Impact Of Notch1on Autophagy And Biological Characteristics In PC12

Notch are widely present and highly expressed in invertebrates and vertebrates. Notch are highly conserved among different species. The functiona of Notch signaling pathway is complex. Notch regulate growth and differentiation of cell, decide cell fate, and involved in all aspects of cell life activities. In mammals, Notch not only involved in hematopoiesis, T cell development, Physiological angiogenesis process etc,but also have close relation with tumor formation.Our laboratory establish a dynamic regulation of gene NICD in PC12cell by Tet-on gene expression system. This cell line overcome the traditional methods that can not accurately regulate the expression of Notchl in cell and is conducive to study the role and molecular mechanisms of notch1in PC12.ObjectiveTetracycline derivative doxycycline (Dox) have a dynamic regulation role on expression of NICD in PC12-Notchl.The purpose is to detecte proliferation, apoptosis and autophagy in PC12-Notchl at different expression levels of NICD and to research the impact of Notchl on autophagy and biological characteristics in PC12. Methods1. Detecting NICD expression in PC12-notchl:Firstly, recove PC12-notch1from liquid nitrogen. Secondly,cells after two generations were plant in six-well plates, adding tetracycline DOX (1ug/ml). According DOX induced time,cells are divided into six different groups (namely, Oh,12h,24h,36h,48h,60h). Observe growth of cell and EGFP-positive cells under inverted fluorescence microscope and calculate the percentage of EGFP-positive cells. Measure EGFP-positive cells in each group by flow cytometry after digesting cells of each group and then draw tendency chart of expresstion of NICD with different induced time.2. Detecting biological characteristics of PC12-notchl for different groups:cells are divided into six different groups (namely, Oh,12h,24h,36h,48h,60h) According DOX induced time. Flow cytometry measure cell cycle and apoptosis rate of different groups after digesting.3. Detecting autophagy:extract protein and detect protein of autophagy:LC3B,Beclinl ATG7, ATG5by westernblot. Calculate gray value and compare expression differences within different groups.4. Detecting signaling pathways of autophagy in PC12-notchl:extract RNA from PC12-notchl in each group and then reverse RNA into CDNA. Q-PCR Detect expression of mRNA of PI3K, AKT1, MTOR and compare expression of mRNA of PI3K, AKT1, MTOR in different groups.Results1. Expression of EGFP-NICD in PC12:cells in Oh group havn’t observed green fluorescence.but from12h group,24h to36h, cells are observed increase green fluorescence and they are20%,55%,71%respectively. Green fluorescent in cells of48h,60h decreased, but higher than Oh group and fluorescence expression rate was63%,59%respectively. In a word, expression of NICD groups with DOX were significantly higher than the non-induced group.2. Cycle and apoptosis:In Dox non-induced group, the apoptosis rate remained low. Starting from Oh group to60h group, the rate of apoptosis was3.3%,9.5%,10.6%,14.0%,18%respectively and the rate of apoptosis is gradually increased with the increased induced time of DOX. Apoptosis rate in DOX induced groups have a significant difference compared to non-induced group. In Dox non-induced group, the rate of S in cell cycle is high, Starting from Oh group to60h group, the rate of of S in cell cycle inPC12-Notchl gradually decreased with the increased induced time of DOX3. Autophagy:westernblot detecte expression of LC3B, Beclinl, ATG7, ATG5in different groups. With software Image for analysis of gray values,we obtain that expression of four protein increased starting from Oh group to36h group and then decreased gradually in group of48h,60h. By analysis of variance,and expression LC3B,Beclinl, ATG7, ATG5in DOX induced groups are significantly higher than non-induced group.4. Signaling pathway of autophagy:Q-PCR detecte expression of PI3K, AKT1, MTOR and the result is that expression of PI3K, AKT1, MTOR in DOX induced groups were significantly lower than non-induced group.Conclusions1. Expression of exogenous NICD in PC12-Notchl can be artificially controled, and the levels of expression is correlated with inducted time.of DOX.2. In PC12-Notchl, high expression of NICD promote the rate of apoptosis and inhibit cell cycle.3. In PC12-Notchl, overexpression of NICD activates autophagy and PI3K-AKT-MTOR pathway is involved in regulation of autophagy...