RASSF1A and SOCS1 genes methylation status as a noninvasive marker for hepatocellular carcinoma

Abstract

BACKGROUND:

DNA methylation status is one of the most prevalent molecular alterations in human cancers. Identification of powerful diagnostic and prognostic biomarkers for hepatocellular carcinoma (HCC) without a biopsy is urgently required.

OBJECTIVE:

The purpose of this study was to determine the methylation status of RASSF1A and SOCS-1genes as a non-invasive biomarker for HCC identification and prognosis.

METHODS:

Methylation specific-PCR technique was performed to recognize the methylation status of RASSF1A and SOCS-1 genes in 100 patients with HCC, 100 patients with liver cirrhosis (LC) but without HCC were considered as cirrhotic liver control group and 100 healthy control.

RESULTS:

Methylation of RASSF1A and SOCS-1 genes were detected in 40% and 38% of HCC patients respectively, 14% and 20% of LC patients respectively. Methylation of SOCS-1 gene in peripheral blood of healthy control was 23%. Methylation of RASSF1A gene was associated with age, tumor size, vascular invasion and $\alpha$ fetoprotein (AFP), while SOCS-1 gene methylation was significantly associated with tumor size and AFP. Furthermore, using RASSF1A/ SOCS-1/ AFP panel improve diagnostic sensitivity for HCC 86% and specificity of 75%.

CONCLUSION:

RASSF1A and SOCS1 genes methylation status may play an important role in the process of hepatocarcinogenesis and may be used as diagnostic and prognostic noninvasive biomarkers for HCC when combined with serum AFP.

Keywords

Hepatocellular carcinoma methylation SOCS-1 RASSF1A

1. Introduction

The incidence of hepatocellular carcinoma (HCC) is dramatically increased in the last few years making it the sixth commonest cancer and the third cause for cancer related mortality [1]. In spite of advances in the diagnosis and treatment of such disease, its prognosis stays poor because of its late detection. At present, serum $\alpha$ -fetoprotein (AFP) test is generally utilized; however it has a moderately low affectability and specificity. Owing to the restrictions of pathological tests, there is a pressing requirement for finding effective diagnostic and prognostic biomarkers for HCC without biopsy [2]. Novel peripheral blood test techniques are important to improve diagnostic sensitivity together with AFP [3].

Table 1
Primers used and the size of amplification products

Gene	Primers	Size (bp)
RASSF1A	MF: 5’-GTGTTAACGCGTTGCGTATC-3’	M $=$ 93 bp
	MR: 5’-AACCCCGCGAACTAAAAACGA-3’	U $=$ 105 bp
	UF: 5’-TTTGGTTGGAGTGTGTTAATGTG-3’
	UR: 5’-CAAACCCCACAAACTAAAAACAA-3
SOCS-1	MF: 5’-TTGTTCGGAGGTCGGATTT-3’	M $=$ 218 bp
promoter	MR 5’-ACTAAAACGCTACGAAACCG-3’
	UF: 5’-TTTTTTGGTGTTGTTTGGAGGTTGGATTTT-3’	U $=$ 243 bp
	UR: 5’-AAAACAAAACAATAAACTAAAACACTACAAAACCA-3’

DNA methylation profile got from blood tests could conceivably be such biomarker [4]. Several studies have postulated that methylation of numerous tumor suppressor genes in HCC may add to the pathogenesis of this disease [5, 6].

The ras association domain family 1A (RASSF1A) gene which is situated on chromosome 3p21.3, is an individual from the ras association domain family that can control the cell cycle and trigger apoptosis. RASSF1A over-expression might be identified with cell cycle arrest [7]. RASSF1A hypermethylation has brought about down regulation of gene expression and postulated new points of view for the detection of the malignant tumor [8].

