Role of Genetic Polymorphism and Expression of Angiopoietin-2 in Patients with Primary Liver Cancer Among the Southeastern Chinese Hans Population

Abstract

Background:

Angiopoietin-2 (Ang2)-mediated angiogenesis plays a crucial role in the pathogenesis of vascular-rich cancers. However, the genetic polymorphism and expression level of Ang2 in patients with primary liver cancer remain unknown.

Methods:

This study included 234 primary liver cancer patients and 199 healthy controls. The expression levels of Ang2 in liver cancer tissues and plasma were determined. Peripheral blood samples were collected to test five ANGPT2 single nucleotide polymorphisms (rs2442598, rs734701, rs1823375, rs11137037, and rs12674822).

Results:

Plasma Ang2 levels in patients with liver cancer were upregulated compared with that in healthy controls. The upregulation of plasma Ang2 levels was significantly associated with vascular invasion, metastasis, and clinical stage. Notably, the transcription level of ANGPT2 was elevated in tumor tissues compared with para-carcinoma tissues. Individuals with the TT genotype at rs2442598 and genotype AC and AC+CC at rs11137037 had higher liver cancer risk compared with healthy controls.

Conclusions:

Upregulated Ang2 levels in blood plasma and cancer tissues of liver cancer patients confirm that Ang2 plays a vital role in the pathogenesis of liver cancer. ANGPT2 rs2442588 and rs11137037 are associated with liver cancer risk, thereby highlighting their role in screening individuals susceptible to liver cancer.

Introduction

Primary liver cancer is mainly composed of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma. HCC accounts for ∼80% (Kim and Park, 2018) of liver cancers and is one of the most common malignant tumors worldwide (Rawla et al., 2018). HCC is the main cancer type in China and an important cause of cancer-related deaths (Feng et al., 2019). In general, patients have no obvious symptoms in the early stages of the disease. Notably, 70-80% of HCC patients are at an advanced stage or have metastases at diagnosis (Liu and Qin, 2019), resulting in poor prognosis.

HCC is a hypervascular malignancy that requires new blood vessels to aid tumor growth and metastasis. Angiopoietin-2 (Ang2; encoded by ANGPT2) is a ligand of the tyrosine kinase receptor Tie, which regulates vascular formation, maintains vascular stability, and is an important angiogenic factor. Abnormal Ang2 expression is closely associated with cancer progression. For instance, Ang2 expression is positively correlated with the clinical stage of lung cancer (Akwii et al., 2019) and promotes breast cancer metastasis (Imanishi et al., 2007). Although studies have postulated that Ang2 is upregulated in the serum of HCC patients and is involved in HCC progression (Scholz et al., 2007; Vanderborght et al., 2020; Ao et al., 2021), its role in tissues and serum of HCC patients remains poorly understood.

Single nucleotide polymorphisms (SNPs) have been widely used for early screening of susceptible tumor genes based on the genetic heterogeneity of tumors (Hemminki and Bermejo, 2005). ANGPT2 polymorphisms are associated with various diseases, including colorectal cancer (Du et al., 2020), lung cancer (Hu et al., 2019), systemic lupus erythematosus (Wang et al., 2021), and rheumatoid arthritis (RA) (Dai et al., 2019). However, the association between ANGPT2 polymorphisms and the risk of liver cancer remains unknown. This study focused on deciphering this association to enhance the knowledge pool.

Materials and Methods

Participants' clinical information and the collection of blood and tissue samples

For this study, patients diagnosed with primary liver cancer by MRI, CT, or histological examination at a tertiary hospital in southeastern China were recruited. Patients involved were of the Chinese Hans race. Participants without evidence of tumors on radiological examination or cancer history were recruited as the control group. Fresh tumor surgical specimens and adjacent normal tissues were collected from patients with liver cancer and stored at −80°C.

Blood samples from all participants were collected in EDTA-containing tubes and centrifuged immediately at 4°C to separate blood cells and plasma, which were subsequently stored at −80°C before use. Laboratory examinations, clinical status, histological type, and other clinical information of all participants were retrieved from their medical records. Liver cancer patients were staged according to the Tumor Node Metastasis classification. All participants provided informed consent before participating in the study. This study was approved by the Ethics Committee and Institutional Review Board of Dongyang People's Hospital (2018-YX-020).

