Aberrant STAT1 methylation as a non-invasive biomarker in blood of HCV induced hepatocellular carcinoma

Abstract

BACKGROUND:

Hepatocellular carcinoma (HCC) is one of the most common types of cancer in the world and a reason behind different oncogenes activation and tumor suppressor genes inactivation. Hyper-methylation of tumor suppressor genes including RASSF1a, GSTP1, p16, and APC cause gene silencing as well as tumor cell invasion. STAT 1 gene is a part of signaling cascade of JAK/STAT and any dysregulation in signaling has been implicated in tumor formation.

OBJECTIVE:

The current investigation focus on the methylation role of STAT1 gene as a non-invasive biomarker in the progression and diagnosis of hepatocellular carcinoma.

METHODS:

STAT1 gene methylation status in 46 HCV induced hepatocellular carcinoma patients and 40 non-HCC controls were examined by methylation specific PCR. STAT1 gene expression was examined by real time PCR and further validated by various bioinformatics tools.

RESULTS:

STAT1 methylation in HCV-induced HCC (67.4%) was significantly higher compared to the non-HCC controls ( $p<$ 0.01). However, mRNA expression of STAT1 gene in methylated groups was significantly lower compared to unmethylated groups ( $p<$ 0.05). Furthermore, insilco analysis of STAT1 validated our results and shown expression of STAT1 mRNA was lower in liver cancer with the median 24.3 ( $p=$ 0.085).

CONCLUSION:

After using peripheral blood samples we observed that STAT1 silencing caused by aberrant methylation could be used as potential non-invasive biomarker for the diagnosis of HCV induced hepatocellular carcinoma. We conclude that blood as a sample source could be used instead of biopsy for early detection of HCC.

Keywords

Hepatocellular carcinoma STAT1 cancer biomarker methylation CpG Island

Figure 1.

Schematic flow of the recruitment of study participants: All the cases were assessed for HCV and HBV viral profile by real time PCR; all selected HCC cases were positive for HCV and negative for HBV. All the subjects were recruited from Bait-ul-Sukon cancer hospital (Karachi, Pakistan). Samples were taken in EDTA tubes and stored at 4 ${}^{\circ}$ C for DNA extraction and further analysis. Commercially available DNA EpiTect (human), methylated and bisulfite converted (Qiagen, Germany) was used as control. Permission for the study was obtained from institution review board of Universi ty of Karachi and written informed consent form was obtained from each patient.

1. Introduction

Hepatocellular carcinoma (HCC) is one of the most common types of cancer in the world and a reason behind different oncogenes activation and tumor suppressor genes inactivation [1, 2]. Various oncogenes like cyclin D1, c-myc and $\beta$ -catenin are most dominating triggers in hepatocarcinogenesis [3, 4, 5]. Moreover, hyper-methylation of tumor suppressor genes including RASSF1a, GSTP1, p16, and APC cause gene silencing and can influence the process of DNA repair, angiogenesis, cell cycle regulation, apoptosis as well as tumor cell invasion [6, 7, 8, 9, 10, 11]. Etiological factors linked with hepatocarcinogenesis are chronic hepatitis B virus or hepatitis C virus, responsible for approximately 80% of HCC cases by accelerating the methylation process [12, 13].

Currently alpha fetoprotein (AFP) assay, tumor biopsy and ultrasonography (US) are considered standard methods for the diagnosis of HCC [14]. But due to low sensitivity, these both are not satisfactory methods for early detection of HCC [15]. Moreover, biopsy is considered invasive technique with a high risk of seeding tumor along the biopsy tract therefore; there is urgent need for the discovery of new effective sample source for the detection of HCC.

STAT 1 gene is a part of signaling cascade of JAK/ STAT [16] and any dysregulation in signaling has been implicated in tumor formation. STAT1 controls several cellular processes, such as the activity of antimicrobials, cell proliferation and has vital role in immune responses [17]. Previous Study shows that promotor hyper-methylation of STAT1 was observed in cervical cancer and gastric cancer patients [18, 19]. STAT1 function is still mystery in cancer biology, most of the research reveals tumor promoter effects of STAT1 while other evidence shows tumor suppressor role in cancer [16]. Another study revealed that STAT1 methylation in tumor tissue cause ovarian cancer [20]. Most of the studies were conducted on tissue samples however no study has been reported to find the correlation between STAT1 hypermethylation and expression in peripheral blood samples of HCV induced HCC. To the best of our knowledge present study is first to demonstrate the STAT1 gene silencing by hypermethylation in peripheral blood samples. That could be used as potential noninvasive biomarker for the diagnosis of HCV induced HCC.