Suppressor of cytokine signaling-1 (SOCS-1) gene which is situated on chromosome 16p12-p13.1, is an intracellular protein that negatively control the JAK/STAT signaling pathway, which is known to assume a vital part in hepatocytes recovery [9]. The SOCS-1 gene has been observed to be silenced by methylation of the CpG islands in human HCC [10]. Previous studies have exhibited that JAK/STAT pathway dysregulation is included in the malignant change for several human malignancies including HCC [11].

The purpose of this study was to access methylation status in SOCS-1 and RASSF1A genes in peripheral blood to elucidate its clinical utility as diagnostic and prognostic non invasive biomarkers for HCC.

2. Patient and methods

Two hundred patients were included in this study. One hundred patients with hepatitis C virus (HCV) infected HCC were chosen by abdominal ultrasound. The diagnosis was confirmed by AFP elevation combined with imaging examination (magnetic resonance imaging (MRI) and/or computed tomography (CT)) and/or pathological examination. The pathological tumor stage was distinguished by 6th edition of the tumor-node-metastasis (TNM) classification of the International Union against Cancer. One hundred patients with LC but without HCC were diagnosed by liver ultrasound, CT, and MRI. These patients were considered as cirrhotic liver control group. In addition to 100 subjects who were enrolled as a healthy control group age and sex matched with the patients were randomly recruited and were seronegative for hepatitis C virus (HCV) and hepatitis B virus (HBV).

Table 2
Clinical features among the different studied groups

	Control ( $n=$ 100)	LC group ( $n=$ 100)	HCC group ( $n=$ 100)
Age	55.9 $\pm$ 8.9	58.2 $\pm$ 7.7	57.6 $\pm$ 3.3
Sex F/M	35/65	38/62	29/71
AST	35.2 $\pm$ 7.4	62.4 $\pm$ 8.2 ${}^{\rm a}$	60.2 $\pm$ 14.5 ${}^{\rm a}$
ALT	36.5 $\pm$ 7.1	66.4 $\pm$ 7.4 ${}^{\rm a}$	75 $\pm$ 17.6 ${}^{\rm a,b}$
AFP ( $\geqslant$ 20 ng/L)	0 (0%)	19 (19%) ${}^{\rm a}$	68 (68%) ${}^{\rm a,b}$

${}^{\rm a}$ : $p<$ 0.001 when compared with control. ${}^{\rm b}$ : $p<$ 0.001 when compared with LC. All data are presented as mean $\pm$ standard deviation or n (%). AFP: Alpha-Fetoprotein; ALT: Alanine Aminotransferase; AST: Aspartate Transaminase; LC: Liver Cirrhosis; HCC: Hepatocellular Carcinoma.

Table 3

Frequency of methylation in HCC, LC and Control

Gene	Control ( $n=$ 100)		LC ( $n=$ 100)		$p$	HCC ( $n=$ 100)		$p$
	M	U	M	U		M	U
RASSF1A	0	100 (100%)	14 (14%)	86 (86%)	$<$ 0.001	40 (40%)	60 (60%)	$<$ 0.001
SOCS-1	23 (23%)	77 (77%)	20 (20%)	80 (80%)	$>$ 0.05	38 (38%)	62 (62%)	0.032

Table 4

The clinicopathological features and gene methylation patterns in HCC patients

Clinicopathological finding	RASSF1A		$p$	SOCS-1		$p$
	M 40	U 60		M 38	U 62
Age
$>$ 50 ( $n=$ 76)	34 (85)	42 (70)		29 (76.3)	47 (75.8)
$<$ 50 ( $n=$ 24)	6 (15)	18 (30)	0.018	9 (23.7)	15 (24.2)	$>$ 0.05
Tumor size
$<$ 5 cm ( $n=$ 46)	10 (25)	36 (60)		11 (28.9)	35 (56.5)
$\geqslant$ 5 cm ( $n=$ 54)	30 (75)	24 (40)	$<$ 0.001	27 (71.1)	27 (43.5)	$<$ 0.001
Pathological grade
Well diff. I–II ( $n=$ 52)	21 (52.5)	31 (51.7)		20 (52.6)	32 (51.6)
Poorly diff. III–IV ( $n=$ 48)	19 (47.5)	29 (48.3)	$>$ 0.05	18 (47.4)	30 (48.4)	$>$ 0.05
Vascular invasion
$-$ ( $n=$ 78)	20 (50)	58 (96.7)		28 (73.7)	50 (80.6)
$+$ ( $n=$ 22)	20 (50)	2 (3.3)	$<$ 0.001	10 (26.3)	12 (19.4)	$>$ 0.05
AFP (ug/L)
$\geqslant$ 20 ( $n=$ 68)	35 (87.5)	33 (55)		31 (81.6)	37 (59.7)
$<$ 20 ( $n=$ 32)	5 (12.5)	27 (45)	$<$ 0.001	7 (18.4)	25 (40.3)	0.001