Determination of Ang2 in the plasma using enzyme-linked immunosorbent assay

Ang2 levels in plasma samples of liver cancer patients and healthy controls were determined using the Human Ang2 Enzyme-Linked Immunosorbent Assay (ELISA) kit (R&D Systems), following the manufacturer's instructions. In brief, 100 μL of the diluted capture antibody was added to each well of the microplate and incubated overnight at room temperature. Plasma and ELISA standards (100 μL) were added to each well and incubated for 2 h at room temperature. The absorbance was measured at 450 nm and 570 nm after adding the detecting antibody, streptavidin-HRP, and substrate solution.

Determination of ANGPT2 expression in tissues of liver cancer patients

Total RNA was isolated from the surgical specimens using the Tissue RNA Purification Kit Plus (ESscience Biotech, China). The RNA samples (1 μg) were reverse-transcribed into cDNA using the PrimeScript™ RT reagent kit (Takara, Japan). ANGPT2 (Liu et al., 2016) and GAPDH (Su et al., 2019) primers were synthesized as described previously (GENEWIZ, Suzhou, China). ANGPT2 expression in tissue samples was detected using a SYBR quantitative real-time polymerase chain reaction (RT-PCR). The PCR mixtures contained 10 μL of 2X TB Green Premix Ex Taq II, 0.4 μM of ANGPT2 primers, 1 μL of cDNA (TB Green^® Premix Ex Taq™ II Kit, Takara), and ddH₂O totaling 20 μL. The reaction mixtures were incubated as followed: initial predenaturation for 30 s at 95°C, followed by 40 cycles of denaturation for 3 s at 95°C and annealing and extension for 30 s at 60°C. GAPDH was used as the internal reference gene.

Genomic DNA extraction and determination of SNPs

Total genomic DNA was extracted from peripheral blood using the AxyPrep Blood Genomic DNA Miniprep Kit (Corning, New York), following the manufacturer's protocol. DNA was quantified using a biodrop (Biochrom, Cambridge, UK) and stored at −20°C.

Five SNP probes were designed as previously described (Dai et al., 2019) and were synthesized by Thermo Fisher Scientific (Waltham, MA). SNPs genotypes were detected using the TaqMan probe quantitative (RT-PCR). The PCR mix contained 1 U TaqMan Genotyping Master Mix (Applied Biosystems), 1 μL genomic DNA (20-100 ng), 0.125 μL probes, and ddH₂O totaling 10 μL. The reaction mixtures were initially denatured for 10 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C, annealing and extension for 1 min at 60°C. Amplification was performed using a QuantStudio™ 5 Real-Time PCR system (Applied Biosystems), and the results were analyzed using the QuantStudio Design & Analysis Software (Applied Biosystems, San Jose, CA).

Statistical analysis

All data analyses were performed using SPSS version 24.0 (IBM, Chicago). Normally distributed data were expressed as mean ± standard deviation and were analyzed using the independent samples t-test. Non-normally distributed data were expressed as the median and analyzed using a nonparametric test. Categorical data were analyzed using the Pearson's χ² test. Logistic regression models were used to analyze ANGPT2 genotypes and the risk of liver cancer. The odds ratio combined with a 95% confidence interval (CI) for liver cancer was determined, followed by the calculation of the adjusted odds ratio (AOR) for alcohol consumption. The significance threshold was set at p < 0.05.

Results

Baseline characteristics of liver cancer patients and healthy participants

The general information and clinical data of 234 patients with liver cancer and 199 healthy participants were obtained. There were no significant differences in age or gender between patients with liver cancer and controls (Table 1). Among the 234 liver cancer patients, 224 (95.7%) were diagnosed with HCC. Notably, liver cancer patients had a higher alcohol consumption percentage (51.3%) compared with healthy controls (24.1%).

Table 1.

Clinical Information of Participants Involved in this Study

Variable	Healthy control N = 199 (%)	Liver cancer N = 234 (%)	p
Age (years, mean ± SD)	59.68 ± 10.87	59.12 ± 10.86	0.597
Gender			0.655
Male	167 (83.9)	200 (85.5)
Female	32 (16.1)	34 (14.5)
Alcohol			<0.001
No	151(75.9)	114 (48.7)
Yes	48 (24.1)	120 (51.3)
Stage
I+II		157 (71.4)
III+IV		63 (28.6)
Vascular invasion
No		159 (68.8)
Yes		72 (31.2)
Metastasis
No		208 (89.7)
Yes		24 (10.3)
Histology
HCC		224 (95.7)
ICC		6 (2.6)
Others		4 (1.7)

HCC, hepatocellular carcinoma; ICC, intrahepatic cholangiocarcinoma; SD, standard deviation.

