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The JAK2/STAT pathway is hyperactivated in many cancers, and such hyperactivation is associated with a poor clinical prognosis and drug resistance. The mechanism regulating JAK2 activity is complex. Although translocation of JAK2 between nucleus and cytoplasm is an important regulatory mechanism, how JAK2 translocation is regulated and what is the physiological function of this translocation remain largely unknown. Here, we found that protease SENP1 directly interacts with and deSUMOylates JAK2, and the deSUMOylation of JAK2 leads to its accumulation at cytoplasm, where JAK2 is activated. Significantly, this novel SENP1/JAK2 axis is activated in platinum-resistant ovarian cancer in a manner dependent on a transcription factor RUNX2 and activated RUNX2/SENP1/JAK2 is critical for platinum-resistance in ovarian cancer. To explore the application of anti-SENP1/JAK2 for treatment of platinum-resistant ovarian cancer, we found SENP1 deficiency or treatment by SENP1 inhibitor Momordin Ic significantly overcomes platinum-resistance of ovarian cancer. Thus, this study not only identifies a novel mechanism regulating JAK2 activity, but also provides with a potential approach to treat platinum-resistant ovarian cancer by targeting SENP1/JAK2 pathway.

Summary Background and aims Immune response against hepatitis B virus (HBV) infection is an important risk factor for the development of hepatocellular carcinoma (HCC). Studies have reported that interleukin 22 (IL-22) exhibits both protective and pathological properties in liver diseases. Our aim was to explore the importance of IL-22 in the development of HCC, and to characterize the relationship between IL-22 levels and the prognosis of HCC. Methods Totally, 136 liver biopsy specimens from 46 patients with chronic hepatitis B (CHB), 37 with atypical hyperplasia (AH), 53 with HCC, patient-matched tumors and peritumoral surgical specimens from 56 HCC patients included in the study. The expression of IL-22 and CD8 was evaluated by immunochemistry. Corresponding serum samples were collected from 30 CHB, 30 AH, and 30 HCC patients. IL-22 expression was determined by an enzyme linked immunosorbent assay. Results Liver-infiltrating IL-22+ cells increased in a stepwise manner from CHB to AH and HCC (CHB vs. AH, P = 0.002; AH vs. HCC, P = 0.010), whereas a decreasing trend was observed for CD8+ T cells (CHB vs. AH, P = 0.031; AH vs. HCC, P = 0.652). Serum IL-22 levels also increased from CHB to AH and HCC (CHB vs. AH, P = 0.024; AH vs. HCC, P = 0.026). Tumor-infiltrating IL-22+ cells and serum IL-22 were associated with histologic grade (P = 0.024 and P = 0.033). Additionally, CD8+ T cells correlated with tumor size (P = 0.032). Furthermore, the high intratumoral IL-22+ cell group and high serum IL-22 group showed lower overall survival (OS; P = 0.001, P = 0.017) and disease-free survival (DFS; P = 0.005, P Conclusions These findings indicate that IL-22 promotes the progression of HCC in CHB patients. High tumor-infiltrating IL-22+ cells and serum IL-22 levels are thought to be unfavorable prognostic indicators for HCC.

Sex as a physiologic factor has a strong association with the features of metabolic syndrome. Our previous study showed that loss of the voltage-gated proton channel Hv1 inhibits insulin secretion and leads to hyperglycemia and glucose intolerance in male mice. However, there are significant differences in blood glucose between male and female Hv1-knockout (KO) mice. Here, we investigated the differences in glucose metabolism and insulin sensitivity between male and female KO mice and how sex steroids contribute to these differences. We found that the fasting blood glucose in female KO mice was visibly lower than that in male KO mice, which was accompanied by hypotestosteronemia. KO mice in both sexes exhibited higher expression of gluconeogenesis-related genes in liver compared with WT mice. Also, the livers from KO males displayed a decrease in glycolysis-related gene expression and an increase in gluconeogenesis-related gene expression compared with KO females. Furthermore, exogenous testosterone supplementation decreased blood glucose levels in male KO mice, as well as enhancing insulin signaling. Taken together, our data demonstrate that knockout of Hv1 results in higher blood glucose levels in male than female mice, despite a decreased insulin secretion in both sexes. This sex-related difference in glucose homeostasis is associated with the glucose metabolism in liver tissue, likely due to the physiological levels of testosterone in KO male mice.