This study was approved by the Faculty of Medicine, Zagazig University Institutional Review Board. All patients read and signed an informed consent form.

2.1 DNA extraction

DNA purification Kit (PROMEGA-USA) was used to extract DNA from peripheral blood samples. All samples were preserved at $-$ 80 ${}^{\circ}$ C until used.

2.2 Methylation-specific PCR analysis (MSP)

Bisulfite modified of genomic DNA was done using the EpiMark Bisulfite conversion Kit (Biolabs, New England). Amplification conditions for SOCS-1 promoter included pre-denatured at 94 ${}^{\circ}$ C for 5 min followed by 35 cycles consisting of 94 ${}^{\circ}$ C for 1 min, 65 ${}^{\circ}$ C for methylation primers and 60 ${}^{\circ}$ C for unmethylation primers for 1 min, 72 ${}^{\circ}$ C for 1 min, followed by a final 5 min extension at 72 ${}^{\circ}$ C [12]. The thermal profile for RASSF1A included an initial denaturation step of 95 ${}^{\circ}$ C for 10 min followed by 40 cycles of 95 ${}^{\circ}$ C for 30 s, 60 ${}^{\circ}$ C for 45 s, and 72 ${}^{\circ}$ C for 45 s, with a final extension step of 72 ${}^{\circ}$ C for 7 min [13]. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. The primers used and the size of amplification products were described in (Table 1).

2.3 Statistical analysis

The results for continuous variables were expressed as means $\pm$ SD. The Chi-square test was used to evaluate the frequency of methylation in HCC patients, LC patients and control, and to evaluate the clinicopathological features and gene methylation patterns in HCC patients. Specificity, sensitivity, accuracy positive predictive value (PPV) and negative predictive value (NPV) were computed for each parameter as well as for each panel. The panel was considered positive for a specific sample when at least one of the parameters was positive in the individual model. A difference was considered significant at $p<$ 0.05. All data were evaluated using SPSS version 10.0 of windows.

3. Results

3.1 Clinical features among the different studied groups (Table 2)

Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were obviously higher in HCC and LC patients compared with the healthy controls ( $p<$ 0.001). The frequency of serum AFP level $\geqslant$ 20 ng/L was higher in patients with HCC than patients with LC ( $p<$ 0.001).

3.2 Frequency of methylation in HCC, LC and control (Table 3)

Methylation of RASSF1A and SOCS-1 genes in peripheral blood were detected in 40% and 38% of HCC patients respectively, 14% and 20% of LC patients respectively. Methylation of SOCS-1 gene in peripheral blood of healthy control was 23%. On the other hand RASSF1A methylation was not detected in the healthy controls.

Table 5
Evaluation of RASSF1A and SOCS-1 genes methylation in detection of HCC

	Sensitivity	Specificity	PPV	NPV	Accuracy
RASSF1A	40%	86%	74.07%	58.90%	63.0%
SOCS1	38%	80%	65.52%	56.34%	59.0%
AFP	68%	81%	78.16%	71.68%	74.5%
RASSF1A/AFP	73%	79%	77.66%	74.53%	76.0%
SOCS1/AFP	75%	78%	77.32%	75.73%	76.5%
RASSF1A/SOCS1/AFP	86%	75%	77.48%	84.27%	80.5%

Positive predictive value; PPV, Negative predictive value; NPV.

