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

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 β -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

3. Results

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 ( 0.01), while mRNA expression level was significantly lower than healthy individuals ( 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 ( 0.01). Whereas low level of HG ( 0.05), platelets and albumin ( 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%), ( 0.01, Fig. 2d). However, STAT1 gene was transcriptionally silenced in 30 HCV induced HCC (65.2%) cases as compared to healthy individuals ( 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.