Research About Cellular Functions And Transcription Activity Of RARα And NLS-RARα

PART I Construction of Recombinant Adenovirus Vector Carrying Human RARa Gene and Its Effect on NB4 Human Promyelocytic Leukemia CellsObjective:To construct and identify an adenovirus vector of containing RARa gene, then detect its expression in NB4 cells and its effect on this cell line treated with ATRA.Method:RARa gene was amplified by PCR using cDNA of NB4 cells as a template and inserted into shuttle plsmid pAdTrace-TO4. The constructed recombinant shuttle plasmid pAdTrace-TO4-RARa was digested with Pme I, and transformed to competent E.coli BJ5183 containing adenovirus backbone plasmid pAdEasy-1. The obtained recombinant adenovirus plasmid Ad-RARa was digested with Pac I and transfected to HEK293 cells for packaging. The obtained recombinant adenovirus Ad-RARa was subjected to three cycles of amplification, then determined titer and identified by PCR and sequencing. NB4 cells were infected by the recombinant adenovirus, with infection rate of 70%. Protein expression, proliferation and differentiation in NB4 cells of RARa were detected by Western blot, CCK-8 and flow cytometry respectively. Cell cycle was measured by Annexin V/PI.Result:Recombinant adenovirus Ad-RARa reached a titer of 5.2 X 105 pfu/mL after three cycles of amplification and recombinant adenovirus Ad-RARa was constructed correctly as proved by PCR and sequencing, and the RARa gene in Ad-RARa was successfully expressed in NB4 cells.Conclusion:Overexpressing NLS-RARa and RARa in leukemia cell line could enhance proliferation and inhibit differentiation.After being treated with a certain concentration of retinoic acid could induce apoptosis and promote cell differentiation and maturation. We concluded that there existed similar functions between NLS-RARa and RARa. NLS-RARa probably infected the cell biological function of leukemic cells through similar effection of RARa.PART II Binding sites and Transcription Regulation of RARa and NLS-RARaObjective:To prove NLS-RARa a variation transcription factor through researching the binding sites of NLS-RARa, compared with RARa’s, comfirm that NLS-RARa possibly competitive binding certain targeted genes of RARa, by which involved in the occurrence of APL.Method:Construct eukaryotic expression vector of RARa, RXRa and NLS-RARa, observed nuclear plasma distribution of RARa, RXRa and NLS-RARa by indirect immunofluorescence (IFF),then immunohistochemistry (Co-IP) was used to detect interaction between RARa, NLS-RARa and RXRa; gel electrophoretic mobility shift assay (EMSA) was used to detect whether RARa, NLS-RARa could bind retinoic acid response element DNA probes; finally measured regulation of retinoic acid response element activity of RARa and NLS-RARa with luciferase reporter experiments.Result:RARa and RXRa eukaryotic expression plasmid were constructed successfully; indirect immunofluorescence results showed that NLS-RARa distributed more in the nucleus compared with RARa; immunoprecipitation showed that NLS-RARa and RXRa existed interaction with each other; gel mobility shift assay and luciferase reporter proved NLS-RARa could bind retinoic acid response element DNA probes and regulated expression of correspondent luciferase plasmid.Conclution:This study constructed RARa, RXRa eukaryotic expression vector successfully and NLS-RARa may serve as a variation transcription factor involved in the occurrence of APL, enriched the signaling networks of APL pathogenesis.