocellular carcinoma (HCC) tissues, p<0.01. *p<0.05 compared to cells cotransfected with wt CASC2 and NC mimic; †p<0.05 compared to cells transfected with pcDNA3.1 vector or si-NC.

• 

  Fig. 5 Overexpression of miR-183 partially relieves cancer susceptibility candidate 2 (CASC2)-mediated inhibition on hepatocellular carcinoma (HCC) cell viability, colony formation, migration, and invasion. The image magnification of transwell assay is ×100. The cells in colony formation assay and transwell assay were stained using crystal violet dye. (A and B) Real-time quantitative PCR (RT-qPCR) assay for miR-183 expression in SMMC-7721 and Huh-7 cells transfected with negative control (NC) mimic or miR-183 mimic. (C-G) SMMC-7721 and Huh-7 cells were co-transfected with pcDNA3.1-CASC2 (CASC2) and NC mimic or miR-183 mimic. (C and D) MTT assay in SMMC-7721 and Huh-7 cells. (E) Colony formation assay in SMMC-7721 and Huh-7 cells. (F) Transwell assay detected the migratory abilities of SMMC-7721 and Huh-7 cells after transfection. (G) Transwell assay detected the invasive abilities of SMMC-7721 and Huh-7 cells after transfection. *p<0.05 compared to cells transfected with pcDNA3.1-vector or CASC2+NC mimic. OD, optical density.

• 

  Fig. 6 Cancer susceptibility candidate 2 (CASC2) regulates the Wnt/β-catenin pathway by downregulating miR-183. (A and B) Western blot assay for C-myc, cyclinD, surviving, and β-catenin expression in SMMC-7721 and Huh-7 cells co-transfected with pcDNA3.1-CASC2 or pcDNA3.1 vector with or without negative control (NC) mimic or miR-183 mimic. *p<0.05 compared to cells transfected with pcDNA3.1 vector; †p<0.05 compared to cells cotransfected with CASC2 and NC mimic.

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