Ang2 expression level in plasma and tissue samples of liver cancer patients

The expression level of plasma Ang2 in 221 patients with liver cancer was significantly higher than that in the 199 healthy participants (Fig. 1A, p < 0.001). Real-time quantitative polymerase chain reaction analysis further revealed upregulation of ANGPT2 transcription in liver cancer tissues (n = 39) compared with the para-cancerous tissues (Fig. 1B, p = 0.0479).

FIG. 1.

Ang2 expression level in liver cancer patients and healthy controls. (A) Comparison of plasma levels of Ang2 between liver cancer patients and healthy controls (labeled as NL group in the figure) by ELISA. (B) Comparison of ANGPT2 expression between liver cancer and para-cancerous tissues by RT-qPCR. Ang2, angiopoietin-2.

The plasma Ang2 levels of patients with vascular invasion and metastasis were higher than those of patients without invasion and metastasis (Table 2). Moreover, the plasma Ang2 levels of stage III+IV patients were higher than those of stage I + II patients.

Table 2.

Association Between Plasma Angiopoietin-2 and Clinical Features in Liver Cancer Patients

Variable	Level of plasma Ang2 (pg/mL)
Variable	No.	Mean ± SD	p
Vascular invasion			<0.001
Yes	64	1901.10 ± 976.75
No	153	1346.38 ± 835.62
Metastasis			0.036
Yes	23	1886.95 ± 1106.69
No	194	1465.29 ± 880.19
Stage			<0.001
I+II	145	1356.29 ± 851.20
III+IV	61	1860.81 ± 977.41

Ang2, Angiopoietin-2.

The distribution frequency of ANGPT2 polymorphisms between liver cancer patients and healthy controls

Table 3 outlines the differences in genotype frequencies of the five ANGPT2 SNPs between liver cancer patients and healthy controls. The AT genotype for ANGPT2 rs2442598 had the highest distribution frequency in both liver cancer patients and healthy controls. Patients with genotype TT at rs2442598 were 1.885 times (AOR: 1.885, 95% CI: 1.066-3.333) more likely to develop liver cancer than healthy controls. Patients with genotype AC at rs11137037 were 2.057 times (AOR: 2.057, 95% CI: 1.338-3.16), whereas those carrying genotype AC+CC were 1.774 times (AOR: 1.774, 95% CI: 1.192-2.64) more likely to develop liver cancer compared with healthy controls. The genotypes with the highest distribution frequencies for rs734701, rs1823375, and rs12674822 were TC, CC, and GT, respectively. Notably, genotype frequencies for rs734701, rs1823375, and rs12674822 in ANGPT2 were not associated with a risk of developing liver cancer.

Table 3.

Genotype Frequencies of Five Single Nucleotide Polymorphisms in the ANGPT2 Gene Between Liver Cancer Patients and the Healthy Controls

Locus	Healthy control (%)	Liver cancer (%)	OR 95% CI	AOR ^a 95% CI
rs2442598
AA	59 (29.6)	56 (23.9)	1.00 (reference)	1.00 (reference)
AT	101(50.8)	118 (50.5)	1.231 (0.783-1.934)	1.23 (0.767-1.974)
TT	39 (19.6)	60 (25.6)	1.621 (0.941-2.793)	1.885 (1.066-3.333)
AT+TT	140 (70.4)	178 (76.1)	0.747 (0.487-1.145)	1.403 (0.897-2.192)
rs734701
TT	60 (30.2)	79 (33.8)	1.00 (reference)	1.00 (reference)
TC	99 (49.7)	121 (51.7)	1.549 (0.879-2.731)	0.908 (0.581-1.417)
CC	40 (20.1)	34 (14.5)	1.438 (0.847-2.44)	0.692 (0.384-1.247)
TC+CC	139 (69.8)	155 (66.2)	0.847 (0.564-1.271)	0.847 (0.555-1.293)
rs1823375
CC	102 (51.3)	110 (47.0)	1.00 (reference)	1.00 (reference)
CG	74 (37.2)	105 (44.9)	1.305 (0.672-2.538)	1.381 (0.908-2.098)
GG	23 (11.5)	19 (8.1)	1.718 (0.873-3.379)	0.846 (0.424-1.689)
CG+GG	97 (48.7)	124 (53.0)	1.185 (0.812-1.731)	1.256 (0.846-1.864)
rs11137037
AA	111 (55.8)	94 (40.2)	1.00 (reference)	1.00 (reference)
AC	62 (31.1)	113 (48.3)	0.815 (0.446-1.493)	2.057 (1.338-3.16)
CC	26 (13.1)	27 (11.5)	1.755 (0.943-3.267)	1.112 (0.592-2.09)
AC+CC	88 (44.2)	140 (59.8)	1.879 (1.281-2.715)	1.774 (1.192-2.64)
rs12674822
GG	62 (31.2)	67 (28.6)	1.00 (reference)	1.00 (reference)
GT	86 (43.2)	108 (46.2)	0.934 (0.561-1.555)	1.175 (0.738-1.871)
TT	51(25.6)	59 (25.2)	1.086 (0.679-1.736)	1.12 (0.659-1.904)
GT+TT	137 (68.8)	167 (71.4)	1.128 (0.747-1.704)	1.155 (0.751-1.775)

