A Preliminary Study On The Roles Of FSHR In Tumor Angiogenesis

Follicle-stimulating hormone receptor(FSHR)is a transmembrane glycoprotein,which belongs to the family of G protein-coupled receptor family.In adults human and animals,FSHR only expressed on the surface of the testicular sertoli cells and ovarian granulosa cells and low expressed on the testicular and ovarian vascular endothelial cells.Gonadotropin-follicle-stimulating hormone(FSH)plays important roles in the process of gonad development by combining with the FSHR expressed in testicular support cells and ovarian granular cell surfaces.In recent years,the study found that FSHR not only expressed in germ cells,but also specifically expressed on most human tumor vascular endothelial cell,suggesting that FSHR may play a role in the formation of tumor angiogenesis.In order to explore the roles of FSHR in tumor angiogenesis,we used human umbilical vein endothelial cells(HUVEC)as a model of vascular formation,to analyze the effects of FSH on the proliferation,migration and tube formation of HUVEC.In addition,we evaluated the effects of a FSHR specific nanobody and FSH antagonist suramin on the proliferation,migration and the tube formation of HUVEC.This results will contribute to clarify the roles of FSHR and benefit to design FSHR-based anti-vascular therapy in the future.There are two main part research contents in the thesis:1.Preliminary illustrate the roles of FSHR in tumor angiogenesis in vitroThe roles of FSH-FSHR in angiogenesis was investigated by HUVEC cells in vitro.Firstly,the expression levels of FSHR on HUVEC cells were determined by RT-PCR,western-blot,cell immunofluorescence microscopy and tube formation.The results showed that FSHR were expressed on HUVEC and Caov-3 cells but not expressed on Skov-3 cells.Secondly,in order to deep analyze FSHR roles in angiogenesis,HUVEC cells were treated with hormone FSH(600 ng/m L)in the presence of different concentrations of FSH antagonists Suramin [0,100,1000 ?g/mL],and the proliferation,migration and tube formation of HUVEC were detected.The results showed that FSH(600 ng/mL)significantly promoted HUVEC cell migration,proliferation and tube formation compared with untreated cells(P<0.05).Furthermore,Suramin showed markedly inhibition on FSH stimulated migration,proliferation and tube formation of HUVEC in a dose-dependent manner(P<0.05).In summary,FSH promotes HUVEC cells tube formation,and Suramin has some inhibitory effect on the migration and proliferation of FSHR positive cell,such as HUVEC and caov-3cell,but has no effect on the FSHR negative CT-26 cells.The results suggest that FSH might make effect through its receptor FSHR.2.Improvement the affinity of anti-FHSR Nanobody VHH06Given the innate defect of naive phage display library,the nanobody VHH-06 exhibited weak binding with FSHR.In order to improve its affinity for well application,two different approaches were employed in remolding the antibody structure.Firstly,site-directed saturated mutagenesis were used to introduce randomized mutation in VHH-06 CDR3 region.A new synthetic VHH-06 CDR3 random mutated DNA sequences were generated and ligated into pMECS vector.The new library with 7.36×108 transformants(namely(NNY)NL-06/?CDR3)was generated by electrotransformation of compentent E.coli TGI with pMECS-VHH-06/?CDR3.By six rounds of enrichment with the antigen His-FSHR234,the antigen specific enrichments were appeared from the point of the output number of phage.In polyclonal phage ELISA examination it was showed that enrichment appeared at the fourth round,but in the subsequent detection by monoclonal phage ELISA with 100 random selected clones,there were no clones with obvious binding with FSHR.For exploring the reasons result in the failure in recovering positive FSHR binders,the diversity and amino acids distribution in CDR3 regions in library were analyzed.It was drawed a conclusion that the poor diversity and detrimental amino acids distribution might contributed a fail results.Secondly,a coiled-coil peptide derived from human cartilage oligomeric matrix protein(COMP48)was used to make a multimeric VHH-06 for improve its affinity.The DNA of 48 amino acids COMP48 peptide was combined with VHH-06 at its carboxyl terminal by a linker with DNA synthesis.The VHH-06-COMP48 DNA fragment was ligated into pET22 b vector and transformed competent BL21 E.coli forexpression and purification.The recombinant VHH-06-COMP48 protein was showed an apparent molecular mass of 110 kD under nonreducing and 25 k D under reducing SDS-PAGE.It indicated a tetrameric VHH-06-COMP48 was formed.Nevertheless,this tetramers give no improved binding activity with FSHR in ELISA detection.In conclusion,we demonstrated in this work that FSH might play a role in vascular formation with increased proliferation,tube formation and migration on a HUVEC cell in vitro.This results will helpful to further clearify the role of FSHR in angiogenesis and give a support to design FSHR targeted therapy for cancer treatment.The trying to improve the affinity of FSHR nanobody VHH-06 with randomized CDR3 mutated library and multimeric VHH-06 approaches were proved not successful.The reason might be ascribed to poor functional diversity and artificial mutation of library,and incorrect protein folding of tetrameric VHH-06-COMP48.