| Background and Objective BLU is a candidate tumor suppressor gene, which is epigenetically inact ivated in many human malignancies. However, the expression and biological functions of BLU in gastric cancer has not yet been reported. In the present study, we investigated the expression of BLU in gastric cancer and explored the mechanism underlying BLU epigenetically inactivation.Methods(1) q RT-PCR analysis was carried out to analys e the m RNA expression of BLU in 52 cases of gastric carcinoma and paired paracancerous tissues, 6 gastric cancer cell lines and GES-1, Western blot was performed to analys e the protein expression of BLU in 7 cell lines.(2) Promoter Scan program software was used to predict core promote of BLU and then verify the prediction outcomes by using luciferase report assays, Methyl Primer Express® Software v1.0 and TFSEARCH were u sed for the prediction of the putative functional Cp G sites in BLU core promoter region.(3) Clonal bisulfite sequencing was performed to investigate the relationship between the methylation level of promoter with expression of BLU in gastric carcinoma, and find the putative functional Cp G.(4) Treated AGS and SGC7901 cells with 5-Aza,q RT-PCR and Western blot analysis were performed to analysis the expression of BLU after demethylation treatment.(5) Luciferase reporter plasmids assay and RNA interference experiments were used to analysis whether BLU is regulated by Sp1.(6) Ch IP assay were performed in AGS and SGC7901 cells to analysis whether Sp1 could bind to BLU promoter, EMSA and luciferase reporter plasmids assay was used to evaluation the effect of methy lated-39 Cp G on the binding ability of Sp1.(7) CCK-8 cell proliferation assay and colony formation assay were performed to analysis the effects of downregulated BLU on cells proliferation and colony formation ability of gastric cancer cells.Results(1) BLU expression was significantly lower in MGC803, SGC7901, BGC823, MKN45, AGS, and HGC27 cells than that in GES-1 cells, BLU m RNA expression was notably reduced in 69.2%(36/52) gastric cancer tissues when compared with their paired adjacent noncancerous tissues,and no significant difference in BLU m RNA level was observed between gastric cancer tissues when classified by various clinicopathologic characteristics.(2) BLU core promoter was putatively at position-181 to +63bp,-85 Cp G and-39 Cp G were predicted to be the putative functional Cp G site.(3) We found a negative correlation between BLU expression and its promoter methylation level(especially methylation of-39 Cp G site) in gastric cancer.(4) BLU expression was restored in AGS and SGC7901 cells after demethylation treatment.(5) Sp1 activates the BLU promoter.(6) Transcription factor Sp1 specially bind to BLU proximal promoter(-43~-35bp), and the methylated-39 Cp G which located within Sp1 binding sites “GC box” could decrease binding ability of Sp1, then repressed transcription and expression of BLU.(7) Knockdown of BLU could promote gastric cancer cell viability and clonogenicity.Conclusion(1) Expression of BLU in gastric cancer was associated with promoter Cp G hypermethylation(especially hypermet hylated-39 Cp G site).(2) Transcription factor Sp1 could activates the BLU promoter,hypermethylated-39 Cp G in BLU proximal promoter directly reduced its binding with Sp1,which may be one of the mechanisms accounting for the inactivation of BLU in gastric cancer.(3) BLU could repress the growth of gastric cancer cells, suggested it might be a potential TSG of gastric cancer and its expression was restored after demethylation treatment. BLU may become a potential diagnostic biomarker and epigenetic therapeutic target for gastric carcinoma. |
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