3.3 The clinicopathological features and gene methylation patterns in HCC patients (Table 4)

Analyzing the correlation between RASSF1A and SOCS-1 genes methylation status and the clinicopathological features revealed close association between promoter methylation of RASSF1A gene and age, tumor size, vascular invasion and AFP. Also, we found that SOCS-1 gene methylation status was significantly associated with tumor size and AFP.

3.4 Evaluation of RASSF1A and SOCS1 genes methylation as a potential HCC diagnostic marker for HCC (Table 5)

RASSF1A gene methylation identified HCC patients with 40% sensitivity, 86% specificity, 74.07% PPV and 58.9% NPV, while SOCS1 gene methylation identified HCC patients with 38% sensitivity, 80% specificity, 65.52% PPV and 56.34% NPV.

Considering the several combinations of both genes methylation and AFP in a panel, the best performance was accomplished by RASSF1A/ SOCS1/ AFP with 86% sensitivity, 75% specificity, 77.48% PPV and 84.27% NPV.

4. Discussion

HCC standout amongst the most widely recognized cancers around the world; its pathogenesis probably involves different environmental and genetic factors. Hepatitis B and C viruses are recognized as the major etiological factors associated with the occurrence of HCC, especially due to their induction of long term chronic inflammation [14].

In our study RASSF1A gene methylation was detected in peripheral blood of 14% of LC patients and 40% of HCC patients. On contrary, no RASSF1A gene methylation was detected in peripheral blood of healthy controls. Previous Chinese study revealed that RASSF1A gene methylation was detected in 17.5% of the cirrhotic Chinese patients [2]. Moreover methylation frequency was 64% among Chinese HCC patients [2], 70% among Taiwan HCC patients [15] and 86% in Thailand HCC patients [16].

Previous studies have revealed that the rate of RASSF1A gene methylation was up to 85, 95 and 100% in HCC tissues [8, 17]. What’s more, RASSF1A promoter methylation has been distinguished in various body fluids of various malignancies incorporating serum in HCC which showed its esteem for early diagnosis of tumors [8].

Interestingly, Jain et al. [18] revealed that the methylated RASSF1A gene could be identified in urine samples and is essentially higher in patients with cirrhosis and HCC, which recommends that RASSF1A gene methylation status in urine, can be used as a potential biomarker for liver oncogenesis.

Our results showed relationship between methylation of the RASSF1A gene promoter and patient age, these data are in comparable to the previous study of Li et al. [19] and Sugawara et al. [20] coworkers who found that methylation of RASSF1A gene was more prominent in old age patients with hepatocellular carcinoma. On the contrary, Xu et al. [21] who used a cutoff of 50 years old found that younger HCC patients showed higher levels of RASSF1A gene methylation.

However, Zhong et al. [17] and Feng et al. [22] found no association between methylation of the RASSF1A gene and age. The portion of hepatocytes which is already methylated with age at the RASSF1A gene promoter may turn out to be more vulnerable to oncogenic transformation [23].

In this study methylation status of the RASSF1A gene promoter in HCC patients was associated with tumor size and vascular invasion which is in agreement with the results of previous studies [21, 24, 25].

Dong et al. [2] found that RASSF1A gene methylation was related to higher histological grading, tumor stage and the occurrence of portal venous invasion. This result speculated that serum RASSF1A gene methylation status could serve as a noninvasive marker for HCC regression or progression.

Our data detected a significant association between the serum level of AFP and RASSF1A methylation status which is in concordance with the findings of Zhang et al. [15] who found a significant correlation between methylation status of tissue RASSF1A gene and serum AFP level in HCC patients.

This disagrees with the previous study of Yeo et al. [25] which showed absence of correlation between serum level of AFP and serum RASSF1A gene methylation status. Moreover, Hu et al. [8] found that there is no association between the serum RASSF1A gene methylation status and clinical biochemical parameters.

