SRp30c Lead To Glucocorticoid Resistance In HEI-OC1 Cells

Objective:Our purpose is to explore the expression level of IL-2,TNFa,SRp30 c,GRβ,GRα after LPS treatment of HEI-OC1 and explore whether SRp30 c reduced the sensitivity of glucocorticoid in HEI-OC1 cells,producing glucocorticoid resistance.Method:The HEI-OC1 cell line was used as the research object.Use different concentrations(50,100ug/ml)of endotoxin lipopolysaccharide-stimulated cochlear hair cells(HEI-OC1).Western blot was used to determine the expression levels of IL-2,TNF-a,SRp30 c,GRβ,and GRα proteins in the 100ug/ml-LPS group,50ug/ml-LPS group and the control group;real-time fluorescent quantitative PCR(q RT-PCR)was used to determine the expressions of IL-2,TNF-a,SRp30 c,GRβ,and GRα mRNA in the three groups of tissues;SRp30c siRNA plasmid and SRp30 c overexpression plasmid were used to transfect HEI-OC1 cells,and Western blot was used to determine GRβ,AP-1,NF-κB and HDAC2 proteins in SRp30 c siRNA group,SRp30 c overexpression group,empty plasmid group and control group;MTT method was used to detect the cell proliferation activity of SRp30 c overexpression group,SRp30 c overexpression+dexamethasone group,dexamethasone group and control group at 0h,24 h,36h,48 h.Result:1.By using the Western blot method,the expressions of IL-2,TNF-a,SRp30 c,and GRβ in the 50ug/ml-LPS group was significantly higher than control,the expressions of GRα in the 50ug/ml-LPS group was significantly lower than control,the expressions of IL-2,TNF-a,SRp30 c,and GRβ in the 50ug/ml-LPS group was significantly higher than 100ug/ml-LPS group,the expressions of GRα in the50ug/ml-LPS group was significantly lower than 100ug/ml-LPS group.2.By using the q RT-PCR method,the expressions of IL-2,TNF-a,SRp30 c,and GRβmRNA in the 50ug/ml-LPS group was significantly higher than control group,the expressions of GRα in the 50ug/ml-LPS group was significantly lower than control,the expressions of IL-2,TNF-a,SRp30 c,and GRβmRNA in the 50ug/ml-LPS group was higher than 100ug/ml-LPS group(P value=0.4342、0.0020、0.1104、0.2428respectively),the expressions of GRα in the 50ug/ml-LPS group was significantly lower than 100ug/ml-LPS group.3.By using the Western blot method,the expressions of GRβ、AP-1、NF-κB、HDAC2 in the SRp30 c overexpression group was significantly higher than control,the expressions of GRβ、AP-1、NF-κB、HDAC2 in the SRp30 c overexpression group was significantly higher than empty plasmid group.4.The expressions of GRβ、AP-1、NF-κB、HDAC2 in the SRp30 c siRNA group was significantly lower than control,the expressions of GRβ、AP-1、NF-κB、HDAC2in the SRp30 c siRNA group was significantly lower than empty plasmid group.5.MTT method was used to determine cell proliferation activity in the SRp30 c overexpression group 、 SRp30 c overexpression + Dexamethasone group 、Dexamethasone group and control group at 0h,24 h,36h,and 48 h,compared with the control group,the cell proliferation activity of the Dexamethasone group was lower,the proliferation activity of the cells was significantly increased after overexpression of SRp30 c,Dexamethasone inhibited the proliferation of HEI-OC1 cells,but its inhibitory effect was weakened due to the transfection of the SRp30 c overexpression plasmid.Conclusion:SRp30c induces glucocorticoid resistance by up-regulating the expression of GRβ in HEI-OCI cells.