The Study On The Function And Mechanism Of Methyltransferase Set7 On Hypoxia-Induced Apoptosis Of Spermatocyte GC-2 Cells

Infertility has become a public health issue of global concern,affecting millions of people of childbearing age.Male infertility accounts for more than 50%of the total.Oligospermia is a common cause of male infertility.Hypoxia environment in high altitude and hypoxia condition in testis or epididymis in the body have negative effects on male reproductive function.Some studies have shown that under hypoxia,the apoptosis rate of spermatogenic cells increases,and the condition in spermatocyte is the most significant.Therefore,clarifying the mechanism of spermatocyte apoptosis induced by hypoxia is conducive to the prevention and treatment of oligospermia.Hypoxia usually causes damage to the body or tissue,resulting in cell apoptosis.Hypoxia inducible factor-1α（hypoxia-inducible factor-1α,HIF-1α）is a highly specific transcription factor under hypoxia,which is stably expressed in tissues and cells under hypoxia,and participates in the transcription of multiple genes that regulate hypoxia response.HIF-1αis associated with decreased spermatogenesis caused by varicocele（VC）,obstructive sleep apnea syndrome（OSAS）,asthma and other diseases.However,the expression of HIF-1αin the process of hypoxia induced spermatocyte apoptosis and its regulatory mechanism still need to be further explored.SET domain containing lysine methyltransferase7（SET7）is one of the methyltransferases containing the SET domain.It participates in the methylation of histones and non-histone proteins,and affects the stability of proteins,gene expression and regulation.Set7 is involved in the pathogenesis of tumor,diabetes,chronic kidney disease and other diseases.Some studies have shown that Set7-mediated lysine methylation has a negative regulatory effect on HIF-1αtranscription activity.To clarify the function and mechanism of methyltransferase Set7 on hypoxia induced spermatocyte apoptosis is important for the prevention and treatment of oligospermia.ObjectiveBy exploring the regulation of Set7 on HIF-1αand its downstream genes,and the mechanism of Set7 on spermatocyte apoptosis under hypoxia,we can provide new targets and basic theoretical support for the prevention and treatment of oligospermia.Methods1.Mouse spermatocyte line GC-2 was treated under hypoxia for 24 hours,the m RNA expression levels of Glut1,Pgk1,Vegf,Pkm1,Pkm2,and Set7 were detected by RT-q PCR.2.After transfection of Set7 plasmid into GC-2 cells for 24 hours,the effect of Set7 on the activity of EPO promoter in hypoxic and normoxic groups was analyzed by luciferase assay.3.Under normoxic,hypoxic or CoCl₂-induced hypoxia conditions,GC-2 cells were transfected with Myc-Set7 plasmids.RT-q PCR was used to detect the m RNA expression level of HIF-1αdownstream genes Glut1,Pgk1,Pkm1 in each group.4.Under normoxic and hypoxic conditions,GC-2 cells were transfected with 1μg or 2μg Myc-Set7 plasmids.Western blot was used to detect the protein expression levels of HIF-1αand Set7 in each group.5.GC-2 cells stably expressing Set7 or its enzymatically dead mutant Set7-H297A were established by lentivirus methods.After hypoxia treatment,the apoptosis rate of each group was analyzed by flow cytometry and the apoptosis of GC-2 cells was observed by fluorescence microscope.Results1.Compared with the normoxia group（21%oxygen concentration）,the m RNA expression levels of Glut1（P<0.001）,Pgk1（P<0.001）,Vegf（P<0.001）,Pkm1（P<0.001）and Pkm2（P<0.001）in the hypoxia group（1%oxygen concentration）were significantly increased;Compared with the normoxia group（21%oxygen concentration）,the expression level of Set7（P<0.001）m RNA in the hypoxia group（1%oxygen concentration）was significantly decreased（P<0.05）.2.In the normoxia group（21%oxygen concentration）,overexpression of Set7 had no significant effects on the activity of EPO promoter（P>0.05）.However,in the hypoxia group,overexpression of Set7 could significantly suppress the activity of EPO promoter（P<0.01）.3.In the CoCl₂-induced hypoxia condition,compared with the empty plasmid,the m RNA expression level of Glut1 in GC-2 cells decreased significantly after transfection of Myc-Set7 plasmid（P<0.05）;In the hypoxia group（1%oxygen concentration）,compared with the empty plasmid,the m RNA expression levels of Glut1（P<0.01）,Pkm1（P<0.001）and Pgk1（P<0.01）were significantly decreased after transfection of Myc-Set7 plasmid.4.In the normoxic group（21%oxygen concentration）and the hypoxic group（1%oxygen concentration）,compared with the empty plasmid,the expression level of HIF-1protein in GC-2 cells transfected with Set7 overexpression plasmid 1μg and 2μg showed no significant difference;However,compared with the normoxia group（21%oxygen concentration）,the expression level of HIF-1αprotein in the hypoxia group（1%oxygen concentration）increased significantly.5.In the normal oxygen group（21%oxygen concentration）,compared with p HAGE,p HAGE-Set7 and p HAGE-Set7-H297A overexpressed cells showed significant difference in the expression level of apoptosis（P>0.05）.In the hypoxia group（1%oxygen concentration）,compared with p HAGE,the apoptosis rate of GC-2cells significantly increased in p HAGE-Set7 overexpressed cells（P<0.001）,but compared with p HAGE-Set7,the apoptosis rate of GC-2 cells significantly decreased in p HAGE-Set7-H297A overexpressed cells（P<0.001）.Conclusion1.After hypoxia induced GC-2 cells,the expression of Glut1,Pgk1,Vegf,Pkm1and Pkm2 m RNA was activated,while the expression of Set7 m RNA was inhibited.2.In hypoxia induced GC-2 cells,the activity of EPO promoter was inhibited upon overexpression of Set7 plasmid in GC-2 cells.3.In CoCl2 hypoxia model,Set7 could inhibit the m RNA levels of Glut1 in GC-2cells;Similarly,under the condition of hypoxia（1%oxygen concentration）,Set7 could inhibit the m RNA levels of Glut1,Pkm1 and Pgk1 in GC-2 cells.4.After hypoxia induced GC-2 cells,Set7 had no effect on HIF-1αprotein expression level in GC-2 cells.5.After hypoxia induced GC-2 cells,Set7 promoted cell apoptosis.