| Testicular germ cell tumor is a male germ cell tumor whose incidence rate has showed a rising trend in recent years.Clinical and epidemiological studies have found cryptorchidism,infertility,testicular atrophy are the risk factors for the occurrence of testicular germ cell tumors.Recent studies in molecular genetics proposed some demonstration about the testicular germ cell tumors occurrence.Though the observation of clinical pathology specimens had found some ideal sensitive and specific markers in testicular germ cell tumor,such as M2A,CD117,OCT-4 and AP-2γ and so on.In recent years,AP-2γ has been found as a new molecular marker in germ cell tumor testis,research had shown that AP-2γ protein had expression in 22 weeks ago normal male fetal testis and the expression of AP-2γ protein will gradually decline in 22 weeks later,AP-2γ protein could not be detected in testis when the fetus birth to normal adulth male,while AP-2γ has high abnormal expression in testicular germ cell tumor,which AP-2γ plays an important role in the process form the benign to the malignant condition of testicular germ cell tumor.Thus,to identify the downstream gene regulated by AP-2y in testicular germ cell tumors will be significant for understanding the genetic mechanism of testicular germ cell tumors.In this paper,firstly,we used immunohistochemistry to analyze AP-2γ protein expression in normal tissues and testicular germ cell tumor tissues,and found that AP-2γ had highly expression in cancerous tissue,whereas no expression in normal tissues.Then we used ChIP-on-chip microarray experiment to find more than 2,000 AP-2γ target genes,which are mostly related to the cell cycle,apoptosis,migration and so on,we combine the signal pathway to analysis and choose SMAD4 as the research object,which is the important member of the TGF-β signaling pathway,we investigated the relationship between AP-2γ and SMAD4 gene.We used western blot to test expression of SMAD4 and AP-2y protein in NTera-2,MCF-7 and HEK293 cell lines.We found that AP-2y had high expression in NTera-2 cell and had expression in MCF-7 cell,no expression in HEK293 cell,SMAD4 had expression in the three cell lines.Then the result of ChIP-PCR showed that AP-2γ can bind to the upstream region of SMAD4 gene promoter in NTera-2 cell.The result of luciferase assay showed AP-2y can inhibit the transcriptional activity of SMAD4 in a dose-dependent manner,while western blot analysis result showed AP-2γ can inhibit SMAD4 in protein expression levels.Immunofluorescence co-localization proved that in NTera-2 cell endogenous AP-2γ had expression in the nucleus,while SMAD4 had expression in the nucleus and cytoplasm and had high expression in the nucleus,endogenous AP-2y and SMAD4 had obvious superposition of signal in NTera-2 nucleus and had a good co-localization.In the colony forming,MTT experiment and the cell counting experiment all showed that the affection of AP-2y to the growth and proliferation of testicular germ cell lines,while the affection may be through up-regulate expression of SMAD4 gene.In the mice cryptorchidism model experiment,we found that AP-2γ can negative regulate the expression of SMAD4 by RT-PCR experiment,the results in vitro were consistent with the results in vitro cell level.There were reports the curcumin can inhibit the expression of AP-2γ,in curcumin drug experiment we also showed that,with the increased concentration of curcumin,the expression of AP-2γ was significantly decreased while SMAD4 expression was significantly increased,experiment also proved that AP-2γ negatively regulate SMAD4.In conclusion,we first proved that SMAD4 gene is the target gene of AP-2y and that SMAD4 expression was negatively regulated by AP-2γ.AP-2γ may regulate the growth and proliferation of testicular germ cells by affecting the expression of SMAD4.These studies lay the foundation for the processes of testicular germ cell tumors occurrence,AP-2γ possibly involves in the regulation through TGF-βsignaling pathway. |
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