miR-455-3p通过靶向BMP2调节地塞米松诱导的软骨细胞自噬性死亡

Effect of miR-455-3p on dexamethasone-induced autophagic death of chondrocytes by targeting BMP2

目的:探究miR-455-3p通过靶向BMP2对地塞米松(Dex)诱导的软骨细胞自噬性死亡的影响。方法:荧光素酶报告实验验证miR-455-3p与BMP2靶向关系。从SD大鼠中分离软骨细胞,Dex处理后的软骨细胞转染miR-455-3p mimic或联合转染pcDNA-BMP2,将软骨细胞分为4组:Control组、Dex组、Dex+mimic组和Dex+mimic+BMP2组。RT-PCR检测BMP2、Ki67、PCNA mRNA水平,流式细胞仪检测细胞凋亡,CCK-8法检测细胞增殖,免疫荧光检测LC3含量,Western blotting法检测BMP2、Caspase-3、Caspase-9、LC3II、LC3I、Beclin1、ATG7蛋白表达水平。结果:miR-455-3p和BMP2存在直接靶向作用关系。与Control组比较,Dex组BMP2 mRNA水平和蛋白水平显著降低,细胞凋亡率显著升高,cleaved Caspase-3/Caspase-3、cleaved Caspase-9/Caspase-9比值显著升高,细胞增殖倍数显著降低,LC3含量显著升高,LC3II/LC3I比值和Beclin1、ATG7蛋白水平显著升高(均P<0.05)。与Dex组比较,Dex+mimic组BMP2 mRNA水平和蛋白水平显著降低,细胞凋亡率显著升高,cleaved Caspase-3/Caspase-3、cleaved Caspase-9/Caspase-9比值显著升高,细胞增殖倍数显著降低,LC3含量显著升高,LC3II/LC3I比值和Beclin1、ATG7蛋白水平显著升高(均P<0.05)。与Dex+mimic组比较,Dex+mimic+BMP2组BMP2 mRNA水平和蛋白水平显著升高,细胞凋亡率显著降低,cleaved Caspase-3/Caspase-3、cleaved Caspase-9/Caspase-9比值显著降低,细胞增殖倍数显著升高,LC3含量显著降低,LC3II/LC3I比值和Beclin1、ATG7蛋白水平显著升高(均P<0.05)。结论:miR-455-3p通过靶向BMP2调节Dex诱导的软骨细胞自噬性死亡。

Objective:To investigate the effect of miR-455-3p on dexamethasone(Dex)-induced autophagic death of chondrocytes by targeting BMP2.Methods:The luciferase reporting assay verified the targeting relationship between miR-455-3p and BMP2.Chondrocytes were isolated from SD rats,and chondrocytes treated with Dex were transfected with miR-455-3p mimic or combined with pcDNA-BMP2 transfection.Chondrocytes were randomly divided into four groups:Control group,Dex group,Dex+mimic group,and Dex+mimic+BMP2 group.The mRNA levels of BMP2,Ki67 and PCNA were detected by RT-PCR.Cell apoptosis was detected by flow cytometry.Cell proliferation was detected by CCK-8 method.LC3 content was detected by immunofluorescence.Western blotting was used to detect the expression levels of BMP2,Caspase-3,Caspase-9,LC3II,LC3I,Beclin1 and ATG7 proteins.Results:The results showed that there was a direct targeting relationship between miR-455-3p and BMP2.Compared with the Control group,the mRNA and protein levels of BMP2 in the Dex group were significantly decreased.The apoptosis rate and the cleaved Caspase-3/Caspase-3,cleaved Caspase-9/Caspase-9 ratios were significantly increased.The cell proliferation multiple was significantly decreased,and the LC3 content was significantly increased.The LC3II/LC3I ratio,Beclin1 and ATG7 protein levels were significantly increased(all P<0.05).Compared with the Dex group,the mRNA and protein levels of BMP2 in the Dex+mimic group were significantly decreased.The apoptosis rate was significantly increased.The cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 ratios were significantly increased.The cell proliferation multiple was significantly decreased,and the LC3 content,LC3II/LC3I ratio,Beclin1 and ATG7 protein levels were significantly increased(all P<0.05).Compared with the Dex+mimic group,the mRNA and protein levels of BMP2 in the Dex+mimic+BMP2 group were significantly increased.The apoptosis rate,cleaved Caspase-3/Caspase-3 and cleaved Caspase-9/Caspase-9 ratios were significantly decreased.The cell proliferation multiple was significantly increased,and the LC3 content,LC3II/LC3I ratio,Beclin1 and ATG7 protein levels were significantly increased(all P<0.05).Conclusion:MiR-455-3p regulates the autophagic death of chondrocytes induced by Dex by targeting BMP2 and promotes proliferation and autophagy of chondrocytes.