Chemokines and their receptors have been proposed to play important roles in tumor progression and metastasis. To investigate their roles in the progression of primary and metastatic malignant liver tumors and their prognosis, we compared expression profiles of CXCL12/CXCR4, CCL20/CCR6, and CCL21/CCR7 in hepatocellular carcinoma (HCC) and colorectal liver metastases (CRLM). Immunohistochemistry was used to analyze the expression levels of the chemokine/chemokine receptor pairs in 29 HCC and 11 CRLM specimens and adjacent non-cancerous tissues, and correlations with clinicopathological variables and overall survival were determined. CCL20/CCR6 expression was higher in HCC than in adjacent non-cancerous tissues. High CCR6 expression in HCC was negatively associated with 5-year survival rate and was an independent prognostic factor for overall survival of HCC patients, whereas differences were not observed between CRLM and adjacent tissues. Furthermore, significantly higher expression of CCL21/CCR7 was found in CRLM than in HCC. In summary, the CCL20/CCR6 axis was elevated in HCC but not in CRLM, whereas the CCL21/CCR7 axis was elevated in CRLM but not in HCC.

LDLRAD4 was previously identified and shown to be connected with psychiatric disorders. The structure of LDLRAD4 protein is similar to that of TMEPAI protein, which is overexpressed in many tumors. However, it is still unknown whether LDLRAD4 is involved in tumorigenesis. In this study, the potential role of LDLRAD4 in tumorigenesis was investigated. LDLRAD4 is elevated in hepatic cancer cells and tumor tissues, and expression of LDLRAD4 promotes hepatic cancer cell HepG2 and SMMC-7721 proliferation and migration. LDLRAD4 interacts Nedd4 to promote cell proliferation and migration and negatively regulates the TGF-β signaling. Furthermore, immunofluorescence microscopy analysis indicates that LDLRAD4 is localized to the lysosome and association with Nedd4 is necessary for its intracellular transport to the lysosome. In addition, depletion of LDLRAD4 in HepG2 liver cancer cells inhibited tumorigenesis in nude mice. These results reveal an oncogenic role of LDLRAD4 in tumorigenesis through its association with Nedd4.

The aim of the study was to explore the clinical significance of let-7 expression in hepatocellular carcinoma (HCC). A PCR array was conducted to screen for let-7 expression in early-stage HCC. Next, the deregulation of let-7 was confirmed by quantitative real-time RT-PCR (qRT-PCR) in another set of liver tissues, including normal control (NC), chronic hepatitis (CH), liver cirrhosis (LC), HCC, and adjacent nontumor (NT) tissues. In addition, as the potential target mRNA of let-7, alpha 2(I) collagen (COL1A2) mRNA was also quantified in the above liver tissues. Finally, an association study comparing let-7 and COL1A2 and their clinical significance in HCC was conducted. PCR array analysis revealed that the expression levels of let-7a/7b/7c were significantly downregulated in early-stage HCC compared to those in NT tissues. As compared to NC samples, qRT-PCR further confirmed that let-7a/7b/7c/7e were significantly upregulated in CH, LC, and NT tissues, while there were no significant differences in expression between the HCC and NC groups. Although COL1A2 may be the target mRNA of let-7, only let-7c expression was inversely correlated with COL1A2 mRNA expression in CH tissues. In HCC tissues, levels of let-7a/7b/7c/7e were positively correlated with that of COL1A2 mRNA. The clinical significance study revealed that elevated let-7a expression was significantly correlated with serosal and vein invasion, while elevated let-7c expression was significantly correlated with vein invasion and advanced TNM stage. Elevated let-7e expression was significantly correlated with vein invasion in HCC. Significantly shorter postoperative overall survival was observed in HCC patients with high let-7c expression. The results suggest that aberrant expression of let-7a/7b/7c/7e occurs in benign liver diseases and HCC. The upregulation of let-7 expression is associated with the progression and poor prognosis of HCC, and further mechanistic studies are warranted.