The present study aimed to investigate the methylation status of STAT1 in peripheral blood of HCV induced HCC patients and cancer free controls by methylation specific polymerase chain reaction (MSPCR). Moreover, explore the correlation between STAT1 methylation and mRNA expression through real time RT PCR and the bioinformatics tool human protein atlas.

2. Materials and methods

2.1 Study subjects

A total of 57 HCC samples were used in this study. Among them, 11 patients were excluded under the exclusion criteria (Fig. 1). Remaining 46 HCV-induced HCC (28 males, 18 females; mean age 51.2 $\pm$ 10.9; range 19–79 years) cases and 40 non-HCC controls (19 males, 21 females; mean age 34.05 $\pm$ 12.08; range 18–62) were included in this study (Fig. 1). All the subjects were recruited from Bait-ul-Sukon cancer hospital (Karachi, Pakistan). Written informed consent form was obtained from each patient and was approved by ethical review committee of University of Karachi.

Table 1
Primer sequences and conditions of STAT1 genes for MSP-PCR

Gene	Primers	Product size	Cycles
MSP Primers
Methylated (sense)	5 ${}^{\prime}$ -TATTTTCGGTATTCGGAATTTTAC-3 ${}^{\prime}$	113	35
Methylated (antisense)	5 ${}^{\prime}$ -GAACAAAAACGACCCAACGC-3 ${}^{\prime}$
Unmethylated (sense)	5 ${}^{\prime}$ -ATTTTTGGTATTTGGAATTTTATGG-3 ${}^{\prime}$	116	35
Unmethylated (antisense)	5 ${}^{\prime}$ -AACCAAACAAAAACAACCCAACA-3 ${}^{\prime}$
Real time RT- Primers
STAT1 Forward	5 ${}^{\prime}$ -GCAGGTTCACCAGCTTTATGA-3 ${}^{\prime}$
STAT1 Reverse	5 ${}^{\prime}$ -TGAAGATTACGCTTGCTTTTCCT-3 ${}^{\prime}$
GAPDH Forward	5 ${}^{\prime}$ -CCACTCCTCCACCTTTGAC-3 ${}^{\prime}$
GAPDH	5 ${}^{\prime}$ -ACCCTGTTGCTGTAGCCA-3 ${}^{\prime}$

2.2 DNA extraction and bisulfite modification

Genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Germany) as per manufacturer protocol. The quantity and purity of extracted DNA checked by NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific, Copenhagen, Denmark) and agarose gel electrophoresis. Extracted DNA (1 $\mu$ g) was subjected to bisulfite treatment for conversion of unmethylated cytosine to uracil without affecting the methyl cytosine bases by using EpiTect Bisulfite Kit (Qiagen, Germany). Briefly, prepare bisulfite reactions by adding extracted DNA, RNase Free water, bisulfate mix, and DNA protect buffer. Performed the bisulfite reactions using C1000™ thermal cycler (Bio Rad, USA), followed by 95 ${}^{\circ}$ C for 5 min, 60 ${}^{\circ}$ C for 25 min, 95 ${}^{\circ}$ C for 5 min, 60 ${}^{\circ}$ C for 85 min, 95 ${}^{\circ}$ C for 5 min, 60 ${}^{\circ}$ C for 175 min, and 20 ${}^{\circ}$ C for 10 min. Bisulfite converted DNA was loaded on 2% agarose gel with a100 bp ladder and result were analyzed by Gel documentation system (Bio-Rad, USA).

2.3 Methylation-specific PCR (MSP)

The bisulfite converted DNA is used as a template using primer specific for either methylated or unmethylated sequences and amplified by using EpiTect MSP kit (Qiagen, Germany) [20] Table 1. Negative and EpiTect positive control DNA (Qiagen, Germany) were included in each set of reaction. Amplified product was loaded on 2% agarose gels and visualized by gel documentation system (Bio-Rad, USA).