Aberrant promoter methylation of the tumor suppressor RASSF1A prompts numerous malignancies, which reveals that it assumes a critical role cancer development [26]. RASSF1A expression down regulation was not related to some conventional etiologies, such as HCV infection and alcohol consumption [27], which postulated that the inactivation of RASSF1A gene may be responsible for development of HCC.

RASSF1 gene is related to cell cycle regulation and cell proliferation. RASSF1A gene methylation may allow the damaged hepatocyte to proceed further into the cell cycle by inhibiting cyclin D1 and escaping G1 phase arrest [28] Also, it leads to the transcriptional silencing and affects its function as a tumor suppressing, therefore resulting in tumor formation [29]. Thus, the hypermethylation RASSF1A gene has been speculated for its important role in hepatocarcinogenesis and its potential as a HCC biomarker [27, 30].

The SOCS family is considered as a negative feedback protein of cytokine-induced signaling path-way [31]. SOCS-1 gene expression was inhibited through aberrant methylation of the CpG islands in various HCC studies [32, 33]. SOCS-1 gene promoter methylation has been documented in in serum samples of ankylosing spondylitis patients [34] and gastric cancer patients [35] which may indicate its value as a non-invasive marker in the diagnosis of tumors.

Our study shown a significant methylation difference of SOCS-1 gene in HCC as compared to control subjects and LC patients, these findings are in comparable to previous studies [6, 11]. Liu et al. [36] suggested that hypermethylation of SOCS-1 gene promoter region may play a crucial role in increasing the risk of HCC.

Nishida et al. [27] and Ko et al. [32] reported that SOCS-1 gene methylation was more predominant in HCV infected HCC compared to non-infected HCC and postulated that chronic infection by HCV may fasten the methylation process during hepatocarcinogenesis.

Okochi et al. [10] and Chu et al. [37] revealed that SOCS-1 gene methylation was frequently observed in HCCs due to cirrhosis rather than in those without cirrhosis. SOCS-1 gene was shown to be a negative regulator of JAK/STAT pathway and its suppression by hypermethylation which accelerates cell growth and HCC transformation of cirrhotic nodules [11].

Concerning correlation between SOCS-1 gene methylation status and clinico-pathological features of HCC, we found that SOCS-1 gene methylation was associated with tumor size and AFP which is in the same line with Ko et al. [32], Chu et al. [37] and Nomoto et al. [38] who found significant correlation between SOCS-1 gene methylation and tumor size. Also, SOCS-1 gene methylation status was correlated with HCC progression [27]. This finding suggested that inactivation of SOCS-1 gene methylation status may be an important factor in HCC carcinogenesis, particularly in cirrhotic patients.

A previous study of Oshimo et al. [39] revealed that SOCS-1 gene methylation was associated with advanced tumor stage and lymph node metastasis in gastric carcinoma.

Saelee et al. [16] found no significant association between gene methylation status and prognostic parameter. This discrepancy might be due to the sensitivity of MSP methods and different CpG sites.

Our finding was in accordance with previous studies of Ye et al. [3] and Allen Chan et al. [40] which revealed the benefit of combining measuring of AFP and RASSF1Al gene methylation status and combining measuring of AFP and SOCS-1 gene methylation status in HCC diagnosis than measuring AFP alone. Combined AFP and RASSF1A gene methylation status showed diagnostic sensitivity and specificity up to 73% and 79%, respectively. Also combined measuring of AFP and SOCS-1 gene methylation status showed diagnostic sensitivity and specificity upto 75% and 78%, respectively. While diagnostic sensitivity and specificity of AFP measurement alone were 68% and 81%, respectively.

Interestingly, three combination panels of RASSF1A /SOCS1/AFP had higher diagnostic performance in detection of HCC when compared with diagnostic performance of each parameter separately as well as any other two parameter combination panel. RASSF1A/SOCS1/AFP panel detected HCC with a sensitivity of 86% and specificity of 75%.