Aims. To develop a noninvasive score model to predict NASH in patients with combined CHB and NAFLD.Objective and Methods. 65 CHB patients with NAFLD were divided into NASH group (34 patients) and non-NASH group (31 patients) according to the NAS score. Biochemical indexes, liver stiffness, and Controlled Attenuation Parameter (CAP) were determined. Data in the two groups were compared and subjected to multivariate analysis, to establish a score model for the prediction of NASH.Results. In the NASH group, ALT, TG, fasting blood glucose (FBG), M30 CK-18, CAP, and HBeAg positive ratio were significantly higher than in the non-NASH group (P<0.05). Multivariate analysis showed that CK-18 M30, CAP, FBG, and HBVDNA level were independent predictors of NASH. Therefore, a new model combining CK18 M30, CAP, FBG, and HBVDNA level was established using logistic regression. The AUROC curve predicting NASH was 0.961 (95% CI: 0.920–1.00, cutoff value is 0.218), with a sensitivity of 100% and specificity of 80.6%.Conclusion. A noninvasive score model might be considered for the prediction of NASH in patients with CHB combined with NAFLD.

Cytokine-induced killer (CIK) cell immunotherapy exhibits significant advantages in the clinical treatment of tumors. This study was designed to compare the biological characteristics of autologous CIK cells from patients with hepatocarcinoma following different procedures for the separation of peripheral blood mononuclear cells (PBMCs). Forty-four hepatocarcinoma patients were enrolled and distributed into two groups. PBMCs were isolated either using a blood cell separator (apheresis method) or Ficoll lymphocyte separation medium (Ficoll method). The total amount, collection efficacy, and cell status of PBMCs in the two groups were determined. According to the number and status of collected PBMCs, different cultivation procedures were used for their amplification and activation and the proliferation ability, phenotype, and killing activity of CIK cells in the two groups were evaluated. Our results indicated that the number of collected PBMCs in the apheresis group was far more than that in the Ficoll group. However, the isolation rate was lower, and more cellular debris was observed in the apheresis group, which may be the cause of some untoward effects. Following in vitro culture, the enrichment time of CIK cells was longer in the Ficoll group, and the percentages of CD3(+)CD4(+) (Th) and CD4(+)CD25(+) (Treg) cells were higher. In the apheresis group, the percentages of CD3(-)CD56(+) (NK) and CD3(+)CD56(+) (NKT) cells were higher, and the CIK cells exhibited a higher cytolytic activity against HepG2 hepatoma cells. In conclusion, different procedures for PBMCs separation can influence the biological activities of CIK cells, and the apheresis method is more effective at enhancing the antitumor efficacy of CIK cells. However, significant attention should be paid to the possibility of adverse reactions in apheresis donors.

Human placental mesenchymal stem cells (hPMSCs), for the treatment of fulminant hepatic failure, have been widely studied. Only a few studies have investigated the effect of the subtype CD200(+)hPMSCs on regeneration of human hepatocytes. CD200(+)hPMSCs can down-regulate activity of several immunocytes and suppress TNF-α secretion from macrophages via the CD200-CD200R axis. We have investigated the influence of CD200-positive human placenta chorionic mesenchymal stem cells (CD200(+)hPCMSCs) on metabolism, proliferation and apoptosis of human hepatocytes in vitro. CD200(+)hPCMSCs promote urea synthesis, albumin secretion and hepatocytes proliferation at co-culture ratios of 1:1 and 3:1. Additionally, CD200(+)hPCMSCs inhibit hepatocyte apoptosis via up-regulation of an anti-apoptotic protein, Bcl-xL. Thus, CD200(+)hPCMSCs can provide supportive benefit for the regeneration of human hepatocytes and also have immunosuppressive properties. Therefore, CD200(+)hPCMSCs may be an ideal candidate for stem cell-based therapy in hepatic failure.

