Effect Of HIPK2 In The Neural Stem Cell Proliferation And Apoptosis

Introduction:The development of central nervous system(CNS) in mammals is a gradual process with complex multi-steps. As the only source of all types of neural cells in the CNS development, neural stem cells(NSCs) play an important role in the formation of brain structures and in the establishment of integrity of neural functions. Previous studies have found that Notch signaling pathway is essential for the process of cell proliferation,self-renewal and differentiation in neural stem. However, the detailed mechanism has not been fully elucidated and need in-depth analysis. Recent studies indicate that Homeodoma-ininteracting protein kinase 2(HIPK2) could be an important player in Notch signaling pathway during regulation of NSCs differentiation. In the pilot study, we specificly knockout site of recombination signal binding protein Jκ(RBP-J) which’s thekey transcription factor in Notch signaling pathway in brain tissue of mice and perform gene chip for the detection of brain tissue. The results of gene chip showed that the expression of Hipk2 was down-regulated when specific blocking Notch signaling pathway,so we hypothesized that Hipk2 may play gulatory role in the process of proliferation,differentiation and apoptosis of NSCs in the regulation of the Notch signaling pathway.We sought to explore the regulatory effect of Notch signaling pathway on Hipk2 and study the effect of Hipk2 on proliferation and apoptosis of NSCs. These studies will open up a new perspective for further understanding of NSCs proliferation, self-renewal and differentiation mechanism.Methods:(1)To detect the expression of Hipk2 by using the method of real-time quantitative polymerase chain reaction(RT-PCR) in neural stem / progenitor cell line(C17.2 cells),primary neural stem cells and the cells which derived from the differentiation of primary neural stem cells induced by 7 days. The expression of Hipk2 was observed by in situ hybridization in brain and retinal tissue in E12.5 and E14.5 day of mouse embryo.(2)The expression plasmid of Hipk2(PCAG-Hipk2-IRES-EGFP) was constructed by using molecular cloning technology and tested by Bacteria Liquid polymerase chain reaction and the reaction of double enzyme digestion. Then the expression plasmid of Hipk2(PCAG-Hipk2-IRES-EGFP) and Vector(PCAG-IRES-EGFP) were transfected into C17.2 cells by using technology of liposome transfection. After C17.2 cells was effected48 hours, cell proliferation was examined by MTT, Flow cytometry methods,immunocytochemistry of Ki67 and Edu. Using Western blot and Flow cytometry methods,we analyzed cell apoptosis under the action of Hipk2.(3)To use GSI(gamma secretase inhibitor) to intervene the growth of C17.2 Cells,RT-PCR and Western blot were used to detect the expression of Hipk2 in C17.2 cells. In order to find the possible site of Notch signaling in Hipk2 promoter, softwares including TRANSFAC, NCBI, promoter scan software were used to analyze Hipk2’s promoter. Weconstructed the luciferase reporter plasmid of PGL3-Basic-Hipk2-promoter reporter gene vector(PGL3-Basic-Hipk2-promoter) by using molecular cloning technology and judged it by Bacteria Liquid polymerase chain reaction and the reaction of double enzyme digestion. Furthermore, PGL3-Basic-Hipk2-promoter and p RL-TK Vector were transfected into C17.2 cells / HT-22 cells / 293 T cells / 3T3 cells by using the technology of liposome transfection, luciferase activity of Hipk2 was detected by luciferase reporter assays in different cells. PGL3-Basic-Hipk2-promoter, p RL-TK Vector and PCAG-GFP were co-transfected into 293 T cells / C17.2 cells by using the technology of liposome transfection, and then detected luciferase activity of Hipk2 by using the technology of liposome transfection in the presence of the intracellular of receptor in Notch signaling pathway. In addition, the same experiment scheme and method were used to study effects of RBP-J / Hes1 on Hipk2.Results:(1)The expression of Hipk2 could be detected in C17.2 cells, NSCs and the cells which derived from the differentiation of NSCs. Compared with the primary neural stem cells, the expression of Hipk2 was decreased in differentiation of primary NSCs after 7days. In situ hybridization histochemistry showed that Hipk2 is widely expressed in brain and retina of embryonic mice. Moreover, Hipk2 has a strong expression in the subventricular zone, retinal neuroblast layer and ganglion cell layer.(2)We successfully constructed plasmid of Hipk2’s gene expression sequence, and examined the effect of Hipk2’s over-expression by MTT, Flow cytometry methods,immunocytochemistry of Ki67 and Edu. The results showed that the expression of Hipk2 could inhibit the proliferation of C17.2 cells. The results from flow cytometric detection of apoptosis and expression levels of activation of caspase-3 and Bcl-2 showed that Hipk2 promoted the apoptosis of C17.2 cells.( 3) RT-PCR and Western blot showed that compared with the control group(dimethyl sulfoxide, DMSO), the expression of Hipk2 in the intervention group of GSIwere decreased in the level of transcription and protein. Bioinformatics analysis from TRANSFAC, NCBI and promoter scan database showed there are two binding sites of RBP-J and nine binding sites of Hes1 in Hipk2 promoter. We successfully constructed the plasmid of PGL3-Basic-Hipk2-promoter, and detected the expression of Hipk2 in different cells. The result of dual luciferase reporter assay showed that the expression of Hipk2 was different in differential cells. Based on the above results, we selected 293 T cells that have a relatively higher expression of Hipk2, and neuron specific C17.2 cells as cell basics of research. The result of dual luciferase reporter assay showed that NICD and RBP-J could interact with promoter of Hipk2 and activate the expression of Hipk2 in 293 T cells and C17.2 cells. However, Hes1 could strongly inhibit expression of Hipk2 promoter in 293 T cells, but this effect was not obvious in C17.2 cells.Conclusion:(1)Hipk2 was highly expressed in neural stem / progenitor cell lines, primary neural stem cells, the brain and retina of embryonic mouse.(2)Hipk2 inhibited proliferation and promoted apoptosis in neural stem / progenitor cells.( 3) NICD and RBP-J of the key molecules in Notch signaling pathway could significantly regulated the expression of Hipk2 promoter.