2.4 RNA extraction and RT PCR

RNA was extracted using QIAamp RNA blood mini Kit (Qiagen, Germany) as per manufacturer protocol. Further DNA digestion was performed using RNase-Free DNase set (Qiagen, Germany), that provides efficient on-column digestion of DNA. Total RNA was reverse transcribed by Revert Aid First Strand cDNA synthesis kit (Thermo Scientic, USA). STAT1 mRNA levels were assessed by real time RT-PCR using syber-green chemistry. After that, cDNA was added to primers and Ultra-Fast SYBR Green (Agilent, USA) and amplified using CFX96 thermal cycler (Bio Rad, USA). In each case cyclic threshold were obtained and expression levels were then evaluated by the $\Delta\Delta$ CT method. Expression of GAPDH was used as the endogenous control. Primers of STAT1 and GAPDH are shown in Table 1 [21]. Non template control and no RT control were included in each set of reaction.

2.5 Insilico analysis of STAT1 gene

STAT-1 genes interacting partners were investigated using String 11 database (http://stringdb.org/) and The SIGnaling Network Open Resource (Singor) 2.0 (https: //signor.uniroma2.it/).

RNA and protein expression of STAT1 gene in different types of cancer including liver cancer and STAT1 gene expression in different types of liver cells were evaluated by the human protein Atlas (https://www. proteinatlas.org/). STAT1 methylation role has been identified by PTMcode2 (https://ptmcode.embl.de/ search.cgi?int=10959&protein=DKFZp686B04100).

Figure 2.

(a) The clinical history of HCC and healthy individuals: Elevated and suppressed levels of aminotransferase (ALT), alkaline Phosphatase (ALP), gama GT (GGT), total bilirubin, direct bilirubin, prothrombin time (PT), hemoglobulin (HG), platelets and albumin were expressed in mean $\pm$ SD. Significant differences were find out by Independent T test, * $p<$ 0.05, ** $p<$ 0.01 as compared to control. (b) Gel analysis of genomic DNA obtained from samples. Lane 1 indicates ladder and lane 2–6 indicate extracted DNA. The whole genomic DNA obtained was run on 1% agarose gel electrophoresis. (c) Gel analysis of Bisulfite converted DNA. Bisulfite converted genomic DNA of HCC and non-tumor samples were run on 2% agarose gel electrophoresis with a 100 bp marker. Where T represented HCC samples: C, non-tumors samples: L, Ladder. (d) STAT1 Gene Methylation specific PCR (MSP): Analysis of 46 primary HCC samples and 40 non-tumor samples was performed by using same set of methylated and unmethylated primers. T represented HCC samples: C, non-tumors samples: L, molecular weight control: NTC, non-template control: P, Positive control of methylated DNA: M, Methylated allele: U, Unmethylated allele.

2.6 Statistical analysis

Data were illustrated as mean $\pm$ SD. Results of clinic-pathological data were analyzed by using Independent T test using SPSS version 20. The chi-square test was used to estimate the frequency of methylation in HCV induced HCC patients and controls. Correlation between the methylation status and mRNA expression levels of STAT1 was analyzed using Mann-Whitney U test. $P<$ 0.05 were taken as statistically significant.

3. Results

3.1 Clinico-pathological study

A total of 46 HCV induced HCC cases and 40 controls were included in this study (Fig. 2a). They underwent clinically indicated blood test and liver function test. Values of alanine aminotransferase (ALT), alkaline phosphatase (ALP), gama GT (GGT), total bilirubin, direct bilirubin and prothrombin time (PT) in HCC patients was higher than that in the control group ( $p<$ 0.01). However, suppressed level of HG ( $p<$ 0.05), platelets ( $p<$ 0.01) and albumin ( $p<$ 0.01) in HCC was observed as compared to control.

Table 2
Methylation results of STAT1 gene in disease and normal condition

Diseases/normal	Total number of samples	Methylated samples (%)	Unmethylated samples (%)	Pearson x2 or Fishers’s exact $p$ value	High (%)	Low (%)	Pearson x2 or Fishers’s exact $p$ value
	STAT1 methylation			STAT1 mRNA expression
HCV-induced HCC	$N=$ 46	31 (67.4)	15 (32.6)	$<$ 0.01	16 (34.7)	30 (65.2)	$<$ 0.01
Healthy controls	$N=$ 40	4 (10)	36 (90)	$<$ 0.01	36 (90)	04 (10)	$<$ 0.01

Figure 3.

STAT1 methylation, Co-evolution and alignment: Schematic diagram showed the domain of STAT1 and its various PTMs.