Circulating free DNA may result from circulating tumor cells or DNA fragments generated by tumor cell necrosis and apoptosis [41]. This hypothesis implies that RASSF1A and SOCS-1 genes methylation may originate in circulating tumor cells, which then leads to tumor metastasis.

In conclusion: This study speculated that methylation status of RASSF1A and SOCS-1 genes may have an important role in the process of human hepatocarcinogenesis and may be used as a non-invasive biomarker for the diagnosis HCC when combined with serum AFP.

Footnotes

Conflict of interest

None.

References

Ferlay

Shin

H.R.

Bray

Forman

Mathers

and Parkin

D.M.

, Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008, Int J Cancer 127 (2010), 2893–2917.

Dong

Zhang

Wang

and Chen

, Combination of serum RASSF1A methylation and AFP is a promising non-invasive biomarker for HCC patient with chronic HBV infection, Diagn Pathol 10 (2015), 133.

Zhu

Zhang

and Li

, The diagnostic value of suppressor of cytokine signaling-1 (SOCS-1) methylation for human hepatocellular carcinoma (HCC): a meta-analysis based on data from 15 studies, Int J Clin Exp Med 9 (2016), 15146–15156.

Mah

W.C.

and Lee

C.G.

, DNA methylation: potential biomarker in Hepatocellular Carcinoma, Biomark Rese 2 (2014), 5.

Lee

H.J.

Kim

J.H.

Lee

H.S.

Jang

J.J.

and Kang

G.H.

, Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis, Am J Pathol 163 (2003), 1371–1378.

Yang

Guo

Herman

J.G.

and Clark

D.P.

, Aberrant promoter methylation profiles of tumor suppressor genes in hepatocellular carcinoma, Am J Pathol 163 (2003), 1101–1107.

Dratviman-Storobinsky

Cohen

Frenkel

Merhavi-Shoham

S.D.

Binkovsky

Pe’er

and Goldenberg-Cohen

, The role of RASSF1A in uveal melanoma, Invest Ophthalmol Vis Sci 53 (2012), 2611–2619.

Chen

and Qiu

, Clinicopathological significance of RASSF1A reduced expression and hypermethylation in hepatocellular carcinoma, HepatolInt 4 (2010), 423–432.

Croker

B.A.

Kiu

and Nicholson

S.E.

, SOCS regulation of the JAK/STAT signaling pathway, Semin Cell Dev Biol 19 (2008), 414–422.

10.

Okochi

Hibi

Sakai

Inoue

Takeda

Kaneko

and Nakao

, Methylation-mediated silencing of SOCS-1 gene in hepatocellular carcinoma derived from cirrhosis, Clin Cancer Res 9 (2003), 5295–5298.

11.

Yoshikawa

Matsubara

Qian

G.S.

Jackson

Groopman

J.D.

Manning

J.E.

Harris

C.C.

and Herman

J.G.

, SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity, Nat Genet 28 (2001), 29–35.

12.

Liu

T.C.

Lin

S.F.

Chang

J.G.

Yang

M.Y.

Hung

S.Y.

and Chang

C.S.

, Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia, Br J Haematol 123 (2003), 654–661.

13.

Wong

Tsang

Lai

P.B.S.

K.F.

and Johnson

P.J.

, Frequent loss of chromosome 3p and hypermethylation of RASSF1A in cholangiocarcinoma, J Hepatol 37 (2002), 633–639.

14.

Jiang

B.G.

Yang

Liu

F.M.

Yang

Zhao

L.H.

Yuan

S.X.

Wang

R.Y.

Zhang

and Zhou

W.P.

, SOCS3 Genetic Polymorphism Is Associated With Clinical Features and Prognosis of Hepatocellular Carcinoma Patients Receiving Hepatectomy, Medicine 94 (2015), e1344.

15.

Zhang

Y.J.

H.C.

Shen

Ahsan

Tsai

W.Y.

Yang

H.I.

Wang

L.Y.

Chen

S.Y.

