| ObjectiveOur aim is to study the regulation of gene associated with retinoid interferon induced mortality 19?GRIM19?on vascular endothelial growth factor?VEGF?in granulosa cells of polycystic ovary syndrome?PCOS?,and to explore its potential molecular regulatory mechanism.MethodsThe primary granulosa cells?PGC?from polycystic ovary syndrome?PCOS?patients and normal human were selected as the research objectto study the regulation of GRIM19 on VEGF in granulosa cells.siRNA silencing technology and plasmid overexpression technology were used to down-regulate or up-regulate GRIM19 gene expression in PGC cells,the relative expression differences of VEGF mRNA and protein was detected before and after treatment.At the same time,PGC cells was incubated with different concentrations of testosterone,the relative expression of GRIM19 and VEGF mRNA and protein were detected in PGC cells to explore the effect of testosterone on the expression of GRIM19 and VEGF in granulosa cell from normal human.Finally,human ovarian granulosa cell line?KGN cells?was selected as our model,and siRNA interference technology was used to down-regulate GRIM19 gene to detect the difference in VEGF mRNA and protein relative expression.In addition,KGN cells overexpressing GRIM19 by transfected pEGFP-GRIM19 plasmid was also executed to detect changes in the relative expression levels of VEGF mRNA and protein after overexpression of GRIM19.Results?.Regulation of VEGF expression by GRIM19 in PGCThe results of real-time quantitative PCR?qPCR?of PGC cells without any treatment were as follows:The expression level of mRNA in GRIM19 in PGC patients was lower than that in normal people?P<0.001?;The expression level of mRNA of GRIM19 in PGC of PCOS patients was 4.13 times lower than that of normal people?P<0.001?;The mRNA expression of VEGF was 4.42 times higher than that of normal people?P<0.001?.The results of Western blot?WB?showed that the expression level of GRIM19 protein in PCOS granule cells was 3.3 times lower than that of normal people?P<0.001?;The protein expression level of VEGF was 4.2times higher than that of normal people?P<0.001?.It is indicated that there is more GRIM19 expression in normal human PGC,and the expression level of VEGF in PGC of PCOSpatients is higher than that of normal people.After silencing the GRIM19 gene of PGC in PCOS patients by specific siRNA,the results of qPCR and WB showed that the mRNA and protein expression levels of VEGF increased by 3.93 times?P<0.001?and 2.47 times?P<0.001?in PGC of PCOS,respectively.Transfection of PGC of PCOS with pEGFP-GRIM19plasmid,the results of qPCR and WB showed that the mRNA and protein expression levels of VEGF were decreased by 2.11 fold?P<0.001?and 2.40 fold?P<0.001?,respectively.?.Effect of testosterone on the expression of GRIM19 and VEGF in PGCWhen normal human PGC were incubated with different concentrations of testosterone,the expression of GRIM19 was decreased with increasing testosterone concentration.At the testosterone concentration of 1×10-4 mmol/L,the expression level of GRIM19 was significantly lower than that of the control group,which was7.01 times?P<0.001?lower at the mRNA level and 7.85 times?P<0.001?lower at the protein level.In contrast,the expression of VEGF and STAT3 increases with increasing testosterone concentration.When the testosterone concentration was 1×10-4mmol/L,the mRNA and protein expression of VEGF increased by 4.54 times?P<0.001?and 4.03 times?P<0.001?,respectively.STAT3 increased by 8.43 times?P<0.001?.The above results indicate that testosterone can increase the expression of VEGF and decrease the expression of GRIM19.This may be because testosterone inhibits the expression of GRIM19,which reduces the inhibition of STAT3 by GRIM19.This demonstrates that testosterone may play an important role in the occurrence and development of PCOS.?.Regulation of VEGF expression by GRIM19 in granulosa cell lines?KGN cells?When the GRIM19 gene of granulosa cell line KGN cells were silenced with siRNA,qPCR and WB results showed that VEGF in KGN cells increased by 3.97times?P<0.001?and 5.11 times?P<0.001?in mRNA and protein levels,respectively.The mRNA levels of STAT3 increased by 3.41 fold?P<0.001?.When KGN cells transfected with pEGFP-GRIM19 plasmid and overexpressed GRIM19,qPCR and WB assays showed that VEGF mRNA and protein decreased by 2.13 fold and 3.09 fold?P<0.001?,respectively.Simultaneously,the protein of p-STAT3 was decreased by 5.47 times?P<0.001?.Conclusion1.In PGC patients with PCOS,VEGF expression was negatively correlated with GRIM19.2.Testosterone can reduce the expression of GRIM19 in PGC,which leads to increased expression of VEGF and p-STAT3.It may be caused by testosterone inhibiting the expression of GRIM19 and activating the STAT3 signaling pathway.3.The expression of VEGF in KGN cells was also negatively correlated with GRIM19,probably due to activation of STAT3 signaling pathway. |
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