3.2 Bisulfite conversion by agarose gel electrophore- sis and Methylation specific PCR of STAT1 gene

All patients’ characteristics were collected from the patients’ record (Fig. 2a) and the DNA was extracted from peripheral blood of HCC patients and healthy controls (Fig. 2b). Extracted genomic DNA was then subjected to bisulfite conversion and run on 2% agarose gel with a 100 bp marker. Bisulfite-converted DNA showed smearing pattern from 1,500 bp down to 100 bp shown in (Fig. 2c). To determine the STAT1 methylation, bisulfite-converted DNA was amplified with primers specific for CpG island promoter region of the STAT1 gene (Fig. 2d). According to our result aberrant DNA methylation in STAT1 was detected in 31 of 46 (67.4%) cases. In contrast 36 (90%) healthy control show no methylation signal Table 2. Result shows that STAT1 methylation was significantly higher in HCV-induced HCC as compared to healthy controls ( $p<$ 0.01). Test was validated by commercially available methylated positive control of STAT1. Each run carried out with non-template control which shows no methylation signal.

3.3 STAT1 methylation by PTMcode2

STAT1 methylation was examined by PTMcode2 on residue R31 with 96% conservation and its association with 6 other PTM. R31 methylation are associated with phosphorylation on residue Y68, S727, Y170; acetylation on residue K410, K413 and ubiquitination on K410 (Fig. 3).

Figure 4.

STAT1 protein expressions in different types of cancer including liver cancer through the human protein Atlas (https://www.proteinatlas.org/). (a) STAT1 Protein expression in different types of cancers against anti-STAT1 rabbit antibody HPA000931. (b) STAT1 protein expression in various cancers against anti-STAT1 HPA000982 antibody in nuclei and cytoplasm. (c) STAT1 protein expression in various cases of most cancer tissues against STAT1 antibody CAB004049.

Figure 5.

(a) Expression of STAT1 RNA in several cancers including liver cancer through the human protein Atlas (https://www.proteinatlas.org/), decreased expression of RNA was noted in liver cancer. (b) Wet lab analysis shows decreased expression of STAT1 mRNA in HCV induced HCC as compared to normal controls, ** $P<$ 0.01. (c) Expression of STAT1 RNA in different cells of hepatocytes through human protein Atlas (https://signor.uniroma2.it/), STAT1 RNA was up regulated in Cholangiocytes c-14.

Figure 6.

(a) Interaction of STAT1 protein with other interacting proteins by STRING 11 (http://stringdb.org/). (b) Interactions of STAT1 protein with their interacting partners by SINGOR 2.0 (https://signor.uniroma2.it/).

3.4 STAT-1 mRNA expression and correlation with methylation

STAT1 RNA expression was examined by real time PCR. Our result shows that STAT1 mRNA expression level in HCV-induced HCC was significantly lower than that in normal individuals ( $p<$ 0.01, Fig. 5c). No RT control ensures that the isolated RNA was DNA free. Furthermore, we analyzed the correlation between methylation of STAT1 and mRNA expression level. The level of STAT1 mRNA was significantly lower in methylated groups as compared to unmethylated groups ( $p<$ 0.05).

3.5 Insilico study of STAT1 protein and RNA expression

RNA and protein expression of STAT1 was evaluated by the human protein atlas. Protein expression in lymphomas, gliomas, melanomas, testis, urothelial, renal, stomach, cervical and breast cancers showed moderate to strong cytoplasmic and nuclear anti-STAT1 rabbit antibody HPA000931 positivity (Fig. 4a and b). Selected cases of most cancer tissues showed moderate to strong cytoplasmic positivity against another STAT1 antibody CAB004049 (Fig. 4c). Prostate, endometrial, thyroid, liver cancer and carcinoid were mainly negative against CAB004049 (Fig. 4c). Over all three Anti-STAT1 antibodies HPA000931, HPA000982, CAB004049 shows decrease expression of STAT1 protein in liver cancer and most abundant in lymphoid tissues (Fig. 4a–c).

STAT1 RNA was highly expressed in head and neck cancer with the median 71.8 ( $p=$ 0.029), while in liver cancer with the median 24.3 ( $p=$ 0.085) (Fig. 5a and b). Among all liver cells STAT1 was highly expressed in cholangiocytes c-14 with $z$ -score 1.04, however its expression was not seen in Erythroid cells c-16 with $z$ -score 0.00 and lower expressed in B-cells c-13 ( $z$ -score 0.54) (Fig. 5c).