Glypican-3 has been reported to be one of the most promising serum markers for hepatocellular carcinoma. This study aimed to assess the clinical utility of serum glypican 3 for the diagnosis of hepatocellular carcinoma. We recruited consecutive patients on a large scale, 283 with hepatocellular carcinoma, 445 with chronic hepatic diseases, and 162 normal controls, to assess the diagnostic accuracy of serum glypican 3 for hepatocellular carcinoma by enzyme-linked immunosorbent assay. In addition, we further analyzed the relationship between the serum levels of α-fetoprotein and glypican-3 in patients with hepatocellular carcinoma. The results indicated that serum glypican 3 was elevated in patients with hepatocellular carcinoma (0 ng/mL, range = 0-14.0 ng/mL, P = .033) and liver cirrhosis (0 ng/mL, range = 0-12.5 ng/mL, P = .001) compared to the levels in normal control (0 ng/mL, range = 0-4.3 ng/mL), but there was no difference between hepatocellular carcinoma and liver cirrhosis ( P = .097). The area under the curve of the receiver–operating characteristics curve for hepatocellular carcinoma versus all controls was 0.519, with a sensitivity of 39.9%, a specificity of 60.6%, and an optimal cutoff value of 0.002 ng/mL. The positive and negative predictive values were 32.0% and 68.3%, respectively. No significant correlation in serum levels was observed between glypican 3 and α-fetoprotein ( P > .05). The diagnostic sensitivity for hepatocellular carcinoma increased to 72.8% (206 of the 283) when glypican 3 was combined with α-fetoprotein. Glypican 3 was not a promising serum maker for the diagnosis of hepatocellular carcinoma alone, but it could be complementary to α-fetoprotein and elevate the sensitivity of hepatocellular carcinoma diagnosis.

Background Abnormally expressed circulating microRNA (miRNA) may serve as a potential biomarker for the diagnosis of cancer patients. Objective We sought to determine the differentially expressed circulating microRNAs in patients with hepatitis B virus (HBV)-positive small hepatocellular carcinoma (HCC) compared to other HBV-positive benign liver diseases. Methods The miScript miRNA PCR Array was used to detect the levels of 84 miRNAs in plasma or serum samples of patients with HBV-related small HCC (23 cases), liver cirrhosis (LC) (20 cases), chronic hepatitis B (CHB) (20 cases) and healthy controls (16 cases). MiRNAs with fold-change values ⩾ 2 or ⩽ 0.5 compared to healthy controls were considered to be deregulated miRNAs. Results The results of duplicate plasma experiments were not reliable. Comprehensive analysis of the two serum experiments showed that the quality controls all met the requirements. We found 18 differentially expressed miRNAs. Relative to healthy controls, nine, three, and 11 miRNAs were up-regulated in the CHB group, LC group and small HCC group, respectively. In contrast, one, three, and three miRNAs were down-regulated in the same patient groups, respectively. Interestingly, miR-195, miR-25 and miR-16 were up-regulated, and miR-205 was down-regulated, in all three experimental groups. Moreover, only in the HCC group, miR-18a, miR-100, miR-145 and miR-223 were up-regulated 3.48-, 2.95-, 2.12- and 3.91-fold, respectively, and miR-200a and miR-222 were down-regulated 2.56- and 2.00-fold, respectively. Conclusions Our study demonstrated the presence of six differentially expressed serum microRNAs in HBV-positive small HCC compared to other benign liver diseases associated with HBV.

Previous studies indicated Squamous Cell Carcinoma Antigen 1 (SCCA1) may be involved in tumorigenesis and progress of various human malignancies by inhibiting cell apoptosis and promoting cell proliferative activity. The aim of the study was to further investigate SCCA1 expression in different extent of liver diseases and evaluate the clinical significance and prognostic value in HCC.Eighty nine patient-matched tumors and peritumoral surgical specimens and 56 liver biopsies specimens from 23 patients with chronic hepatitis B (CHB), 19 with dysplastic nodule (DN), and 14 with HCC were enrolled. An additional four normal liver (NL) samples were used as controls. SCCA1 expression in liver tissue was measured by immunochemistry. Another 28 HCC specimens and paired non-tumor tissues were used for SCCA1 detection by Western blot. The prognostic value of SCCA1 expression in HCC was evaluated by the Cox proportional hazards regression model analysis.Western blot analysis showed SCCA1 positive rate in HCC was higher than the matched adjacent noncancerous tissues (p0.001). Immunohistochemistry revealed that SCCA1-positive rate increased gradually from NL, CHB, PNT to DN and HCC (p0.05). Clinicopathological analysis showed that SCCA1 expression was positively associated with tumor differentiation (p = 0.043) and patients' Child-Pugh score (p = 0.021). The SCCA1-poistive group showed better overall survival than the negative group (p = 0.029). Importantly, SCCA1 expression was an independent prognostic factor for the overall survival of HCC patients (hazard ratio = 3.757, p0.001).SCCA1 expression pattern may relate to the progression of chronic liver diseases. Furthermore, our study supports a potential association of negative SCCA1 expression with poor outcome in HCC.

