lncRNA CADM1-AS1 inhibits cell-cycle progression and invasion via PTEN/AKT/GSK-3β axis in hepatocellular carcinoma

Figures & data

Table 1 Associations of clinicopathological characteristics with CADM1-AS1 expression in hepatocellular carcinoma

	Variables	CADM1-AS1 expression		Total	χ^2	P-value
High	Low
Age (year)					0.002	0.962
	≤50	19	21	40
	>50	24	26	50
Sex					0.674	0.412
	Female	6	4	10
	Male	37	43	80
Grade					0.004	0.947
	1/2	25	27	52
	3	18	20	38
T stage					5.436*	0.020
	t1	33	25	58
	T2/T3	10	22	32
TNM stage					5.436*	0.020
	Ι	33	25	58
	II/III	10	22	32
Cirrhosis					0.657	0.418
	Negative	6	3	9
	Positive	37	43	80
	Dull			1
HBsAg					0.180	0.671
	Negative	10	9	19
	Positive	33	37	70
	Dull			1
AFP					0.024	0.877
	Negative	18	20	38
	Positive	25	26	51
	Dull			1

Abbreviations: HBsAg, hepatitis B surface antigen; AFP, alpha-fetoprotein.

Figure 1 CADM1-AS1 expression and transfection efficiency in HCC cell line. (A) CADM1-AS1 expression in cancerous and non-cancerous tissues, determined in ISH (n=90). (B) Representative images for ISH detecting CADM1-AS1 expression in cancerous and non-cancerous tissues. (C) Kaplan-Meier survival analysis of overall survival in HCC patients based on CADM1-AS1 expression. Differences were assessed by the log-rank test (n=90). (D) Levels of CADM1-AS1 in HCC (HepG2, BEL-7402 and Huh-7) and normal hepatocyte (LO2) cell lines, detected by qRT-PCR. (E) Lentivirus transfection efficiency and cell morphology were observed under an inverted fluorescence microscope using bright and GFP field channels (x100 and x400 magnification). (F) RT-qPCR were performed to detect the expression levels of CADM1-AS1, with GAPDH as a reference control. Representative images from experiments performed three times are shown (***P<0.001).

Abbreviations: HCC, hepatocellular carcinoma; ISH, In situ hybridization; GFP, Green Fluorescent Protein; RT-qPCR, real-time quantitative PCR; CADM1-AS1, cell adhesion molecule 1 antisense RNA 1.

Table 2 Univariate and multivariate analyses of factors associated with overall survival in patients with hepatocellular carcinoma

variables	Univariate analysis			Multivariate analysis
	HR	95%CI	P-value	HR	95%CI	P-value
Expression	0.386	0.204–0.730	0.003*	0.368	0.191–0.712	0.003*
Sex	1.460	0.452–4.718	0.527
Grade	1.970	1.123–3.458	0.018*	2.077	1.169–3.691	0.013*
Age	1.421	0.777–2.598	0.254
T stage	1.661	1.024–2.694	0.040*	0.900	0.119–6.833	0.919
TNM stage	1.679	1.008–2.796	0.047*	1.360	0.161–11.502	0.777
Cirrhosis	1.487	0.532–4.159	0.450
HBsAg	1.065	0.511–2.219	0.866
AFP	1.428	0.772–2.642	0.256

Note: Pearson’s chi-square tests or Fisher exact tests were used. *P<0.05

Abbreviations: HBsAg, hepatitis B surface antigen; AFP, alpha-fetoprotein; CI, confidence interval; HR, hazard ratio.

Figure 2 Effects of CADM1-AS1 on HCC cell proliferation in vitro. (A) Growth curves for HepG2 and BEL-7402 cells after transfection were determined by the CCK-8 assay. (B) The EDU assay was performed to determine the proliferation ability after transfection. (C) Colony formation assay was performed to assess the colony forming ability after transfection. Representative images from experiments performed three times are shown (*P<0.05; **P<0.01; ***P<0.001).

Abbreviations: CADM1-AS1, cell adhesion molecule 1 antisense RNA 1; HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; EDU, 5‐Ethynyl‐2′‐deoxy-uridine.

Figure 3 Effects of CADM1-AS1 on migration, invasion and cell-cycle on HCC cells in vitro. (A) Transwell invasion assay was performed to determine the invasive ability. (B) Transwell migration assay was performed to determine the migratory ability. (C) The scratch-wound assay was performed to detect the migratory ability. (D) Cell cycle progression was evaluated by flow cytometry. Representative images from experiments performed three times are shown (*P<0.05; **P<0.01; ***P<0.001).

Abbreviations: CADM1-AS1, cell adhesion molecule 1 antisense RNA 1; HCC, hepatocellular carcinoma.

Figure 4 Mechanism of CADM1-AS1 on the AKT/GSK-3β signaling pathway and HCC cell-cycle progression in vitro. PTEN, total AKT, total GSK-3β, p-AKT and p-GSK-3β levels were examined by Western blot in HepG2 cell after transfection. GAPDH was used as the loading control. PTEN, total AKT, total GSK-3β, p-AKT and p-GSK-3β levels were examined by Western blot in BEL-7402 cell after transfection. GAPDH was used as the loading control. P15, P21, P27, CDK2, CDK4, CDK6, cyclinD and cyclinE protein levels were determined in HepG2 cells after transfection. P15, P21, P27, CDK2, CDK4, CDK6, cyclinD and cyclinE protein levels were determined in BEL-7402 cells after transfection. Representative images from experiments performed three times are shown (*P<0.05; **P<0.01; ***P<0.001).

Abbreviations: CADM1-AS1, cell adhesion molecule 1 antisense RNA 1; HCC, hepatocellular carcinom.

Figure 5 CADM1-AS1 overexpression suppresses HepG2 cell growth in vivo. (A) Tumor volumes and weighs of Xenograft tissues. (B) The expression of CADM1-AS1 was detected by qRT-PCR in xenograft tumor tissues. GAPDH was used as a loading control. (C) PTEN/AKT/GSK-3β signaling pathway and cell cycle regulators protein levels were examined by Western blotting in xenograft tumor tissues. GAPDH was used as a loading control. (D) Representative IHC staining images of P15, P21, P27, CDK2, CDK4, CDK6, cyclinD and cyclinE expression levels in xenograft tumor tissues Representative images from experiments performed three times are shown (**P<0.01; ***P<0.001).

Abbreviations: CADM1-AS1, cell adhesion molecule 1 antisense RNA 1; RT-qPCR, real-time quantitative PCR; IHC, immunohistochemical.