3.6 Identification of the target gene of STAT1 constructing its regulatory network

Functional association network of STAT-1 with neighboring partner was developed through STRING 11 and Signor 2.0 interaction network. STAT1 gene interacts with activated signaling proteins of the tyrosine signaling including JAK family kinases. The predicted functional partners of STAT1 gene with confidence score are JAK2-1 (0.998), IRF9 (0.997), MX1 (0.996), CREBBP (0.995), ISG15 (0.997), TYK2 (0.994), IFNAR1 (0.993), IFIT1 (0.992), DDX58 (0.992) (Fig. 6a), FGFR4 (0.1354), FGFR3 (0.1364), KIT (0.5622), MAPK14 (0.2197), PIAS4 (0.3534), SRC (0.3586), CAMK2B (0.2197), ISGF3 COMPLEX (0.2197), RELA (0.783), STAT2 (0.971), STAT6 (0.2197), JAK1/STAT1/ STAT3 (0.1364), MAPK8 (0.1364), PTPRG (0.1364), PTPN11 (0.3586), PIAS1 (0.5653), CAMK2G (0.2197), IRF2 (0.1364), PTPN2 (0.4576), PRKCD (0.2197), STAT1/STAT3 (0.1364), NfKb-p65/p50 (0.1364), EGFR (0.2817), IKBKE (0.337) and IFNGR2/IFNGR1 (0.2197). Indirect interactions were observed with CNTFR (0.1364), TBX21 (01364.), ORF6 (0.1364), Host translation inhibitor nsp1 (0.2197), M1 polymerization (0.1364), FANCC (0.379), non-structure protein 6 (0.4203) (Fig. 6b).

4. Discussion

Hypermethylation of tumor suppressor genes such as RASSF1a [9], SMPD3 [22], DOK1 [9] and P15-INK4b [23] causes gene silencing, which leads to the induction of HCC [24, 25]. STAT1 is major gene in the Janus Kinase/signal transducer and activator of transcription signaling pathway [26]. JAK/STAT pathway has potential role in different tumors [27, 28]. Studies have shown that STAT1 expression is decreased in numerous cancers including head and neck cancer, breast cancer, leukemia and multiple myeloma [29]. In addition, STAT1 has been reported to prevent the growth of neoplastic and benign cells by regulating the expression of anti-proliferative genes, for instance BCL-xL and caspases [30].

Our data showed that, promoter methylation in HCV induced HCC individuals was significantly higher ( $p<$ 0.01), while mRNA expression level was significantly lower than healthy individuals ( $p<$ 0.01). Previously it was reported that STAT1 gene act as tumor suppressor in HBV associated squamous cell carcinoma and silencing of STAT1 gene through promoter methylation results in squamous cell carcinoma [31].

We divided our study in two steps (1) wet lab analysis and (2) insilico analysis. Firstly, we compile clinicopathological parameters data of HCV associated HCC patients and healthy individuals. Total bilirubin, ALT, ALP, GGT, direct bilirubin and PT was significantly higher in HCV induced HCC patient ( $p<$ 0.01). Whereas low level of HG ( $p<$ 0.05), platelets and albumin ( $p<$ 0.01) in cases was observed in comparison to healthy individual (Fig. 2a and b).

After that we performed major part of research by extracting genomic DNA from HCV associated HCC patients and healthy controls. Then bisulfite conversion of DNA leads to DNA fragmentation and limited base pairing that results in appearance of smearing pattern from 1500 bp to 100 bp on agarose gel shown in Fig. 2a–c. Finally, methylation in promoter region of STAT1 gene and its expression was detected through MSP PCR and real time PCR respectively. Aberrant methylation in STAT1 gene was detected in 31 (67.4%) cases in contrast to healthy controls 4 (10%), ( $p<$ 0.01, Fig. 2d). However, STAT1 gene was transcriptionally silenced in 30 HCV induced HCC (65.2%) cases as compared to healthy individuals ( $p<$ 0.01) (Fig. 5b). Dry lab analysis provided healthy support for compiling our research which shows that STAT1 mRNA expression was significantly lower in liver cancer with a median 24.3 ( $p=$ 0.085) as compared to other cancers (Fig. 5a).

