FKBP52 Regulates The Balance Of Estrogen Receptor ER And Androgen Receptor AR By FOXA1

[Objectives] ER and AR belong to the members of the steroid hormone receptor superfamily, the balance of which is essential to the normal growth and development of organisms. The expression amount of ER and AR in healthy women is considerable, and some ER and AR imbalance in the incidence of disease within the organization is significantly higher than the normal tissue, indicating the importance of its balance in the human body. FKBP52 gene knockout male mice in the presence of mammary gland development, as a co chaperone protein FKBP52 through the participation in the formation of hormone receptor complexes play a function. Previous studies have found that ER/AR has an effect on the balance of FKBP52, but the specific molecular mechanism is still lack of understanding. This paper through the analysis of the results of RNA-seq, combined with immunohistochemical results suggested that FKBP52 may affect the balance between AR and ER through FOXA1, and explore the specific molecular mechanism.This study has important implications for understanding the role of FOXA1 in ER and AR balance, It is significant for us to realize the function of FOXA1 and its role in the occurrence and development of tumors.[Methods] 1) Phenotype analysis and behavioral experiment. By measuring mutation males, wild females and male the second and fourth finger length to calculate the ratio. Observed the change of sexual orientation in mutation males.2) RNA seq. We extracted RNA of nipple tissue in female WT and FKBP52 KO mice and skin tissue in WT male mice. Then, the samples of RNA were sent to sequence by company. We want to find some genes related to mammary gland development according to sequence results.3) Real-time PCR. The result show that the expression of ER, AR and some related genes changes in mammary gland.4) Immunohistochemistry. The expression and distribution of the related genes in the mammary gland development were detected; 5) Cell experiments. Detection of FOXA1 changes by interference and overexpression of FKBP52 gene in human breast cancer cell lines; (6) The detection protein stability treatment with the CHX; 7) Co-IP experiments. To explore the interaction between FKBP52 and FOXA1 in protein level.[Results] 1) Mutant male mice was different from wild males,which exhibits feminization characteristics.2) RNA sequencing results showed that the gene expression profile of mutant male mice is similar to the wild female. FOXA1, CITED1, Wnt5a, Co14a2, Sox-15, Sox-7, krt6-b, Ihh, BTC, Igfbp2 genes were significantly changed.3) Real-time PCR results are consistent with RNA-seq.4) The results of immunohistochemistry showed that the expression of ER was increased in FKBP52 gene knockout male mice. And the expression of ERaare similar to FOXA1. 5)In ER positive cell line MCF-7 downregulation FKBP52 cause FOXA1 increased upregulation FKBP52 cause FOXA1 decreased,but in ER negative cell line SK-BR-3 no changes.So FKBP52 and FOXA1 have a direct regulatory relationship, And this regulation relies on estrogen receptor ER.6) CHX experiments revealed that FKBP52 decreased the stability of FOXA1 protein.7) Co-IP experiments show that FKBP52 and FOXA1 have direct interaction at the protein level.[Conclusions] The lack of FKBP52 caused FOXA1 stability enhanced, thereby affecting the FOXA1 expression leads to ER activity enhancement ultimately changing the balance between ER and AR, which may produce abnormal development of the mammary gland.[Objective] To establish a rapid method for synthesis and detecting of gRNA.[Methods] According to the sequence of Nkp46 gene, the gRNA primers were designed. After Nkp46 gRNA DNA fragment was amplified by PCR technology using the synthesized gRNA core fragment as the template. The gRNA was in vitro transcripted. The efficiency and specificity of gRNA and Cas9 were detected by an in vitro reaction.[Results] The specificity and activity of Nkp46 gRNA are high, which could cut the target double-stranded DNA at the designed site successfully.[Conclusions] This protocol is a rapid method for synthesis and detecting of gRNA successfully.