LncRNA CRNDE通过miR-181a-5p/SOX6轴调节脂多糖诱导人肺泡上皮细胞的炎症反应和细胞凋亡

LncRNA CRNDE regulates lipopolysaccharide-induced inflammatory response and apoptosis in human alveolar epithelial cells via miR-181a-5p/SOX6 axis

目的探索长链非编码RNA(LncRNA)CRNDE通过微小RNA-181a-5p(miR-181a-5p)/转录因子性别决定区Y-box 6(SOX6)轴对脂多糖(LPS)诱导人肺泡上皮细胞炎症反应和细胞凋亡的影响。方法将所有质粒和寡核苷酸分别转染A549细胞,并进行LPS处理,并以未经处理的A549细胞为对照。qRT-PCR检测细胞中CRNDE、miR-181a-5p以及SOX6 mRNA的表达水平;流式细胞仪、ELISA法检测细胞凋亡及肿瘤坏死因子-α(TNF-α)、炎症因子白细胞介素-1β(IL-β)和IL-6水平;双荧光素酶实验分别验证CRNDE和miR-181a-5p以及miR-181a-5p与SOX6的靶向关系;Western blot检测磷酸化(p)-核因子κB(NF-κB)p65/NF-κB p65、SOX6、活化的半胱氨酸天冬氨酸酶3(cleaved caspase-3)的表达水平。两组间比较采用独立样本t检验;各组数据均满足正态分布,当方差齐时采用单因素方差分析,进一步两组间比较采用SNK-q检验,方差不齐时采用Games-Howell法检验。结果与对照比较,LPS的细胞凋亡率[(41.11±4.11)%比(6.11±0.61)%]、TNF-α[(353.21±35.31)比(200.15±20.02)pg/mg]、IL-1β[(286.91±28.71)比(35.76±3.58)pg/mg]、IL-6[(642.31±64.23)比(32.64±3.26)pg/mg]、CRNDE(0.66±0.06比0.33±0.03)、SOX6(0.68±0.06比0.28±0.03)、cleaved caspase-3(0.76±0.07比0.24±0.02)、p-NF-κB p65/NF-κB p65(0.85±0.08比0.26±0.03)升高,miR-181a-5p表达(0.32±0.03比0.64±0.06)降低(P<0.05);与si-NC组相比,干扰CRNDE降低LPS诱导细胞的凋亡率[(15.58±1.55)%比(41.09±4.09)%]、TNF-α[(200.15±20.02)比(356.51±35.65)pg/mg]、IL-1β[(99.32±9.94)比(288.88±28.89)pg/mg]、IL-6[(211.34±21.14)比(639.89±63.99)pg/mg]、CRNDE(0.35±0.03比0.65±0.06)、SOX6(0.39±0.04比0.64±0.07)的表达(P<0.05),干扰SOX6降低LPS诱导细胞的凋亡率[(15.34±1.53)%比(41.09±4.09)%]、TNF-α[(192.37±19.24)比(356.51±35.65)pg/mg]、IL-1β[(100.02±10.01)比(288.88±28.89)pg/mg]、IL-6[(204.86±20.49)比(639.89±63.99)pg/mg]、SOX6(0.36±0.03比0.64±0.07)的表达(P<0.05);与miR-NC相比,过表达miR-181a-5p降低LPS诱导细胞的凋亡率[(15.34±1.53)%比(40.89±4.09)%]、TNF-α[(189.45±19.01)比(356.68±35.67)pg/mg]、IL-1β[(95.37±9.54)比(288.67±28.87)pg/mg]、IL-6[(209.84±20.98)比(637.93±63.81)pg/mg]水平,miR-181a-5p(0.55±0.05比0.35±0.03)表达增加(P<0.05)。抑制miR-181a-5p表达可逆转干扰CRNDE对LPS诱导A549细胞的保护作用;上调SOX6可逆转干扰CRNDE、过表达miR-181a-5p对LPS诱导A549细胞的保护作用。结论干扰CRNDE可以抑制LPS诱导A549细胞的炎症反应和细胞凋亡,可能与调控miR-181a-5p/SOX6轴有关。