Furthermore, it is evaluated that STAT1 protein expression in liver cancer was lower with HPA000931, HPA000982 and CAB004049 antibodies (Fig. 4a–c). According to the results of human protein atlas, 2 of 12 patients with liver cancer have medium expression of STAT1 protein, 1 patient has low expression while 9 patients have no STAT1 protein expression (Fig. 4a–c).

Among all liver cells expression of STAT1 gene was higher in cholangiocytes c-14 with $z$ -score 1.04, and lower expression was detected in B-cells c-13 ( $z$ -score 0.54) however, no expression was seen in erythroid cells c-16 with $z$ -score 0.00 (Fig. 5c).

Moreover, the predicted functional partner of STAT1 gene as by String 11 and Singor 2.0 with high confidence score were JAK2, JAK1, IRF9, MX1, CREBBP, ISG15, TYK2, IFNAR1, IFIT1 and DDX58., FGFR4, FGFR3, KIT, MAPK14, PIAS4, SRC, CAMK2B, ISGF3 COMPLEX, RELA, STAT2, STAT6, JAK1/STAT1/STAT3, MAPK8, PTPRG, PTPN11, PIAS1, CAMK2G, IRF2, PTPN2, PRKCD, STAT1/STAT3, NfKb-p65/p50, TYK2, EGFR, IKBKE and IFNGR2/IFNGR1 (Fig. 6a and b). Cellular responses to cytokine (KITLG/SCF), interferon and other growth factors are mediated by the STAT1 signal transducer and transcription activator. Interferon binding to cell surface receptors activates protein kinases, resulting in the activation of Jak kinases and the phosphorylation of tyrosine to form a complex called ISGF3 transcription factor complex (Fig. 6). This complex reaches the nucleus and the phosphorylated STATs dimerize and interact with ISGF3G/IRF-9. ISGF3 binds to the IFN Stimulated Response Element (ISRE) allowing an antiviral transcription of IFN-stimulated (ISG) genes that drive the cell. STAT1 is phosphorylated on tyrosine and serine residues in response to type II IFN (IFN-gamma). It then forms an IFN-gamma-activated factor (GAF) homodimer, moves into the nucleus and links to the IFN gamma-activated sequence (GAS). This accelerates target gene expression, inducing an antiviral cellular state and making it responsive towards KIT and KITLG/SCF signaling. Furthermore, it mediates cellular responses to stimulated FGFR gene family that directly belongs to the STAT family transcription factors [32]. The ERK/MAPK pathway generally involves a series of adaptors (Shc, GRB2, Crk, etc.) that connect the receptor to a guanine nucleotide exchange factor (SOS, C3G, etc.). This transmits the signal to small proteins that attach to GTP (Ras, Rap1), which in turn activates the central cascade unit composed of MAPK family (Raf). They enter the cytosol and relocate in nucleus may cause phosphorylation of numerous transcription elements, resulting in variable gene expression [33]. Furthermore, in many cytokine receptor systems, JAKs and STATs are critical components; they regulate development, survival, differentiation, and pathogen resistance [34, 35]. Our finding demonstrates that aberrant methylation status of STAT1 may results in transcriptionally silencing of gene and STAT1 promoter methylation may be a potential biomarker for HCC diagnosis.

5. Conclusion

Present study provided strong evidence that aberrant promoter methylation is the primary mechanism of STAT1 silencing in HCV induced HCC. Analysis of STAT1 methylation could be a potent noninvasive biomarker for HCC diagnosis. For future studies, it is suggested that the probability of tumor formation in liver might be reduced by using demethylating reagents for reducing the STAT1 hypermethylation.

Abbreviations

Hepatocellular carcinoma (HCC), Methylation specific PCR (MSP PCR)

Acknowledgments

We thank the staff of the Molecular Genetics Laboratory, Dr. Ziauddin hospital for helpful discussions and support.

Author contributions

RK and FN Conceived and designed the experiments, UZ and NNS performed the experiment. UZ and FN analyzed and interpreted the data, RK and NNS revised for important intellectual content and contributed reagents, material, analysis tools or data, UZ and RK prepare the manuscript.

Footnotes

Conflict of interest

None.

References

Kanda

Sugimoto

and Kodera

, Genetic and epigenetic aspects of initiation and progression of hepatocellular carcinoma, World J Gastroenterol 21 (2015), 10584–10597.

