Preliminary Study Of Mechanism About Beagle ERβ And Splice Isoforms ERβ₄₁₉, ERβ₄₃₁

Estrogen regulate physiological activities through specific intracellular estrogen receptor(ERS) signaling pathway, which classified two different types, commonly known as genomic and non genomic pathway. This research makes a primary exploration on the mechanism of interaction between ERβ, ERβ419, ERβ431 and extrogen, based on beagle ERβ and two splice isoforms ERβ419 and ERβ431, which we have successfully obtained before.(1) lentiviral vectors which express ERβ, ERβ419 and ERβ431 were constructed and packed : full-length genes of ERβ,ERβ419 and ERβ431 are amplified by using recombinant plasmid pEGFG-N1-ERβ,pEGFG-N1-ERβ419 and pEGFG-N1-ERβ431 as template, combined to pMD18-T vector throμgh gel extraction and purfication and then linked to LV5 and Lenti-OE-Flag vector seperately. The constructed recombinant lentiviral vector LV5-ERβ, Lenti-OE-Flag-ERβ419,Lenti-OE-Flag-ERβ431 are transfected into HEK293 T cells to obtain lentivirus eventually.(2)Cell lines of stable expression were established. HEK293 T cells are cultured two weeks with antibiotics in culture media, which have been infected by recombinant lentivirus. Screen three cell lines of which target protein steady expressed, and verify the results via Western Blotting.(3)The subcellular localization of target protein. Dye membrane by PKH26 and nucles in Hoechst33258, observe the localization of target protein with GFP under laser scanning confocal microscope.(4) The transcriptional activity of ERβ, ERβ419 and ER431 expressed vector were detected: 6 groups were conducted as follows, control groups, LV5-ERβ groups, LV5-NC groups,Lenti-OE-Flag-ERβ431 groups, Lenti-OE-Flag-ERβ419 groups, Lenti-OE-Flag-NC groups. Each group was further divided into two groups, each group of cells transiently transfected with the recombinant plasmid ERE-LUC and RLUC, In each one group with a final concentration of 10μmoL/L E2, while the other group with an equal volume of ethanol. The luciferase activity of each group was tested after adding the stimulation.Results:(1) Full-length genes of ERβ, ERβ419 and ERβ431 are amplified, and then cloned to LV5 and Lenti-OE-Flag vector successfully. Recombinant lentivirus plasmid was obtained by using recombinant plasmid LV5-ERβ, Lenti-OE-Flag-ERβ431 and Lenti-OE-Flag-ERβ419.(2)After infected HEK293 T cell by lentivirus, cell lines that stably expressed ERβ, ERβ419 andERβ431 were successfully established through puromycin screen.(3) Cells dyed by PKH26 and Hoechst33258, successfully localized three target proteins. ERβ and ERβ419 are located in nucleus and cytoplasm, while ERβ431 is located in cytoplasm.(4) ERE-LUC and RLUC recombinant plasmid was succesfully constructed. Compared with anhydrous ethanol group, The transcriptional activity of Beagle ERβ with estrogen treatment was significantly increased(P<0.01), while there was no significant change neither ERβ419 nor ERβ431(P>0.05).Conclusion: Beagle ERβ and ERβ419 are located in nucles and cytoplasm. ERβ431 is located in cytoplasm. This is probably due to lack of NSL, leading to ERβ431 not transporting to nucles;succesfully constructed transcriptional activation system of Beagle ERβ, but the partly lack of LBD lead to ERβ419 and ERβ431 can’t combine with E2, then transcriptional activity could not be activated.