AOR was the odds ratio adjusted by alcohol assumption and the bold values were statistically significant (p < 0.05).

AOR, adjusted odds ratio; CI, confidence interval; OR, odds ratio.

Association of ANGPT2 SNPs with alpha fetoprotein, aspartate aminotransferase, and alanine aminotransferase in liver cancer patients

Among the liver cancer patients, the alpha fetoprotein (AFP) level of patients with genotype AA of rs11137037 was significantly higher than that of patients with genotype AC+CC (p = 0.039, Table 4). In contrast, the alanine aminotransferase (ALT) levels of liver cancer patients with genotype AA of rs2442598 were significantly lower than those of patients with genotype AT+TT (p = 0.015). Notably, the aspartate aminotransferase (AST) levels of liver cancer patients were not associated with any genotype (Table 4). There was also no association between genotype frequencies and AST and ALT levels in liver cancer patients for rs734701, rs1823375, rs11137037, and rs12674822.

Table 4.

Association Between Single Nucleotide Polymorphisms Genotypes and Alpha Fetoprotein, Aspartate Aminotransferase, and Alanine Aminotransferase in Liver Cancer Patients

Locus	Genotype	AFP (ng/mL)	AST (U/L)	ALT (U/L)	AST/ALT
rs2442598	AA	15.3 (4, 92.9)	46.3 ± 49.4	34.9 ± 23.4	1.4 ± 0.6
	AT+TT	20.1 (4.3, 462)	55.0 ± 70.9	48.8 ± 62.5	1.3 ± 0.7
	p	0.177	0.39	0.015	0.523
rs734701	TT	23.5 (3.9, 329.0)	63.2 ± 94.9	52.7 ± 73.0	1.4 ± 0.7
	TC+CC	20.1 (4.9, 362.6)	47.6 ± 44.8	41.7 ± 44.6	1.3 ± 0.6
	p	0.811	0.172	0.22	0.601
rs1823375	CC	13.9 (3.7, 199.3)	46.5 ± 41.3	39.6 ± 29.0	1.3 ± 0.5
	CG+GG	38.7 (5.6, 377.5)	58.7 ± 82.3	50.6 ± 71.7	1.4 ± 0.8
	p	0.101	0.147	0.12	0.09
rs11137037	AA	41.1 (7, 612.3)	58.4 ± 83.4	47.4 ± 57.6	1.4 ± 0.8
	AC+CC	14.35 (4.1, 183.0)	49.3 ± 52.1	44.1 ± 55.0	1.3 0.5
	p	0.039	0.304	0.658	0.321
rs12674822	GG	14.7 (4, 215.0)	53.9 ± 64.8	46.3 ± 59.8	1.4 ± 0.8
	GT+TT	20.1 (4.5, 383.7)	52.5 ± 67.2	45.1 ± 54.5	1.3 ± 0.6
	p	0.23	0.885	0.879	0.529

Discussion

HCC is a hypervascular malignancy and the most common primary liver cancer type. Ang2 cooperates with Ang1 and vascular endothelial growth factor to regulate vascular formation, stabilization, and remodeling (Kaseb et al., 2014). Ang2-mediated angiogenesis plays an important role in the proliferation and progression of HCC cells (Ji et al., 2012; Diaz-Sanchez et al., 2013; Vanderborght et al., 2020). In this study, ANGPT2 rs2442598 and rs11137037 were associated with the risk of liver cancer and AFP and ALT levels in the plasma. Ang2 expression in plasma and cancer tissues of liver cancer patients was upregulated and associated with clinical staging, vascular invasion, and metastasis.

