Screening LRP16 interacting proteins and functional regulation of
|PLA Postgraduate Medical School
|Pathology and Pathophysiology
|LRP16 Protein Interactions Yeast two-hybrid technology Androgen receptor Coactivator Proliferation
Objective: ① Application yeast two-hybrid screening human breast cancer cell line MCF-7 cDNA library with LRP16 interacting protein; ② confirm LRP16 with AR and other nuclear receptors and NF-kB interaction; ③ Research LRP16 the AR transcriptional regulation of the activity and its effect on prostate cancer cell lines with different concentrations of androgen LNCap stimulated proliferation; ④ observation of androgen on the regulation of LRP16 expression. Methods: ① pLPC-LRP16 plasmid was digested agarose gel electrophoresis, recovery, get LRP16 gene fragments, connect it to the yeast two-hybrid system bait vector pGBKT7, the insert by digestion verify their correct orientation, it will re bait plasmids were transformed into yeast AH109 and Y187, and detect the presence or absence of self-activation in yeast and toxicity. Subsequently transformed yeast extract total protein by Western blot analysis bait plasmid expression in yeast to identify it as a bait protein is feasible. ② the bait plasmid pGBKT7-LRP16, MCF-7 ds cDNA library of fragments and linearized plasmid pGADT7-Rec co-transformed yeast AH109, ??in nutrient-deficient medium (SD /-Leu /-Trp /-His /-Ade) on screened positive colonies. Positive clones were extracted plasmid PCR, the PCR products were sequenced, and the yeast plasmid was transformed into E. coli Top10 get interacting with a single bait plasmid library plasmid and sequenced reply verify their interaction in yeast. ③ with co-immunoprecipitation (Co-IP) and GST pull-down and other in vivo, in vitro method validation LRP16 with ART-27 interaction; further confirmed using GST pull-down LRP16 with AR and other nuclear receptors and NF-kB directly interactions and verified LRP16 interacts with AR domain structure; ④ luciferase reporter gene assay LRP16 transcriptional activation of the AR and AR on LRP16 promoter regulation; ⑤ using MTT assay inhibition of endogenous LRP16 in Different concentrations of androgen stimulation on LNCap proliferation; ⑥ at different concentrations and different time points testosterone stimulation, detected by Western blot LRP16 protein expression levels and the prostate cancer cell lines in different LRP16 expression. Results: ① The recombinant plasmids were digested bait verification, fragment size and direction of insertion is correct, after it was transformed into yeast no self-activation and toxicity, Western blot confirmed bait plasmid in yeast can properly express recombinant human LRP16 protein; ② obtain eight candidate proteins interacting with LRP16, select 4 in yeast reply verify, which has three positive clones can be grown; ③ ART-27 interacts with LRP16 and AR, ERβ, PPARα, PPARγ and other nuclear Receptor and NF-kB were a direct interaction with LRP16; ④ LRP16 by a unique C-terminal domain and the AR LBD domain interactions; ⑤ in the role of testosterone, the AR overexpression LRP16 enhancement of transcriptional activity, inhibition of endogenous expression of LRP16 can weaken the AR transcriptional activation; ⑥ reduce the expression of endogenous LRP16 inhibit androgen levels LNCap different cell proliferation; ⑦ low concentrations of testosterone can LRP16 protein expression increased, the role of testosterone ie 3h LRP16 protein expression can multiply, and AR for LRP16 promoter activity enhancement effect exists; ③ AR-positive prostate cancer cell lines LNCap than AR-negative prostate cancer cell lines DU145 in LRP16 protein content. Conclusion: ① Application yeast two-hybrid screening a family of basic functions explicitly interacting with LRP16 candidate proteins; ② LRP16 with AR, NF-kB direct interaction, and this interaction is not dependent on the presence of ART-27; ③ LRP16 is part of the nuclear receptor family interacting protein factor; ④ androgen target gene LRP16 feedback reactivity enhancement of AR transcriptional activation is a coactivator of AR; ⑤ LRP16 may be regulated through interaction with AR androgen-sensitive prostate cancer cell growth.