Effect Of SP1 On Transcriptional Regulation Of Cancer Testis Antigen OY-TES-1 In Glioma

Background:Glioma is the most common primary brain tumor with highest mortality rate.Although it has been recieved the comprehensive treatment including surgery,radiotherapy and chemotherapy,the survival rate of glioma patients has not been significantly improved.It is urgent to look for a new method to treat glioma at present.In recent years,immunotherapy is being considered as an attractive therapy for cancer patients,which has advantage of low toxicity,the potential for avoiding drug-resistance and the potential for persistence of the antitumor effect.However,the crucial step of cancer immunotherapy is to identify the appropriate cancer-specific target antigens.Cancer/testis antigens(CTA)are tumor-specific and highly expressed in tumors,especially certain CTA tumor vaccines can also improve the prognosis of cancer patients.Thus,CTAs are considered to be potential ideal target antigens for cancer immunotherapy.OY-TES-1,as a member of the CTA family,also has tumor-specific characteristics.Our previous study found that OY-TES-1 is highly expressed in glioma with the relation to the WHO grade and can also affect the prognosis ofglioma.However,the mechanism of OY-TES-1 expression in glioma has not been yet clear,and it is rarely reported that the transcription of OY-TES-1 is controlled by other factors.We have previously found by bioinformatics analysis that there are multiple Sp1 transcription factor binding sites in the upstream of the OY-TES-1 gene translation initiation site.It is hypothesized that Sp1 may involve in participating the expression of OY-TES-1 in glioma.Objective:To investigate the relationship between the expression of Sp1 and OY-TES-1in glioma,the effect of Sp1 on the expression of OY-TES-1 in glioma cells and to explore the transcriptional activation role of Sp1 in the OY-TES-1 expression in glioma.Method:(1)30 cases of different grade glioma were selected.The expression of SP1 and OY-TES-1 were detected by PCR and immunohistochemistry.Then,the relationship between the expression of Sp1 and OY-TES-1 in glioma was statistically analized by bioinformatics software Onconmine.(2)Glioma cell lines U87,U251 and SHG44 were cultured and used to detect the expression of Sp1.And the overexpression and silencing Sp1 were performed in tumor cells.The effect of Sp1 on OY-TES-1 expression was observed from the cell level.(3)The OY-TES-1 promoter gene reporter vector was constructed.The relative core region of OY-TES-1 promoter was screened by double luciferase assay,and then the Sp1 transcription factor binding sites in OY-TES-1 promoter was confirmed with bioinformatics analysis The activation of Sp1 on the promoter was established with the double luciferase activity detection.The binding of Sp1 to OY-TES-1 promoter was confirmed by Chi P-PCR.Result:(1)Sp1 and OY-TES-1 were highly expressed in glioma tissues.The positive expression rates of Sp1 mRNA and protein were 87%(26/30)and 90%(27/30)respectively in 30 glioma specimens,while the positive expression rates of OY-TES-1 mRNA and protein were 83%(25/30)and 60%(18/30)respectively.The correlation analysis between the expression of Sp1 and OY-TES-1 in gliomas showed that they were not only positively correlated with mRNA expression(P<0.001),but also positively correlated with protein expression(P = 0.033).(2)The expression of Sp1 was highest in SHG44,U251 times,U87 lowest.Overexpression of Sp1 in U87 and U251 cells can lead to increased expression of Sp1 mRNA and protein,and the expression of OY-TES-1 mRNA and protein was also increased.Down-expression of Sp1 in U251 and SHG44 can lead to decreased expression of Sp1 mRNA and protein,and the expression of OY-TES-1 mRNA and protein was also decreased.(3)The four-phase interrupted OY-TES-1 promoter reporter vector was transfected into U87 cells and the OY-TES-1 promoter region from-184 bp to+67bp was determined as core promoter by the double luciferase.The binding site of Sp1 in the OY-TES-1 gene core promoter region was analized by bioinformatics software.Compared with the classical binding site of Sp1,the OY-TES-1 core promoter region contained two classic Sp1 binding sites.The different Sp1 mutation sites of OY-TES-1 promoter gene reporter vector were transfected into U87,U251 and SHG44 cells,respectively.The results showed that both single and double mutated Sp1 binding sites can decrease the activity of the promoter.The double mutated Sp1 binding sites can significantly reduce the activity of the promoter.Chromatin immunoprecipitation confirmed that Sp1 could bind to the OY-TES-1 promoter region.Upregulation of Sp1 could enhance the activity of OY-TES-1 promoter,and down-regulation of Sp1 could decrease the activity of OY-TES-1 promoter and all of them were statistically significant.Conclusion:(1)SP1 and OY-TES-1 were highly expressedin the glioma tissue,their expression were positively correlated.(2)Sp1 could affect the expression of OY-TES-1 in glioma cells.(3)Sp1 could bind to OY-TES-1 core promoter region and positive regulate OY-TES-1 promoter.