Study On The Regulatory Mechanism Of Sox2and Pax6to The ErbB Family Genes

Objective: There are similarity and distinctions between normal stem cells and cancerstem cells. Both of them are regulated by various intracellular transcription factors andextracellular growth factors. Transcription factor and growth factor are the two importantregulatory parts of the stem cell behavior, for example, self-renew, proliferation anddifferentiation. ErbB family growth factor receptors play an important role in regulating theproliferation and differentiation, not only in normal stem cells, but also in cancer and cancerstem cells. Epithelial Growth Factor Receptor (EGFR), one of the four members of ErbBfamily, is crucial for the growth and differentiation of the neural stem cells. It is veryimportant to investigate their interaction and compare the regulatory mechanism between thenormal stem cells and the cancer stem cells. This study is the first step on this way.In our previous study, two transcription factors initiate the interest of us. The first is theSox genes（SRY related HMG-Box genes）, which are very important in regulating the celldifferentiation.Sox2is one of the specific markers of stem cells, which is critical in regulatingself-renew multi-potential of neural stem cells. We found previously that Sox2could take partin the regulation of neural stem cell self-renew through up-regulation EGFR. Sox2over-expression increases the EGFR expression in neurospheres. Therefore we ask thefollowing questions:(1)If there is the similar Sox2-EGFR mechanism in cancer or cancerstem cells?(2)Do Sox2and Sox family transcription factors have similar regulatorymechanisms on ErbB family genes? The second transcription factor we are interested is the Pax6(paired box6), a crucialtranscription factor in the genesis of the EGF-responsive neural stem cells in the cerebralcortex. But whether Pax6could regulate the expression of EGFR is still unknown. So wedesigned a series of experiments to answer this question.There are two parts of work in this study:(1) the regulatory mechanism of Sox2on theErbB genes;(2) the regulatory mechanism of Pax6on the EGFR expression.Part1.The regulatory mechanism of Sox2on the ErbB genesMethods: Establish the Sox2and Sox10over-expression plasmid and adenovirus in vitro.Transfect Sox2or Sox10into breast cancer cell line MCF-7, neuroglioma cell line U87, bonemarrow mesenchymal stem cells (MSC). Screen the mRNA expression changes of the fourErbB members（EGFR、ErbB2、ErbB3、ErbB4） by Real-Time PCR. Other stem and cancerstem cell markers (CD133, S100b, p75NTR, nestin, GFAP, olig2) are investigated, too.Immunofluorescent and Western-blot analysis were used to confirm the expression changes inprotein level. Luciferase reporter gene assay was designed to test the possible regulation ofSox2on the ErbB promoters.Results:(1)48hours after the transfection of Sox2into breast cancer cell line MCF-7,the ErbB2and ErbB3mRNA levels up-regulated obviously, while not changed in glioma cellline U87and mescenchymal stem cells. In all the three cell lines, the EGFR expression didnot changed. The cancer stem cell marker CD133up-regulated slightly in MCF-7.Othermarkers did not changed in all the three tested cell lines, including S100b, p75NTR, nestin,GFAP and olig2.(2)The protein level changes of ErbB2was further confirmed by westernblot. The co-localization of Sox2and ErbB2was found in some MCF-7cells.(3) By thedual-luciferase reporter assay, Sox2was found to increase the ErbB2and ErbB3promotermediated luciferase activity in a dose-dependent way.Conclusions: Sox2can upregulate the expression of ErbB2in MCF-7cells. Part2.The regulatory mechanism of Pax6on EGFRMethods:(1) Using Pax6mutant mouse, we compared the neurosphere formation abilityof wild type and mutant brain cells. Cells were dissected from the dorsal part of the cortex andthan seeded directly into96wells. The EGFR expression of the formed neurospheres wastested by western blot and by Real-Time PCR.(2)To test the biological function of EGFR onthe neural stem cells, the EGFR over-expression adenovirus was transfected into theneurospheres. After7days transfection, the neurite outgrowth of the differentiated neuralstem cells was compared.Results:(1) Neurosphere formed less by cells from Pax6mutant brain cortex. The EGFRexpression was also down-regulated in these neurospheres.(2)EGFR tranfection results thedecrease of the neurite outgrowth of neurospheres.Conclusions: Pax6mutant could down-regulate the EGFR expression and inhibitsproliferation of neural stem cells; In addition, EGFR overexpression could inhibit the neuriteprolong of neural stem cell, maintain multiple potentiality behavior.