Objective To explore the effects of long non-coding RNA(lncRNAs)CRNDE on lipopolysaccharide(LPS)-induced inflammatory response and apoptosis of human alveolar epithelial cells via the microRNA-181a-5p(miR-181a-5p)/transcription factor sex-determining region Y-box 6(SOX6)axis.Methods All plasmids and oligonucleotides were transfected into A549 cells and treated with LPS.Untreated A549 cells were used as the control.QRT-PCR was performed to detect the expression levels of CRNDE,miR-181a-5p and SOX6 mRNA in cells;flow cytometry and ELISA were performed to detect cell apoptosis and the levels of tumor necrosis factor-α(TNF-α),inflammatory factors interleukin-1β(IL-1β)and IL-6;dual luciferase experiment was performed to verify the targeting relationship between CRNDE and miR-181a-5p,and the relationship between miR-181a-5p and SOX6;Western blot was performed to detect the expression levels of phosphorylated(p)-nuclear factorκB(NF-κB)p65/NF-κB p65,SOX6 and cleaved caspase-3.Results Compared with the control,the apoptosis rate(41.11%±4.11%)vs(6.11%±0.61%),TNF-α(353.21±35.31)vs(200.15±20.02)pg/mg,IL-1β(286.91±28.71)vs(35.76±3.58)pg/mg,IL-6(642.31±64.23 vs 32.64±3.26)pg/mg,the expression of CRNDE(0.66±0.06 vs 0.33±0.03),SOX6(0.68±0.06 vs 0.28±0.03),cleaved caspase-3(0.76±0.07 vs 0.24±0.02),p-NF-κB p65/NF-κB p65(0.85±0.08 vs 0.26±0.03)were visibly raised in the LPS group,and the expression of miR-181a-5p(0.32±0.03 vs 0.64±0.06)was visibly decreased(P<0.05);Compared with the si-NC group,knockdown CRNDE decreased the apoptosis rate(15.58%±1.55%vs 41.09%±4.09%),TNF-α[(200.15±20.02)vs(356.51±35.65)pg/mg],IL-1β[(99.32±9.94)vs(288.88±28.89)pg/mg],IL-6[(211.34±21.14)vs(639.89±63.99)pg/mg],CRNDE(0.35±0.03 vs 0.65±0.06),SOX6(0.39±0.04 vs 0.64±0.07)induced by LPS(P<0.05),Knockdown SOX6 inhibited the apoptosis rate(15.34%±1.53%vs 41.09%±4.09%),TNF-α[(192.37±19.24)vs(356.51±35.65)pg/mg],IL-1β[(100.02±10.01)vs(288.88±28.89)pg/mg].The expression of IL-6[(204.86±20.49)vs(639.89±63.99)pg/mg],SOX6(0.36±0.03 vs 0.64±0.07)induced by LPS(P<0.05);Compared with miR-NC,overexpression of miR-181a-5p reduced apoptosis(15.34%±1.53%vs 40.89%±4.09%),TNF-α[(189.45±19.01)vs(356.68±35.67)pg/mg],IL-1β[(95.37±9.54)vs(288.67±28.87)pg/mg],IL-6[(209.84±20.98)vs(637.93±63.81)pg/mg]induced by LPS,and the expression of miR-181a-5p(0.55±0.05 vs 0.35±0.03)was significantly increased(P<0.05);inhibiting the expression of miR-181a-5p could reverse the protective effect of CRNDE on LPS-induced A549 cells;up-regulation of SOX6 could reverse the protective effects of CRNDE interference and miR-181a-5p overexpression on LPS-induced A549 cells.Conclusion Interfering with CRNDE could inhibit the inflammatory response and apoptosis of A549 cells induced by LPS,which may be related to the regulation of the miR-181a-5p/SOX6 axis.