Hepatitis B virus related acute-on-chronic liver failure is a serious condition with a high mortality. Oxidative stress, inflammation, necrosis and apoptosis may play an important role in it. This study is to investigate whether serum AOPP, S100A12, HMGB1 and sRAGE can provide diagnostic or prognostic information in HBV-related ACLF.We measured serum S100A12, HMGB1 and sRAGE levels in 50 patients with HBV-related ACLF, 35 patients with liver cirrhosis (LC), 35 patients with chronic hepatitis B (CHB) and 35 healthy controls by enzyme-linked immunosorbent assay. AOPP measured by spectrophotometry.Significantly higher AOPP, S100A12, HMGB1 and sRAGE levels on admission were found in patients with ACLF compared with LC, CHB and healthy controls (P0.001). In ACLF patients, they were higher in nonsurvivors than survivors (P0.001). They had a positive relationship with total bilirubin and MELD scores. AOPP, S100A12 and HMGB1 concentrations continually declined in survivors while increased in nonsurvivors, sRAGE concentrations did not change in survivors, but gradually increased in nonsurvivors during hospitalization. ROC curve analysis showed that the four biomarkers had a higher AUC than TBIL. Multivariate Cox regression analysis demonstrated that S100A12, AOPP and sRAGE were independent risk factors for poor prognosis.Serum AOPP, S100A12 and sRAGE maybe reflect the oxidation stress, inflammation levels in HBV-related acute-on-chronic liver failure. Increased AOPP, S100A12 and sRAGE may serve as important biological markers of worse outcome.

Background and Aims The diagnostic value of serum GPC3 in patients with hepatocellular carcinoma (HCC) remains controversial. Thus, we performed a systematic review and meta-analysis to assess the diagnostic accuracy of serum GPC3 for HCC. Methods A systematic search was performed for the related studies. Sensitivity, specificity and other measures regarding the accuracy of serum GPC3 and alpha-fetoprotein (AFP) in the diagnosis of HCC were pooled using random-effects models. Summary receiver operating characteristic curve (sROC) analysis was used to summarize the overall test performance. Results Nineteen studies were included in this meta-analysis. Pooled sensitivity, specificity and 95% confidence interval (CI) of serum GPC3 for the diagnosis of HCC were 55.2% (52.9–57.4%) and 84.2% (82.2–86.0%), respectively. When combining GPC3 with AFP, pooled sensitivity, specificity, and 95% CI were 75.7% (71.8–79.4%) and 83.3% (79.6–86.6%), respectively. The area under sROC (AUC) and 95% CI for AFP combined with GPC3 were 0.762 (0.649–0.875). For diagnosis of early HCC, pooled sensitivity and specificity of serum GPC3 were 55.1% (47.9–66.2%) and 97.0% (95.2–98.2%), respectively. The AUC of GPC3 for early HCC was 0.793 (0.668–0.917). Conclusions This meta-analysis indicates that serum GPC3 has a comparable accuracy to AFP for the diagnosis of HCC, and there is an elevation in the sensitivity of diagnosis when GPC3 was combined with AFP. Diagnostic accuracy of serum GPC3 for early HCC is still unsatisfactory.

PURPOSE: Tumor-infiltrating FoxP3+ T cells have been reported in various human tumors, which impaired cell-mediated immunity and promoted disease progression. However, its prognostic value for survival in patients with different gastrointestinal cancers [hepatocellular carcinoma (HCC), colorectal cancer (CRC), gastric cancer (GC)] remains controversial. METHODS: Relevant literature was searched using PubMed, Embase, Cochrane, Ovid Medline and Chinese wanfang databases. A meta-analysis was conducted to estimate pooled survival and recurrence ratios. The odds ratio (OR) and 95% confidence intervals (CI) were calculated employing fixed- or random-effects models depending on the heterogeneity of the included trials. RESULTS: For HCC and GC, the overall survival at 1, 3 and 5-year of high FoxP3+ T cells infiltration patients were lower than low FoxP3+ T cells infiltration patients (P0.05). CONCLUSIONS: Our findings suggested that tumor-infiltrating FoxP3+ T cells were a factor for a poor prognosis for HCC and GC, but a good prognosis for CRC.

