Long non-coding RNA CCAT2 is associated with poor prognosis in hepatocellular carcinoma and promotes tumor metastasis by regulating Snail2-mediated epithelial–mesenchymal transition

Figures & data

Table 1 Primers used for qRT-PCR and siRNA oligonucleotides

Genes	Sequences
Primers used for qRT-PCR
CCAT2
Sense	5′-CCCTGGTCAAATTGCTTAACCT-3′
Antisense	5′-TTATTCGTCCCTCTGTTTTATGGAT-3′
Vimentin
Sense	5′-GAGAACTTTGCCGTTGAAGC-3′
Antisense	5′-GCTTCCTGTAGGTGGCAATC-3′
E-cadherin
Sense	5′-TACGCCTGGGGACTCCACCTA-3′
Antisense	5′-CCAGAAACGGAGGCCTGAT-3′
Snail2
Sense	5′-TGTGCTTACCAACCAAATAGTATG-3′
Antisense	5′-GGCTCTGGGCGGGTACAAAG-3′
GADPH
Sense	5′-TGGTATCGTGGAAGGACTCAT-3′
Antisense	5′-GTGGGTGTCGCTGTTGAAGTC-3′
siRNA-CCAT2
siRNA1
Sense	5′-GCUAAGAGGAAACCACCUUTT-3′
Antisense	5′-AAGGUGGUUUCCUCUUAGCTT-3′
siRNA2
Sense	5′-GCUCCACCUCUGACCAAUUTT-3′
Antisense	5′-AAUUGGUCAGAGGUGGAGCTT-3′
siRNA3
Sense	5′-CCAGGCAAUAACUGUGCAATT-3′
Antisense	5′-UUGCACAGUUAUUGCCUGGTT-3′
Negative control
Sense	5′-UUCUCCGAACGUGUCACGUTT-3′
Antisense	5′-ACGUGACACGUUCGGAGAATT-3′

Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA; CCAT2, colon cancer-associated transcript 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 1 Increased expression of CCAT2 in HCC tissues and cell lines and its prognostic significance.

Notes: (A) qRT-PCR analysis of CCAT2 expression levels in human normal liver cell line (MIHA) and HCC cell lines (HepG2, SMMC7721 and MHCC97H). GAPDH expression was assessed as an internal control. (B) The expression of CCAT2 was detected in 96 paired human HCC and adjacent normal tissues via qRT-PCR. The level of CCAT2 in tumor tissues was significantly higher than that in normal tissues. (C) Kaplan–Meier analysis of overall survival based on CCAT2 expression in all HCC patients. Patients with high levels of CCAT2 expression showed reduced survival times compared with patients with low expression (P=0.005, log-rank test). *P<0.05, **P<0.01.
Abbreviations: CCAT2, colon cancer-associated transcript 2; HCC, hepatocellular carcinoma; qRT-PCR, quantitative real-time polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Cum survival, cumulative survival.

Table 2 Association of CCAT2 expression with clinicopathological characteristics of HCC

Clinical parameters	CCAT2 expression		Chi-squared test P-value
	Low*	High*
Age (years)			0.102
≤54	29	21
>54	19	27
Sex			0.820
Male	34	35
Female	14	13
Alcoholism			0.041
Negative	30	20
Positive	18	28
Smoking			0.682
Negative	27	25
Positive	21	23
TNM stages			0.041
I	31	21
II/III	17	27
Vessel invasion			0.026
Absence	6	15
Presence	42	33
Tumor size (cm)			0.214
≤5	23	17
>5	25	31

Note:

* Patients were divided into high (above the median) and low (below the median) CCAT2 expression groups according to the median expression level (1.89) of CCAT2.

Abbreviations: CCAT2, colon cancer-associated transcript 2; HCC, hepatocellular carcinoma; TNM, tumor–node–metastasis.

Table 3 Univariate and multivariate Cox regression analyses of CCAT2 for overall survival of patients with HCC (n=96)

Variables	Univariate analysis		Multivariate analysis
	HR (95% CI)	P-value	HR (95% CI)	P-value
Age (years) (≤54 vs >54)	1.152 (0.682–1.946)	0.598
Sex (male vs female)	0.752 (0.410–1.380)	0.358
Smoking (positive vs negative)	0.581 (0.333–1.012)	0.055
Alcoholism (positive vs negative)	1.518 (0.898–2.567)	0.119
Tumor size (≤5/>5)	1.548 (0.900–2.661)	0.114
TNM stages (I vs II/III)	2.333 (1.371–3.969)	0.002	1.992 (1.045–3.795)	0.036
Vessel invasion (absence vs presence)	0.451 (0.252–0.809)	0.008	0.880 (0.427–1.815)	0.730
CCAT2 expression (high vs low)	2.118 (1.245–3.603)	0.006	1.849 (1.064–3.213)	0.029

Abbreviations: CCAT2, colon cancer-associated transcript 2; HCC, hepatocellular carcinoma; HR, hazard ratio; CI, confidence interval; TNM, tumor–node–metastasis.

Figure 2 Effect of CCAT2 on HCC cells migration and invasion.

Notes: (A) The expression of CCAT2 in HepG2 and MHCC97H cells transfected with siRNA against CCAT2 was detected by qRT-PCR. (B and C) Wound-healing assay and Transwell assay indicated that siRNA-mediated silence of CCAT2 significantly inhibited cell migration and invasion, compared with negative control. Original magnification ×40. **P<0.01.
Abbreviations: CCAT2, colon cancer-associated transcript 2; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control.

Figure 3 qRT-PCR analysis expression of snail2, vimentin and E-cadherin in HCC cells treated with siRNA-CCAT2.

Notes: The expression level of snail2 (A) and vimentin (B) was significantly reduced and that of E-cadherin (C) as upregulated in HCC cells after knockdown of CCAT2. **P<0.01.
Abbreviations: qRT-PCR, quantitative real-time polymerase chain reaction; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; CCAT2, colon cancer-associated transcript 2; NC, negative control.