Epigenetic Changes Of SOX17 In Lung Carcinoma And Functional Study

BackgroundLung cancer is the leading cause of cancer-related death worldwide. Although various therapies have developed these years, including surgical resection, chemotherapeuticsis,radiation and targeted therapy,the overall 5-year survival rate is still limited. The development of lung cancer is a complex process,involving multiple genes and many factors. Both genetics and epigenetic alterations has played an important role in the tumorigenesis. Many studies have shown that, methylation of the promoter CpG islands (CGIs) resulted in down regulating or silencing of tumor suppressor genes, which recognized as an important epigenetic mechanism, plays an important role in multiple human carcinoma.SRY-box containing gene 17 (SOX 17) belongs to the Sox gene family. Sox 17 binds to the target DNA sequence 5'-(A/T)(A/T)CAA(A/T)G-3'and regulates a series of target genes,involving in embryonic development and maintenance of fetal blood stem cells. A growing body of evidence suggests that SOX17 plays an important role in the initiation and progression of numbers of human carcinoma,including colorectal Cancer, hepatoma and breast carcinoma, via the interaction with canonical WNT pathway, and has been recognized as a potential tumor suppressor gene.The epigenetic character and funtion of SOX17 in lung cancer remains unclear.Objective 1. Detection of the expression and methylation change of SOX17 in lung cancer cell lines.2. Analysis of the relationship between SOX17 expression and methylation change in lung cancer and corresponding adjacent lung tissue, based on the clinical and pathological. 3. Functional study of SOX17 in lung cancer cell line.Methods.1.RT-PCR and Western blot were Performed to detect SOX17 expression in lung cancer cell lines before and after 5-Aza- dC treatment.2.Methylation specific PCR(MSP) was used to detect DNA methylation of SOX17 in lung cancer cell lines and tissues.3.Immunohistoehemisty was performed to examine SOX17 expression in lung cancer and corresponding adjacent tissues.4. Transient transfect eukaryotic expression vector into lung cancer cell line,and G418 was used for cell selection.5. Colony formation assay was performed to evaluate inhibitory effect of SOX17 on clonogenicity of lung cancer cell.Results1. The expression of was detected by Semi-quantitative RT-PCR in lung cancer cell lines (H23,A549,H446 and 95D). As shown in Figure(1-1), SOX17 was expressed in H446 and 95D cell lines and weakly in A549. No expression was detected in H23 cell lines.The same results were further confirmed by western blot assay as shown in figure(1-2). Meanwhile, SOX17 gene promoter region was completely methylated in H23 and partial methylated in A549,H446 and 95D(shown in figure 1-3).2. Promoter methylation were detected in 53 of 83 cancer tissues,while only 5 of 31 adjacent tissues were methylated(P<0.05). Immunohistochemistry (IHC) was performed on 29 cases paraffin sample of lung cancer and adjacent tissues. Reduced or loss expression of SOX17 was shown in 25 cases of cancer tissues,whereas normally expression was found in their matched adjacent tissues. In these negatively expression samples,22 cases were promoter methylated(88.0%). Clinical association study found that the methylation status was significantly associated with sex and tumor differentiation.(P<0.05)3. To further evaluate the function of SOX17 in lung cancer, colony formation assays were performed with H23 cells. As shown in figure3-2, the colony formation efficiency of H23 cells was significantly inhibited by restoration of SOX 17 expression.ConclusionMethylation of SOX17 gene promoter region as a mechanism of down regulating SOX17 expression frequently takes place in lung cancer. SOX17 methylation is associated with sex and tumor differentiation. Restoration of SOX17 inhibites colony formation of H23 cells, indicating that SOX 17 might be a potential tumor suppressor gene in lung cancer.