Promoter aberrant methylation status of ADRA1A is associated with hepatocellular carcinoma

Figures & data

Table 1. Patients characteristics.

Parameters	Categories	Number
Gender	Female	29
	Male	131
Age	<55	81
	≥55	79
Hypertension	Yes	37
	No	123
Diabetes	Yes	20
	No	140
Hyperlipemia	Yes	37
	No	121
Alcohol habit (≥50mg/day)	Yes	55
	No	105
Family history	Yes	20
	No	140
AFP	Positive	53
	Negative	104
CEA	≤5	132
	>5	24
CA199	Positive	17
	Negative	138
HBV-related HCC	Yes	136
	No	22
Preoperative anti-HBV treatment	Yes	19
	No	141
Preoperative TACE	Yes	9
	No	151
Tumour number	1	145
	2	12
	3	3
Tumour diameter (cm)	≤3	38
	>3, ≤5	48
	>5, ≤10	60
	>10	14
Tumour thrombus	Yes	29
	No	131
Liver cirrhosis stage	0	13
	1	36
	2	50
	3	11
Encapsulation	Complete	108
	Not	52
Tumour necrosis	Yes	29
	No	131
Satellite Tumour	Yes	15
	No	145
Microvascular invasion	Yes	31
	No	129
Extrahepatic metastasis	Yes	9
	No	151
TNM Stage	I-II	121
	III-IV	38

AFP, alpha-fetoprotein; CEA, carcinoembryonic antigen; CA199, carbohydrate antigen 19–9; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; TACE, transcatheter arterial chemoembolization; TNM, tumour node metastasis.

Figure 1. The flow diagram of ARs screening.

Figure 2. The expression and methylation levels of adrenoreceptors (ARs) in HCC from inhouse and TCGA data-set. (a) The expression levels of ARs from our inhouse data-set. (b) The expression levels of ARs from TCGA data-set. (c) The methylation levels of ADRA1A from inhouse data-set. (d) The methylation levels of ADRA1A from TCGA data-set. (e) The methylation levels of ADRA2B from inhouse data-set. (f) The methylation levels of ADRA2B from TCGA data-set. FPKM: fragments per kilobase million; *P < 0.05, **P < 0.001, ***P < 0.0001, NS: not significant.

Figure 3. The expression levels of ADRA1A in HCC cell lines and tissues. (a) The expression levels of ADRA1A protein in HCC and LO2 cell lines; Ratio refers to the result of the grey values ADRA1A bands and GAPDH bands. (b) The mRNA expression levels of ADRA1A in HCC and LO2 cell lines. (c) The representative immunohistochemical results of ADRA1A in HCC tissue and paired non-cancerous tissue. Scale bars = 200μm. (d) The score of immunohistochemical results in all 20 paired HCC samples. (e) The proportion of ADRA1A expression regulation in all 20 paired HCC samples. (f) The overall survival (OS) data of ADRA1A from the Gene Expression Profiling Interactive Analysis website (http://gepia.cancer-pku.cn), a network analysing TCGA data online. *P < 0.05.

Figure 4. The identification of transcriptional activity and correlation of CpG sites in ADRA1A promoter. (a) The schematic diagram and sequence of CpG sites of ADRA1A promoter amplicon. (b) The verification of transcriptional activity in selected amplicon via luciferase assay. (c) The methylation levels of all CpG sites in ADRA1A promoter region from our study. (d) Heatmap of correlation analysis between the methylation levels of different CpG sites in ADRA1A. Upper-right triangle: Heat map of Pearson r values. Lower-left triangle: Heat map of P values. *P < 0.05, ***P < 0.0001, NS: not significant.

Table 2. Methylation status (%) of ADRA1A in hepatocellular carcinoma patients.

CpGs	Group	HCC			HBV-related HCC
		Mean (%)	△Mean (%)	P value	Mean (%)	△Mean (%)	P value
CpG_1.2	Normal	23.9	−9.6	<0.001	24.0	−10.0	<0.001
	Tumour	33.5			34.0
CpG_3.4	Normal	12.2	−8.9	<0.001	12.1	−9.1	<0.001
	Tumour	21.1			21.2
CpG_5	Normal	24.8	−11.3	<0.001	24.7	−10.6	<0.001
	Tumour	36.0			35.3
CpG_7	Normal	18.0	−8.1	<0.001	18.2	−8.4	<0.001
	Tumour	26.1			26.6
CpG_8	Normal	23.9	−9.6	<0.001	24.0	−10.0	<0.001
	Tumour	33.5			34.0
CpG_13	Normal	10.8	−5.2	<0.001	10.9	−5.0	<0.001
	Tumour	16.0			15.9
CpG_14	Normal	10.7	−1.9	0.107	10.6	−1.7	0.183
	Tumour	12.7			12.3
CpG_15.16	Normal	13.6	−9.4	<0.001	13.9	−9.3	<0.001
	Tumour	23.0			23.1
CpG_17	Normal	18.0	−8.1	<0.001	18.2	−8.4	<0.001
	Tumour	26.1			26.6
CpG_18	Normal	14.5	−7.8	<0.001	14.4	−7.8	<0.001
	Tumour	22.2			22.2
CpG_19	Normal	9.7	−8.8	<0.001	9.8	−8.6	<0.001
	Tumour	18.5			18.5
Mean	Normal	17.0	−8.2	<0.001	17.1	−8.2	<0.001
	Tumour	25.2			25.3

HBV, hepatitis B virus; HCC, hepatocellular carcinoma.

Figure 5. The correlation between methylation status and expression levels of ADRA1A from inhouse and TCGA data-set and the expression levels of ADRA1A for DAC treatment. (a) The correlation between the methylation of cg09557462 and ADRA1A gene expression from inhouse data-set. (b) The correlation between the methylation of cg02409177 and ADRA1A gene expression from inhouse data-set. (c) The correlation between the methylation of cg20303399 and ADRA1A gene expression from the TCGA data-set. (d) The correlation between the methylation of cg07645844 and ADRA1A gene expression from the TCGA data-set. (e) The mRNA expression levels of ADRA1A in LM3 and SMMC-7721 cell lines with different DAC concentration treatment. (f) Gel image of ADRA1A promoter Methylation specific PCR (MSP). M: methylated; U: unmethylated.

Figure 6. The association between the methylation levels of ADRA1A and clinicopathological features. (a) The association between the mean methylation levels of ADRA1A and alcohol intake or AFP. (b) The expression difference of four isoforms of ADRA1A between normal and alcohol hepatitis patients. (c) The association between the methylation levels of CpG sites and liver cirrhosis stage. (d) The ROC curves of methylation levels in 14 CpG sites of ADRA1A promoter between HCC tissues and ANT. *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 7. Differential gene expression between ADRA1A knockdown group and control group in HCC cell lines by RNA-sequencing. (a) Representative western blot of ADRA1A in PLC/PRF/5 and LM3; Ratio refers to the result of the grey values ADRA1A bands and GAPDH bands. (b) Detection of cell proliferation for shADRA1A and shCtrl in two HCC cell lines. (c) Top 20 signalling pathways obtained by RNA-sequencing between PLC/PRF/5-shADRA1A and PLC/PRF/5-shCtrl (n = 3). (d, e, f) Heat map of differentially expressed genes for pathways in caner (d), cytokine-cytokine receptor interaction pathway (e) and drug metabolism-cytochrome P450 pathway (f).