Llinàs-Arias

and Esteller

, Epigenetic inactivation of tumor suppressor coding and non-coding genes in human cancer: An update, Open Biol [Internet] 7 (2017), 170152.

Stine

Z.E.

Walton

Z.E.

Altman

B.J.

Hsieh

A.L.

and Dang

C.V.

, MYC, metabolism, and cancer, Cancer Discov 5 (2015), 1024–1039.

S.Y.

Lan

S.H.

S.R.

Chiu

Y.C.

Lin

X.Z.

I.J.

et al., Hepatocellular carcinoma-related cyclin D1 is selectively regulated by autophagy degradation system, Hepatology 68 (2018), 141–154.

Khalaf

A.M.

Fuentes

Morshid

A.I.

Burke

M.R.

Kaseb

A.O.

Hassan

et al., Role of Wnt/β-catenin signaling in hepatocellular carcinoma, pathogenesis, and clinical significance, J Hepatocell Carcinoma 5 (2018), 61–73.

Anestopoulos

Voulgaridou

G.P.

Georgakilas

A.G.

Franco

Pappa

and Panayiotidis

M.I.

, Epigenetic therapy as a novel approach in hepatocellular carcinoma, Pharmacol Ther 145 (2015), 103–119.

Pfeifer

, Defining driver DNA methylation changes in human cancer, Int J Mol Sci 19 (2018), 1166.

Wang

L.H.

C.F.

Rajasekaran

and Shin

Y.K.

, Loss of tumor suppressor gene function in human cancer: An overview, Cell Physiol Biochem 51 (2018), 2647–2693.

Araújo

O.C.

Rosa

A.S.

Fernandes

Niel

Villela-Nogueira

C.A.

Pannain

et al., RASSF1A and DOK1 promoter methylation levels in hepatocellular carcinoma, cirrhotic and non-cirrhotic liver, and correlation with liver cancer in Brazilian patients, PLoS One 11 (2016), e0153796.

10.

Cai

Chen

and Mao

, Clinical significance and association of GSTP1 hypermethylation with hepatocellular carcinoma: A meta-analysis, J Cancer Res Ther 14 (2018), S486–S489.

11.

Zhang

Huang

Sui

Zhong

Yang

et al., Association between gene methylation and HBV infection in hepatocellular carcinoma: A meta-analysis, J Cancer 10 (2019), 6457–6465.

12.

Ramadori

Cubero

Liedtke

Trautwein

and Nevzorova

, Alcohol and hepatocellular carcinoma: Adding fuel to the flame, Cancers (Basel) 9 (2017), 130.

13.

Han

Zhou

Xie

Sun

Wei

Nie

et al., Association between the methylation of the STAT1 and SOCS3 in peripheral blood and gastric cancer, J Gastroenterol Hepatol 35 (2020), 1347–1354.

14.

Omata

Cheng

A.L.

Kokudo

et al., Asia-Pacific clinical practice guidelines on the management of hepatocellular carcinoma a 2017 update, Hepatology International 11 (2017), 317–370.

15.

Tzartzeva

Obi

Rich

N.E.

et al., Surveillance imaging and alpha fetoprotein for early detection of hepatocellular carcinoma in patients with cirrhosis: A meta-analysis, Gastroenterology 154 (2018), 1706–1718.

16.

Zhang

and Liu

, STAT1 in cancer: Friend or foe? Discov Med 24 (2017), 19–29.

17.

Meissl

Macho-Maschler

Müller

and Strobl

, The good and the bad faces of STAT1 in solid tumours, Cytokine 89 (2017), 12–20.

18.

Jha

A.K.

Sharma

Nikbakht

Jain

Sehgal

Capalash

et al., A comparative analysis of methylation status of tumor suppressor genes in paired biopsy and serum samples from cervical cancer patients among north indian population, Russ J Genet 52 (2016), 226–230.

19.

Han

Zhou

Xie

Sun

Wei

Nie

et al., Association between the methylation of the STAT1 and SOCS3 in peripheral blood and gastric cancer, J Gastroenterol Hepatol 35 (2020), 1347–1354.

20.

Chang

M.C.

Chiang

Y.C.

C.M.

Chen

Y.L.

Chen

C.A.

Cheng

W.F.

et al., New primers for methylation-specific polymerase chain reaction enhance specificity of detecting STAT1 methylation, Taiwan J Obstet Gynecol 51 (2012), 43–49.

