孕酮和骨化三醇通过miR-590-5p靶向RelA调控子宫内膜癌细胞Ishikawa的细胞因子分泌

Progesterone and calcitriol use miR-590-5p to target RelA to regulate cytokine secretion in endometrial cancer cells Ishikawa

目的:探讨孕酮和骨化三醇抑制子宫内膜癌细胞Ishikawa细胞因子分泌的分子机制。方法:孕酮和骨化三醇分别处理Ishikawa细胞,检测细胞因子IL-1β、TNF-α、CXCL1及CXCL2的mRNA水平,NF-κB亚基RelA、RelB、c-Rel、p50、p52蛋白水平。荧光素酶报告检测RelA启动子活性。高通量测序检测孕酮和骨化三醇处理Ishikawa细胞后miRNA表达差异。过表达孕酮和骨化三醇共同调控的差异miRNA,检测RelA表达。结果:孕酮和骨化三醇分别处理Ishikawa细胞后,细胞因子IL1β、TNF-α、CXCL1、CXCL2分泌水平和表达均显著降低(P<0.05),NF-κB亚基RelA表达下调,NF-κB亚基RelB、c-Rel、p50、p52表达无改变。孕酮和骨化三醇分别处理Ishikawa细胞,RelA启动子活性变化差异无统计学意义(P>0.05),但RelA mRNA水平降低(P<0.05)。孕酮和骨化三醇分别处理Ishikawa细胞后,有110种miRNAs表达上调,骨化三醇处理后有57种miRNAs表达上调,其中14种miRNAs两种处理后均上调。过表达两种处理后均上调的miRNAs,发现过表达miR-590-5p可降低NF-κB亚基RelA mRNA水平(P<0.05),且靶向NF-κB亚基RelA-3'端非编码区(P<0.05)。此外,过表达miR-590-5p后RelA蛋白水平下降,敲低后NF-κB亚基RelA蛋白水平上升。过表达miR-590-5p后,细胞因子IL-1β、TNF-α、CXCL1、CXCL2表达均显著降低(P<0.05),敲低后细胞因子IL-1β、TNF-α、CXCL1、CXCL2表达均显著升高(P<0.05)。但同时过表达或敲低miR-590-5p和RelA时,细胞因子IL-1β、TNF-α、CXCL1、CXCL2表达无明显变化(P>0.05)。结论:孕酮和骨化三醇处理Ishikawa细胞后miR-590-5p表达升高,miR-590-5p靶向NF-κB亚基RelA-3'端非编码区,降低RelA蛋白水平,降低NF-κB复合体活性,最终下调细胞因子IL1β、TNF-α、CXCL1、CXCL2表达。本研究为孕酮和骨化三醇治疗子宫内膜癌的分子机制提供新依据,为孕酮和骨化三醇降低机体炎症反应提供理论依据。

Objective:To investigate molecular mechanism of progesterone and calcitriol inhibiting secretion of cytokines in endometrial cancer cells Ishikawa.Methods:Ishikawa cells were treated with progesterone and calcitriol,respectively,the mRNA levels of cytokines IL-1β,TNF-α,CXCL1 and CXCL2,and the protein levels of NF-κB subunits RelA,RelB,c-Rel,p50,and p52 were detected.Luciferase reporter was used to detect RelA promoter activity.High-throughput sequencing was used to detect differences in miRNA expression in Ishikawa cells treated with progesterone and calcitriol.Overexpression of miRNA co-regulated by progesterone and calcitriol was used to detect RelA expression.Results:Ishikawa cells were treated with progesterone and calcitriol,respectively,the secretion levels and expressions of cytokines IL-1β,TNF-α,CXCL1,CXCL2 were significantly decreased(P<0.05),and the expression of NF-κB subunit RelA was significantly decreased(P<0.05).NF-κB subunits RelB,c-Rel,p50,p52 expression did not change significantly.Ishikawa cells were treated with progesterone and calcitriol,respectively,and there was no significant change in the activity of RelA promoter(P>0.05),but the level of RelA mRNA was decreased(P<0.05).The expression of 110 miRNAs was up-regulated after progesterone and calcitriol treatment in Ishikawa cells,respectively,and 57 miRNAs were up-regulated after calcitriol treatment,of which 14 miRNAs were up-regulated after both treatments.Overexpression of miRNAs that were up-regulated after both treatments showed that overexpression of miR-590-5p could reduce the level of NF-κB subunit RelA mRNA(P<0.05),and targeted the NF-κB subunit RelA-3'end non-coding region(P<0.05).In addition,the protein level of RelA was decreased after overexpression of miR-590-5p,and the protein level of NF-κB subunit RelA was increased after knockdown.After overexpression of miR590-5p,the expressions of cytokines IL-1β,TNF-α,CXCL1,and CXCL2 were significantly decreased(P<0.05);after knocking down miR-590-5p,it was found that expression levels of cytokines IL-1β,TNF-α,CXCL1 and CXCL2 were increased significantly(P<0.05).However,when miR-590-5p and RelA were overexpressed or knocked down at same time,expression levels of cytokines IL-1β,TNF-α,CXCL1 and CXCL2 did not change significantly(P>0.05).Conclusion:The expression of miR-590-5p increases after progesterone and calcitriol treatment in Ishikawa cells.miR-590-5p targets the NF-κB subunit RelA-3'end non-coding region,reduces the protein level of RelA,and reduces the activity of NF-κB.complex,finally down-regulates the expression of cytokines IL-1β,TNF-α,CXCL1 and CXCL2.This study provides a new basis for the molecular mechanism of progesterone and calcitriol in the treatment of endometrial cancer,and provides a theoretical basis for progesterone and calcitriol to reduce the body's inflammatory response.