Chen

C.J.

and Santella

R.M.

, Predicting hepatocellular carcinoma by detection of aberrant promoter methylation in serum DNA, Clin Cancer Res 13 (2007), 378–384.

16.

Saelee

Chuensumran

Wongkham

Chariyalertsak

Tiwawech

and Petmitr

, Hypermethylation of Suppressor of Cytokine Signaling 1 in Hepatocellular Carcinoma Patients, Asian Pacific J Cancer Prev 13 (2012), 3489–3493.

17.

Zhong

Yeo

Tang

M.W.

Wong

Lai

P.B.

and Johnson

P.J.

, Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas, Clin Cancer Res 9 (2003), 3376–3382.

18.

Jain

Xie

Boldbaatar

Lin

S.Y.

Hamilton

J.P.

Meltzer

S.J.

Chen

S.H.

C.T.

Block

T.M.

Song

and Su

Y.H.

, Differential methylation of the promoter and first exon of the RASSF1A gene in hepatocarcinogenesis, Hepatol Res 45 (2015), 1110–1123.

19.

Zhang

Yang

Hao

and Li

, Promoter hypermethylation of DNA damage response genes in hepatocellular carcinoma, Cell Biol. Int 36 (2012), 427–432.

20.

Sugawara

Haruta

Sasaki

Watanabe

Tsunematsu

Kikuta

and Kaneko

, Promoter hypermethylation of the RASSF1A gene predicts the poor outcome of patients with hepatoblastoma, Pediatr Blood Cancer 49 (2007), 240–249.

21.

Wang

Han

Luo

and Zheng

, Quantitative analysis of RASSF1A promoter methylation in hepatocellular carcinoma and its prognostic implications, Biochem Biophys Res Commun 438 (2013), 324–328.

22.

Feng

Stern

J.E.

Hawes

S.E.

Jiang

and Kiviat

N.B.

, DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection, Exp Mol Pathol 88 (2010), 287–292.

23.

Di Gioia

Bianchi

Destro

Grizzi

Malesci

Laghi

Levrero

Morabito

and Roncalli

, Quantitative evaluation of RASSF1A methylation in the non-lesional: Regenerative and neoplastic liver, BMC Cancer 6 (2006), 89.

24.

Agathanggelou

Cooper

W.N.

and Latif

, Role of the Ras-association domain family 1 tumor suppressor gene in human cancers, Cancer Res 65 (2005), 3497–3508.

25.

Yeo

Wong

W.L.

Lai

P.B.

Zhong

and Johnson

P.J.

, High frequency of promoter hypermethylation of RASSF1A in tumor and serum of patients with hepatocellular carcinoma, Liver Int 25 (2005), 266–272.

26.

Pfeifer

G.P.

Yoon

J.H.

Liu

Tommasi

Wilczynski

S.P.

and Dammann

, Methylation of the RASSF1A gene in human cancers, Biol Chem 383 (2002), 907–914.

27.

Nishida

Nagasaka

Nishimura

Ikai

Boland

C.R.

and Goel

, Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma, Hepatology 47 (2008), 908–918.

28.

T.H.

Kim

B.K.

Kim

M.S.

Kim

K.S.

Jung

and Park

Y.N.

, Aberrant CpG island hypermethylation in dysplastic nodules and early HCC of hepatitis B virus-related human multistep hepatocarcinogenesis, J Hepatol 54 (2011), 939–947.

29.

Wang

Chen

and Bi

, The prognostic value of RASSF1A promoter hypermethylation in non-small cell lung carcinoma: A systematic review and meta-analysis, Carcinogenesis 32 (2011), 411–416.

30.

Chan

K.C.A.

Lai

P.B.S.

Mok

T.S.K.

Chan

H.L.Y.

Ding

Yeung

S.W.

and Dennis Lo

Y.M.

, Quantitative Analysis of Circulating Methylated DNA as a Biomarker for Hepatocellular Carcinoma, Clinical Chemistry 54 (2008), 1528–1536.