21.

Chen

Wang

Xie

and Wang

, STAT1 negatively regulates hepatocellular carcinoma cell proliferation, Oncol Rep 29 (2013), 2303–2310.

22.

Kwee

S.A.

Peplowska

Tiirikainen

Song

M.A.

and Wong

L.L.

, Abstract 3316: Hypermethylation at the SMPD3 promoter is frequently detected in hepatocellular carcinoma tumor tissue and corresponding plasma circulating DNA, and potentially differs by tumor subtype, Cancer Res 78(13 Supplement) Supplement (2018), 3316–3316.

23.

Dong

and Wang

, Aberrant DNA methylation in hepatocellular carcinoma tumour suppression review, Oncology Letters 8 (2014), 963–968.

24.

Anestopoulos

Voulgaridou

G.P.

Georgakilas

A.G.

Franco

Pappa

and Panayiotidis

M.I.

, Epigenetic therapy as a novel approach in hepatocellular carcinoma, Pharmacol Ther 145 (2015), 103–119.

25.

Kim

S.Y.

Han

Y.K.

Song

J.M.

Lee

C.H.

Kang

J.M.

et al., Aberrantly hypermethylated tumor suppressor genes were identified in oral squamous cell carcinoma (OSCC), Clin Epigenetics 11 (2019), 116.

26.

Murray

P.J.

, The JAK-STAT signaling pathway: Input and output integration, J Immunol 178 (2007), 2623–2629.

27.

Pencik

Pham

H.T.

and Schmoellerl

, JAK-STAT signaling in cancer: From cytokines to non-coding genome, Cytokine 87 (2016), 26–36.

28.

Tang

Yuan

Song

Chen

Tan

and Li

, Association analyses of the JAK/STAT signaling pathway with the progression and prognosis of colon cancer, Oncol Lett 17 (2019), 159–164.

29.

Calò

Migliavacca

Bazan

Macaluso

Buscemi

Gebbia

et al., STAT proteins: From normal control of cellular events to tumorigenesis: Stat proteins in tumorigenesis, J Cell Physiol 197 (2003), 157–168.

30.

Battle

and Frank

, The role of STATs in apoptosis, Curr Mol Med 2 (2002), 381–392.

31.

Bing

Zhu

Liu

and Li

, Glucocorticoid-induced S-adenosylmethionine enhances the interferon signaling pathway by restoring STAT1 protein methylation in hepatitis B virus-infected cells, J Biol Chem 289 (2014), 32639–32655.

32.

Arteaga

C.L.

and Engelman

J.A.

, ERBB receptors: From oncogene discovery to basic science to mechanism-based cancer therapeutics, Cancer Cell 25 (2014), 282–303.

33.

Romeo

Zhang

and Roux

P.P.

, Regulation and function of the RSK family of protein kinases, Biochem J 441 (2012), 553–569.

34.

Sansone

and Bromberg

, Targeting the interleukin-6/Jak/ stat pathway in human malignancies, J Clin Oncol 30 (2012), 1005–1014.

35.

Seif

A.A.

Aly

H.H.

Elzoghby

D.M.

Elbreedy

A.M.

and Lotfy

, Aberrant p16 methylation as an early diagnostic marker in blood of hepatocellular carcinoma patients, Egypt J Med Hum Genet 20 (2019), 27.

Aberrant STAT1 methylation as a non-invasive biomarker in blood of HCV induced hepatocellular carcinoma

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

2. Materials and methods

2.1 Study subjects

Table 1 Primer sequences and conditions of STAT1 genes for MSP-PCR

2.3 Methylation-specific PCR (MSP)

2.4 RNA extraction and RT PCR

2.5 Insilico analysis of STAT1 gene

3. Results

3.1 Clinico-pathological study

Table 2 Methylation results of STAT1 gene in disease and normal condition

3.3 STAT1 methylation by PTMcode2

3.5 Insilico study of STAT1 protein and RNA expression

3.6 Identification of the target gene of STAT1 constructing its regulatory network

4. Discussion

5. Conclusion

Abbreviations

Acknowledgments

Author contributions

Footnotes

Conflict of interest

References

Table 1
Primer sequences and conditions of STAT1 genes for MSP-PCR

Table 2
Methylation results of STAT1 gene in disease and normal condition