The Expression Of SOX7 MRNA And Methylation Of SOX7 DNA In Hepatocarcinoma Cell Lines And Hepatocarcinoma Tissues

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, which is the main type of parimay liver cancer. It accounts for 85-90 percent of primary liver cancer. The incidence of HCC is higher and rapidly increasing in China. The most common causes of HCC include HBV, HCV or both chronic infection, alcohol and high intake of foods containing aflatoxin intake. The occurrence of HCC is a long-term, complex, multi-factor interaction and mutual participation in the continuous process. The molecular biologic mechanism of HCC is not completely clear. Wnt signaling pathway playing an important role in the cancer development was focused by scientists in recent years. Wnt signaling pathway has been reported to be crucial in mammalian embryonic development as a morphogen, which modulated cell proliferation and differentiation. Recently, many studies on tumorigenesis and progression suggested that Wnt signaling pathway plays the crucial role in the cancer development. As the terminal modulator of Wnt pathway, SOX genes which are common pathway of Wnt signaling pathway may play key roles in tumorigenesis and progression by modulating many downstream target genes. SOX7 is the key member of SOX F subgroup, which modulates the target genes through combined with transcription factor TCF/LEF. The recent researches show that the abnormal expression of SOX7 is involved in the tumorigenesis and development of many kinds of human tumors. Li found that the expression level of SOX7 mRNA is down-regulated in 48%(10/21) prostate tissues and 11 kinds of prostate cancer cells. Yu etc pointed out that the expression level of SOX7 mRNA in 80%(16/20) colon cancer tissues and 9 colon cancer cell lines. The expression level of SOX7 mRNA was also obviously down-regulated in primary breast cancer, the primary kidney and primary lung cancer. The relationship between SOX7 expression level and HCC has little studies.Objectives1 To investigate the expression of SOX7 mRNA in Human hepatocarcinoma cell lines and paired primary HCC and their matched adjacent hepatic tissues, and whether the expression levels of SOX7 was correlated with clinical and pathological parameters of HCC patients.2 To study the methylation of SOX7 in Human hepatocarcinoma cell lines and HCC tissues and their matched adjacent hepatic tissues.Methods1 Hepatocarcinoma cell linesHepG2, Snu449, SMMC-7721, Snu182, SK-Hep-1, Huh-1, Huh-7, Plc-PRF-5 and Hep3B were provided by Department of the Pathogenic Microbiology, Peking University.2 Subjects and samplesThe subjects were HCC patients treated in Henan Tumor Hospital from 2009 to 2010. The cancer tissues and matched adjacent tissues of the subjects were collected after operation. All samples were stored in the icebox at-80℃within half an hour. The patients'clinical data such as age, portal vein tumor thrombus, intrahepatic metastasis, microvascular invasion, level of cirrhosis, extrahepatic metastasis, tumor size and tumor location were collected. These data were statistically analyzed.3 Expression of SOX7 mRNA in samplesReal-time PCR was used to determine the mRNA expression level of SOX7 in the hepatocarcinoma cell lines, HCC tissues and matched adjacent tissues. Ct value represents the amounts of mRNA. For each sample,△Ct (Ctsox7-CtHMBs) value represents the difference mRNA expression level between SOX7 and HMBS, which is the internal reference gene.4 Methylation level of SOX7 in samplesWe quantified the methylation level of SOX7 promoter in 7 hepatocarcinoma cell lines,19 cases of paired HCC tissues by using methylation PCR assay. The sample was defined as methylation positive when the level of high methylation was more than 10%, else as methylation negative.5 Data analysis We used Fisher's Exact Test. Spearman Rank Correlation analytical method has used to compare the expression level of SOX7 mRNA between HCC tissues and matched adjacent tissues. The statistical significance level was 0.05. All statistical analyses were performed using the program SPSS 16.0.Results1 The expression level of SOX7 mRNA in Hepatocarcinoma cell linesThe expression level of SOX7 mRNA was significantly lower than that in normal adjacent liver tissues.2 Expression of SOX7 mRNA in paired HCC tissuesThe expression of SOX7 mRNA is down-regulated in 19 (47.5%) cases of HCC tissues compared with the matched adjacent tissues.The expression level of SOX7 mRNA was significantly lower than that in matched adjacent tissues (z=-1.978, P< 0.05).3 Relationship between expression level of SOX7 mRNA in HCC and the patients clinical pathological characteristicsThe difference of SOX7 mRNA expression level of the different age groups is significant. The expression level of SOX7 mRNA was significantly down-regulation in high stage tumors compared to low stage tumors (χ2=8.319, P<0.05). There were no association between the expression level of SOX7 mRNA and intrahepatic metastasis, portal vein tumor thrombus and level of cirrhosis.4 Methylation results of quantitative PCRMethylation of SOX7 DNA promoter was found in the liver cancer cell lines Hep3B, SK-Hep-1, HepG2, Huh-1 and only 3 cases of 19 liver tissues in which SOX7 mRNA was down regulated.Conclusions1 The expression of SOX7 mRNA level in 7 HCC cell lines was significantly lower than that in the normal liver tissues. The expression level of SOX7 mRNA was significantly lower than that in matched adjacent tissues.2 The difference of SOX7 mRNA expression level among the different age groups is significant. The expression level of SOX7 mRNA was significantly down-regulation in high stage tumors compared to low stage tumors.3 Methylation of SOX7 DNA promoter was found in the liver cancer cell lines Hep3B, SK-Hep-1, HepG2, Huh-1 and less cases in which SOX7 mRNA was down regulated.