miR-495-3p通过调控HDAC9表达对缺氧/复氧诱导的神经细胞凋亡及炎症反应的影响

Effect of miR-495-3p on apoptosis and inflammatory response of neurons induced by hypoxia/reoxygenation through the regulation of HDAC9 expression

目的探讨微小RNA-495-3p(miR-495-3p)对缺氧/复氧(H/R)诱导的神经细胞凋亡及炎症反应的影响及机制。方法体外培养神经母细胞瘤细胞SK-N-SH,建立H/R损伤细胞模型。实验分组:Con组、H/R组、H/R+miR-NC组、H/R+miR-495-3p组、H/R+si-NC组、H/R+si-HDAC9组、H/R+miR-495-3p+pc DNA组、H/R+miR-495-3p+pc DNA-HDAC9组。采用q RT-PCR与Western blot分别检测细胞中miR-495-3p、组蛋白去乙酰化酶9(HDAC9)的表达;采用膜联蛋白V(AnnexinV)-异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染法检测细胞凋亡情况;采用酶联免疫吸附(ELISA)法检测炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素6(IL-6)水平;双荧光素酶报告基因检测miR-495-3p与HDAC9的靶向关系;Western blot检测B淋巴细胞瘤-2相关蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白表达水平。结果与Con组比较,H/R组神经细胞中miR-495-3p的表达水平显著降低(P<0.05),HDAC9的表达水平显著升高(P<0.05);H/R处理后细胞凋亡率升高(P<0.05),细胞中Bax蛋白表达水平升高(P<0.05),TNF-α、IL-1β、IL-6水平升高(P<0.05),而Bcl-2蛋白表达水平降低(P<0.05);miR-495-3p过表达或抑制HDAC9表达后细胞凋亡率降低(P<0.05),Bax蛋白表达水平降低(P<0.05),TNF-α、IL-1β、IL-6水平降低(P<0.05),而Bcl-2蛋白表达水平升高(P<0.05);miR-495-3p可靶向结合到HDAC9的3′UTR区,并下调HDAC9的表达;HDAC9过表达可逆转miR-495-3p过表达对H/R诱导的神经细胞凋亡及炎症因子表达水平的抑制作用。结论miR-495-3p可通过靶向下调HDAC9的表达抑制H/R诱导的神经细胞凋亡及抑制炎性因子的产生进而对神经细胞发挥保护作用。

Objective To investigate the effect and mechanism of microRNA-495-3p(miR-495-3p)on hypoxia/reoxygenation(H/R)-induced apoptosis and inflammatory response of neurons.Methods The neuroblastoma cells SK-N-SH were cultured in vitro to establish a H/R injury cell model.There were 8 experimental groups as following;Con group,H/R group,H/R+miR-NC group,H/R+miR-495-3p group,H/R+si-NC group,H/R+si-HDAC9 group,H/R+miR-495-3p+pc DNA group,H/R+miR-495-3p+pcDNA-HDAC9 group.Real-time quantitative polymerase chain reaction(q RT-PCR)and Western blot were used to detect the expression of miR-495-3p and histone deacetylase 9(HDAC9),respectively.Apoptosis was detected by Annexin V-FITC/PI double staining.The levels of inflammatory factors TNF-α,IL-1βand IL-6 were detected by ELISA.Dual luciferase reporter assay was used to verify the targeting relationship of miR-495-3p to HDAC9.Western blot was used to detect the expression levels of Bax and Bcl-2 proteins.Results As compared with the Con group,the expression level of miR-495-3p was significantly decreased in the H/R group(P<0.05)and the expression level of HDAC9 was significantly increased(P<0.05).The apoptosis rate(P<0.05),the expression of Bax protein(P<0.05),and the levels of TNF-α,IL-1βand IL-6(P<0.05)were increased after H/R treatment,but the expression level of Bcl-2 protein was decreased(P<0.05).The apoptosis rate(P<0.05),Bax protein expression level(P<0.05)and TNF-α,IL-1β,IL-6 levels(P<0.05)were decreased after over-expression of miR-495-3p or the inhibition of HDAC9 expression,while Bcl-2 protein expression level was increased(P<0.05).miR-495-3p bound to the 3’UTR region of HDAC9 and down-regulated HDAC9 expression.Overexpression of HDAC9 reversed the inhibitory effect of miR-495-3p overexpression on H/R-induced apoptosis and inflammatory factor expression levels of neurons.Conclusion miR-495-3p can protect neurons by inhibiting H/R-induced apoptosis and the production of inflammatory factors through down-regulating HDAC9 expression.