The aim of the study was to detect the expression level of thymosin β4 (Tβ4) in serum and tissues of patients with chronic hepatitis B (CHB) combined nonalcoholic fatty liver disease (NAFLD). The effects of Tβ4 in hepatic steatosis, chronic inflammation, and fibrosis development in CHB combined NAFLD patients were also discussed. The study included 46 patients in the case group with CHB and NAFLD and 42 patients in the control group with CHB. ELISA was applied to detect serum Tβ4 and TNF-α level. Furthermore, the correlation analysis of Tβ4 levels with biochemical index, pathological index, and TNF-α level was performed. The Tβ4 immunohistochemical levels of different inflammation fibrosis levels were compared, and the correlation analysis with TNF expression was performed. The Tβ4 levels in patients with CHB combined NAFLD showed no statistical difference when compared to the control group. In patients with CHB combined NAFLD group, the Tβ4 level had no correlation with ALT, AST, TG, FGP, hepatitis B virus (HBV)-DNA levels, and fat grading, but had negative correlation with inflammation score and fibrosis score (P

Recent studies have shown that imbalance between tumor-infiltrating interleukin (IL)-17(+) T cells and regulatory T cells (Tregs) is an important regulator of progression in various cancers, but little is known regarding this imbalance in hepatocellular carcinoma (HCC). This study explored the role of imbalance between IL-17(+) T cells and Tregs in the immunopathogenesis of HCC in patients with chronic hepatitis B (CHB) infection.Fifty-six of patient-matched tumors and peritumoral surgical specimens from 56 patient with HCC and 136 liver biopsies specimens from 46 patients with CHB, 37 with atypical hyperplasia (AH), and 53 with HCC were enrolled. The expressions of IL-17, FoxP3, CD4, and CD8 in liver tissue were measured by immunochemistry for the evaluation of liver-infiltrating lymphocytes.The density of liver infiltrated FoxP3(+) Tregs was increased in a stepwise manner from CHB to AH then HCC, while there was a decreasing trend for the density of IL-17(+) T cells and CD8(+) T cells. In surgical specimens of less differentiated HCC, the quantity of tumor-infiltrating FoxP3(+) Tregs was significantly lower and IL-17(+) T cells and CD8(+) T cells were significantly higher. Additionally, peritumoral IL-17(+) T cells were increased in poorly differentiated HCC. High intratumoral FoxP3(+) Tregs with high intratumoral IL-17(+) T cells showed a significantly lower overall survival (OS) and disease-free survival (DFS) compared with other groups (OS, P = 0.033; DFS, P = 0.004). High intratumoral FoxP3(+) Tregs with high peritumoral IL-17(+) T cells showed a significantly lower survival rate compared with other groups (OS, P0.001 and DFS, P0.001).Our findings suggest that intrahepatic IL-17(+) T cells and FoxP3(+) Tregs may cooperate to promote the progression of HCC.

Background Recent studies have focused on regulatory T cells (Tregs) in chronic hepatitis B (CHB) and hepatocellular carcinoma (HCC) and they were also conducted independently of each other. Aims This study tried to characterize Tregs in blood and tumour infiltration, and to explore the correlations between Tregs and the context of chronic hepatitis B in HCC patients. Methods The liver-resident Tregs and CD8+ T cells on core biopsy were investigated using immunohistochemistry staining in individuals (n = 209) with CHB (n = 47), HCC (n = 137) or healthy controls (n = 25). Circulating Tregs were detected in the above patients with CHB (n = 27) or HCC (n = 101) by flow cytometry. Results The number of tumour-infiltrating and circulating FoxP3+ Tregs was significantly high in patients with CHB (P

To establish a quantitative technique for assaying gene methylation in hepatocellular carcinoma (HCC) and evaluate its feasibility for clinical application.Following bisulfite modification and PCR amplification, the fragments of CDKN2A and ACTB were cloned into plasmids to generate calibration curves using SYBR Green quantitative PCR, and then these two genes were quantitatively analyzed in 41 cases of HCC specimen.The amplification curve, dissociation curve, calibration curve and electrophoresis analysis showed that SYBR Green fluorescent quantitative PCR could assay 10(2)-10(8) copies/microL of recombinant plasmids with high specificity, high sensitivity and a wide detection range. The tests on 41 cases of HCC specimens further confirmed its feasibility for quantitative analysis of methylation.SYBR Green fluorescent PCR is an easy, fast and high-throughout quantitative tool, and it can be used for methylation analysis in basic research or clinical assay.