Construction Of Mouse HSP10 SiRNA And Overexpression Adenovirus Vector And Investigation Of Its Role On The Apoptosis Of Mouse Ovarian Granulosa Cells

Objective: HSP10(Heat Shock Protein 10,HSP10)also known as CPN10, GROES or HSPE1, a member of micromolecule HSPs family. HSP10 has been considered mainly as a chaperonin, in coordination with HSP60, promoting the effect on protein folding. Our previous studies showed that HSP10 was highly expressed in normal ovaries compared to those from polycystic ovary syndrome. Recent data suggest that HSP10 may be not only a component of the folding machine but also an active player of the cell signaling network, influencing cell cycle, nucleocytoplasmic transport, and metabolism, with putative roles in the lack of cell differentiation and in the inhibition of apoptosis. Howerver, the role of HSP10 in the development of polycystic ovary syndrome have not been documented before. In this study, We constructed HSP10 siRNA expression and HSP10 over-expression recombinant adenoviruses. Then, HSP10 recombinant adenovirus vector was transfected into mouse follicular granulosa cells. The aim of this study was to explore the effects of HSP10 on the apoptosis of mouse follicular granulosa cells and further demonstrate the role and mechanism of HSP10 on the etiology and pathophysiology development of polycystic ovary syndrome at molecular level, which will provide strategies focusing on prevention and treatment of polycystic ovary syndrome.Methods: (1) The two complementary 64 nt oligonucleotides was designed and synthesized according to the target sequence. These oligonucleotides were annealed and ligated to pShuttle-H1 to get plasmid pShuttle-H1-HSP10 and confirmed by EcoRI digestion. The inserted sequences were confirmed by dideoxy sequencing. Plasmid pShuttle-H1-HSP10 was digested with HindIII and Not? and subcloned into the same sites of pAdTrack-CMV. The ligation product was linearized by PmeI and transformed into competent E.coli BJ-5183 cells; backbone plasmid was then transformed into these cells. The resulting homologously recombined adenoviral plasmid, pAdTrack-H1-siRNA/HSP10, was verified by PacI digest and sequencing. The recombinant vector was amplified in DH5αE. coli. Control virus was generated using pAdTrack-H1-siRNA/control vector. AD-293 cells were transfected with 4μg of PacI-linearized recombinant and 20μl of lipofectin reagent . After 10 days, the cells were harvested and lysed by four cycles of freeze/thaw to obtain viral supernatants. The virus was amplified by repeated infection of AD-293 cells. (2) HSP10 cDNA was amplified by RT-PCR from mouse ovary and was cloned into PGEM-T vector. HSP10 cDNA was then subcloned into the shuttle vector pAdTrack-CMV for the construction of pAdtrack- HSP10. The shuttle vector was linearized with PmeⅠand subsequently transformed into E.coli BJ-5183 cells with adenovirus backbone plasmid pAdEasy-1 to achieve homologous recombination. After digestion with PacⅠ, it was transfected into AD-293 cells for packaging and amplification. The appearance of recombinant adenovirus were determined by GFP expression. The expression of HSP10 gene was confirmed by PCR analyses. (3) Ovaries of immature PMSG-treated mice were cleaned of surrounding connective tissue and placed into DMEM/F12 supplemented with 10% fetal bovine serum (FBS). DMEM/F12 also contained 2mM glutamine, penicillin (100 units/ml), and streptomycin (100 lg/ml). Granulosa cells were harvested by needle puncture of ovarian follicles. Cells were re-covered by centrifugation at 1000g for 5 min, washed twice, inoculated into 6-well culture dishes at a density of 5×105 cells/ml, and cultured at 37℃in a humidified atmosphere of 95% air and 5% CO2. Granulosa cells were infected with recombinant AdCMV-H1-siRNA/HSP10 and AdCMV-HSP10 adenovirus.After infecting for 48 h. Knocking down and overexpression of HSP10 gene in mouse granulosa cells was demonstrated by western blot. The granulosa cells were collected to compare the difference of apoptosis level between HSP10-knocking down group and control group by flow cytometry and TUNEL assay.Results: (1) It was confirmed by restriction endonuclease analysis and sequencing that oligonueleotide sequence was inserted into pShuttle-H1 successfully., further pShuttle-H1-siRNA connected with pAdTrack-CMV after digestion, and then built a new shuttle plasmid pATC-H1-siRNA. Then, recombinant adenovirus vector AdCMV-siRNA/HSP10 was constructed successfully. After transfected it into AD-293 cells in which the recombinant adenovirus are packaged and amplified, the high titer recombinant adenoviruses were harvest. (2) A 309bp cDNA was amplified by RT-PCR from mouse ovary with correct sequence. Then, recombinant adenovirus vector AdCMV-HSP10 was constructed successfully. After transfected it into AD-293 cells in which the recombinant adenovirus are packaged and amplified, the high titer recombinant adenoviruses were harvest. (3) Mouse granulosa cells culture approach was established successfully. HSP10 recombinant adenovirus vector was transfected into mouse granulosa cells by this platform. Compared with control group, the expression of HSP10 was decreased in mouse granulosa cells infected with AdCMV-siRNA/HSP10, and increased in mouse granulosa cells infected with AdCMV-HSP10(p<0.05).Finally, The granulosa cells were collected to compare the difference of apoptosis level between HSP10-knockdown group and control group, as well as HSP10-overexpression group and control group by flow cytometry and TUNEL assay. The result indicates that knockdown of HSP10 in mouse granulosa cells significantly induced apoptosis(p<0.05 ),while overexpression of HSP10 in mouse granulosa cells significantly suppressed apoptosis(p<0.05).Conclusion:(1)The recombinant adenovirus exerting RNA interference to HSP10 gene and mouse HSP10 recombinant adenovirus have been constructed successfully, which offers a basis for the research into the pathogenesis of PCOS.(2) Our experiments have proved that knockdown of HSP10 induced apoptosis of mouse Ovarian Granulosa Cells. while overexpression of HSP10 in mouse granulosa cells suppressed apoptosis. This suggested HSP10 might has some effects on the apoptosis of mouse ovarian granulosa cells. The decreased expression of HSP10 in PCOS may contribute to the development of ovulation failure.