Study Of Sp3Related Gene Expression Profiling And Effect On β-catenin Transcription

Specificity protein (Sp) family are special transcription factors regulatingthe intracellular transcription of a broad and diverse spectrum of genes. Thisfamily contain9members from Sp1to Sp9, among which, Sp3plays a dual-rolein transcription regulation which shows activated or suppressive function indiverse tumors and different periods. The role of Sp3in human hepatocellularcarcinoma (HCC) remains unclear. Our previous study has suggested that Sp3may promote the pathogenesis of HCC. However its molecular mechanismneeds further study.β-catenin is a crucial molecule in Wnt signaling pathway. β-catenincontributes not only to cytoskeleton, cell differenciation and cadherin-basedadheren junction, but also plays as the key nuclear effector of canonical Wntsignaling pathway regulating the expression of proto-oncogene such as CyclinD1, C-myc, etc.There exists intense association of Sp3and β-catenin. Our previous studyindicated that the expression of β-catenin was significantly up-regulated in HCCtissues and HepG2cells and β-catenin was down-regulated post Sp3knockdownby RNA interference (RNAi). Sp3may have a crosstalk with β-catenin and potentially regulate the transcription of β-catenin directly or indirectly. However,the mechanism remains unknown and needs further study.Part1. Study of HCC gene expression profiling post Sp3knockdownby RNAiObjective: To analyze the alteration of gene expression profiling with Sp3knockdown in HCC cell line HepG2, to discover the target genes and pathwaysinfluenced by Sp3and initially to confirm the association of Sp3andWnt/β-catenin. Methods: RNAi was performed to knockdown Sp3gene inHepG2cells and NimbleGen Human Gene Expression Microarray was assayedfor gene expression profile analysis. Furthermore, real-time quantitative PCR(qRT-PCR) was employed to confirm several differentially expressed genesrelated to cell cycle. The cell cycle was analyzed by flow cytometry. Results:The mRNA level of Sp3was significantly down-regulated. The expressionalteration of1789genes was revealed by microarray, including1007up-regulated and782down-regulated genes post Sp3downregulation. Thesegenes were involved in most of cellular biological process such as proliferation,differentiation, programmed death, adhesion, metabolic process, and so on.Several molecules of Wnt/β-catenin pathway including β-catenin were alsodown-regulated. qRT-PCR confirmed that the mRNA level of CCND1, CCNE2,TGFB1was down-regulated while the mRNA level of CDKN2A wasup-regulated post Sp3downregulation. Flow cytometry analysis showed that thepercentage of cells in G1phase increased significantly post Sp3downregulation.Conclusion: Gene expression profiling alters with Sp3downregulation inHepG2. Downregulation of Sp3induces cell-cycle arrest in G0/G1phase inHepG2cells. Sp3may play an essential role in the pathogenesis ofhepatocellular carcinoma as a transcription factor, via regulating cell cycle progression and Wnt/β-catenin pathway.Part2. Effect of Sp3on regulation of the transcription of human β-cateninObjective: To investigate the effect of Sp3on the regulation of the transcriptionof the promoter of human β-catenin and to reveal the association of Sp3andWnt/β-catenin. Methods: Sp1, Sp3expression plasmids and β-catenin reporterplasmid (-410/-1bp) were transiently transfected into HEK293T cells. DualLuciferase Reporter Gene Assay was employed to detect the transcriptionactivity of β-catenin gene. Results: Sp1expression plasmid (0.4μg) induced2.4folds increase of the promoter activity while Sp3expression plasmid (0.4μg)significantly induced5.3folds increase. The promoter activity remainedunchanged with the increasing of Sp3/Sp1post0.4μg Sp1and Sp3expressionplasmids co-transfection. Co-transfection of Sp1expression plasmid (0.3μg) andSp3expression plasmid (0.1μg) induced3.5folds increase of the promoteractivity, stronger than the individual transfection. Conclusion: Sp3may activatethe transcription of β-catenin gene (-410/-1bp) while Sp1shows weakeractivated function. Co-transfection of Sp1and Sp3gains little effect on thetranscription while there may exist synergistic effect between Sp1and Sp3. Sp3may influence Wnt/β-catenin pathway via interaction with β-catenin.