Our study confirmed elevated levels of Ang2 in plasma and tissues from liver cancer patients, which was consistent with previous findings (Scholz et al., 2007; Diaz-Sanchez et al., 2013; Chen et al., 2018). Furthermore, liver cancer patients with vascular invasion and metastasis had higher plasma levels of Ang2, thereby indicating that Ang2 plays a potential role in tumor metastasis and growth. Ang2 mediates HCC metastasis in an epithelial-mesenchymal transition-independent manner (Fang et al., 2015) and promotes lung cancer cell metastasis through epithelial-mesenchymal transition (Dong et al., 2018). Although the modalities of Ang2-regulated tumor cell metastasis vary among tumor types, Ang2 plays an important role in tumor progression. In this study, patients in the later clinical stages had higher plasma levels of Ang2, thus suggesting that Ang2 imbalance is closely associated with poor prognosis (Llovet et al., 2012; Ao et al., 2021).

SNPs in intron regions are genetic variations that can regulate gene transcription or translation, thereby affecting cancer susceptibility (Nahon and Zucman-Rossi, 2012; Qiu et al., 2020). Herein, there were five loci of ANGPT2 involved, but no association between genotypes and plasma expression levels was observed (data not shown). Individuals with the ANGPT2 rs2442598 TT genotype exhibited a higher risk of liver cancer. In our previous studies, the TT genotype of rs2442598 was associated with an increased risk of RA (Dai et al., 2019) and a lower risk of coronary artery disease (Lan et al., 2021).

The CC genotype at the same locus (rs2442598) is associated with increased susceptibility to systemic sclerosis in Polish Caucasians (Michalska-Jakubus et al., 2021). In contrast, the AA genotype is associated with the risk of diabetic retinopathy in patients with type 1 diabetes mellitus in a Brazilian population (Dieter et al., 2021). In addition, there is an increase in the frequency of the CT+TT genotype in patients with psoriasis vulgaris (He et al., 2015). However, there is no relationship between rs2442598 and the risk of systemic lupus erythematosus (Wang et al., 2021), colorectal cancer (Du et al., 2020), and lung cancer (Hu et al., 2019).

The findings of this study suggested that individuals with the rs11137037 AC genotype have a higher risk of liver cancer. Moreover, the rs11137037 AC genotype may be a susceptibility locus for RA (Dai et al., 2019) and coronary artery disease (Lan et al., 2021). In contrast, the other three SNPs (rs734701, rs1823375, and rs12674822) were not significantly correlated with liver cancer susceptibility but are closely associated with RA risk (Dai et al., 2019). Therefore, the genotypes of ANGPT2 differ in different diseases and ethnicities.

ANGPT2 rs2442598 and rs11137037 in liver cancer patients were positively associated with ALT and AFP levels in the plasma. ALT mainly exists in liver cells and is the main serological index for clinically evaluating liver function. Abnormally elevated ALT levels suggest hepatocyte injury and an increased risk of liver cancer (Suruki et al., 2006; Sun et al., 2021). AFP, a glycoprotein, is a diagnostic marker for liver cancer (Wang and Wang, 2018). ANGPT2 SNPs can thus predict the severity of liver damage in liver cancer patients.

Despite the interesting findings, this study had several limitations. The study was conducted in a single tertiary hospital in southeast China, with a relatively small sample size. Moreover, the participants in this study were all Chinese Han people and did not include other ethnic groups.

Conclusion

Plasma Ang2 levels are associated with liver injury in liver cancer patients and thus play a vital role in prognosis. ANGPT2 rs2442588 and rs11137037 are associated with a higher liver cancer risk, thereby highlighting their role in screening individuals susceptible to liver cancer.

Footnotes

Authors' Contributions

Conceptualization (lead), funding acquisition (lead), writing—review and editing (equal) by B.W. Resources (lead), conceptualization (supporting), writing—review and editing (supporting) by Y.L. Software (lead), investigation (equal), resources (equal) by S.Y. Investigation (lead), formal analysis (lead), writing—original draft (lead) by J.Z. Conceptualization (equal), methodology (lead), writing—review and editing (lead), and writing—original draft (equal) by X.P.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Zhejiang Province Public Welfare Technology Application Research Project (#2019KY761) and the Science and Technology Bureau of Jinhua (#2020-4-139).

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