31.

Dimitriou

I.D.

Clemenza

Scotter

A.J.

Chen

Guerra

F.M.

and Rottapel

, Putting out the fire: coordinated suppression of the innate and adaptive immune systems by SOCS1 and SOCS3 proteins, Immunol Rev 224 (2008), 265–283.

32.

Kim

S.J.

Joh

J.W.

Park

C.K.

Park

and Kim

D.H.

, CpG island hypermethylation of SOCS-1 gene is inversely associated with HBV infection in hepatocellular carcinoma, Cancer Lett 271 (2008), 240–250.

33.

Miyoshi

Fujie

Moriya

Shintani

Tsutsumi

Makuuchi

Kimura

and Koike

, Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma, J Gastroenterol 39 (2004), 563–569.

34.

Lai

N.S.

Chou

J.L.

Chen

G.C.

Liu

S.Q.

M.C.

and Chan

M.W.

, Association between cytokines and methylation of SOCS-1 in serum of patients with ankylosing spondylitis, Mol Biol Rep 41 (2014), 3773–3780.

35.

Chan

M.W.

Chu

E.S.

K.F.

and Leung

W.K.

, Quantitative detection of methylated SOCS-1, a tumor suppressor gene, by a modified protocol of quantitative real time methylation-specific PCR using SYBR green and its use in early gastric cancer detection, Biotechnol Lett 26 (2004), 1289–1293.

36.

Liu

Cui

L.H.

C.C.

and Zhang

, Association of APC, GSTP1 and SOCS1 promoter methylation with the risk of hepatocellular carcinoma: A meta-analysis, Eur J Cancer Prev 24 (2015), 470–483.

37.

Chu

P.Y.

Yeh

C.M.

Hsu

N.C.

Chang

Y.S.

Chang

J.G.

and Yeh

K.T.

, Epigenetic alteration of the SOCS1 gene in hepatocellular carcinoma, Swiss Med Wkly 140 (2010), w13065.

38.

Nomoto

Kinoshita

Kato

Otani

Kasuya

Takeda

Kanazumi

Sugimoto

and Nakao

, Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma, Br J Cancer 97 (2007), 1260–1265.

39.

Oshimo

Kuraoka

Nakayama

Kitadai

Yoshida

Chayama

and Yasui

, Epigenetic inactivation of SOCS-1 by CpG island hypermethylation in human gastric carcinoma, Int J Cancer 112 (2004), 1003–1009.

40.

Allen Chan

K.C.

Lai

P.B.S.

Mok

T.S.K.

Chan

H.L.Y.

Ding

Yeung

S.W.

and Dennis Lo

Y.M.

, Quantitative analysis of circulating methylated DNA as a biomarker for hepatocellular carcinoma, Clinical Chemistry 54 (2008), 1528–1536.

41.

Philipp

A.B.

Nagel

Stieber

Lamerz

Thalhammer

Herbst

and Kolligs

F.T.

, Circulating cell-free methylated DNA and lactate dehydrogenase release in colorectal cancer, BMC Cancer 14 (2014), 245.

RASSF1A and SOCS1 genes methylation status as a noninvasive marker for hepatocellular carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

Table 1 Primers used and the size of amplification products

Table 2 Clinical features among the different studied groups

2.2 Methylation-specific PCR analysis (MSP)

2.3 Statistical analysis

3. Results

3.1 Clinical features among the different studied groups (Table 2)

3.2 Frequency of methylation in HCC, LC and control (Table 3)

Table 5 Evaluation of RASSF1A and SOCS-1 genes methylation in detection of HCC

3.4 Evaluation of RASSF1A and SOCS1 genes methylation as a potential HCC diagnostic marker for HCC (Table 5)

4. Discussion

Footnotes

Conflict of interest

References

Table 1
Primers used and the size of amplification products

Table 2
Clinical features among the different studied groups

Table 5
Evaluation of RASSF1A and SOCS-1 genes methylation in detection of HCC