人环状RNA＿0114428对脂多糖诱导的人肾小管上皮HK-2细胞损伤的作用及其机制

目的：探讨人环状RNA＿0114428 (circ＿0114428)对脂多糖(LPS)诱导的人肾小管上皮HK-2细胞损伤的作用，阐明circ＿0114428通过调节微小RNA-139-5p (miR-139-5p)/转化生长因子β受体2 (TGFBR2)轴改善HK-2细胞损伤的可能分子机制。方法：体外培养HK-2细胞，双荧光素酶报告基因实验验证miR-139-5p与circ＿0114428和TGFBR2的靶向调控关系，CCK-8法筛选合适的LPS干预浓度和时间。给予10 mg·L^-1 LPS干预12 h构建HK-2细胞损伤模型，HK-2细胞分为对照组、LPS组、LPS+circ＿0114428沉默对照(LPS+si-NC)组、 LPS+circ＿0114428沉默(LPS+si-circ＿0114428)组、LPS+circ＿0114428沉默+miR-139-5p抑制剂对照(LPS+si-circ＿0114428+in-NC)组和LPS+circ＿0114428沉默+miR-139-5p抑制剂(LPS+si-circ＿0114428+in-miR-139-5p)...

Effect of human circular RNA_0114428 on lipopolysaccharide-induced injury of human renal tubular epithelial HK-2 cells and its mechanism

Objective To investigate the effect of human circular RNA_0114428 （circ_0114428） on the lipopolysaccharide （LPS）-induced injury of human renal tubular epithelial HK-2 cells， and to clarify the possible molecular mechanism of circ_0114428 in improving the injury of HK-2 cells by regulating microRNA-139-5p （miR-139-5p）/transforming growth factor β receptor 2 （TGFBR2） axis. Methods The HK-2 cells were cultured in vitro， the dual luciferase reporter gene experiment was used to verify the targeted regulation relationship between miR-139-5p and circ_0114428 or TGFBR2， and the CCK-8 method was used to screen the appropriate LPS intervention time and concentration. The HK-2 cell injury model was established by administering 10 mg·L^－1 LPS for 12 h， and the HK-2 cells were divided into control group， LPS group， LPS+circ_0114428 silence control （LPS+si-NC） group， LPS+circ_0114428 silence （LPS+si-circ_0114428） group， LPS+circ_0114428 silence+miR-139-5p inhibitor control （LPS+si-circ_0114428+in-NC） group， and LPS+circ_0114428 silence+miR-139-5p inhibitor（LPS+si-circ_0114428+in-miR-139-5p） group. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of circ_0114428， miR-139-5p and TGFBR2 mRNA in the cells in various groups， flow cytometry was used to detect the apoptotic rates of the cells in various groups， and Western blotting method was used to detect the expression levels of TGFBR2， B-cell lymphoma-2 （Bcl-2） and Bcl-2 associated X protein （Bax） proteins in the cells in various groups；enzyme linked immunosorbent assay（ELISA） was used to detect the levels of interleukin-6（IL-6）， tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） in the cell supernatant in various groups. Results The results of dual luciferase reporter gene experiment showed that miR-139-5p had a targeted regulatory relationship with circ_0114428 and TGFBR2 （t=10.171， P<0.05；t=7.682， P<0.05）.The CCK-8 method results showed that the concentration of LPS induction was 10 mg·L^－1 and the optimal time was 12 h.Compared with control group， the expression levels of circ_0114428， expression levels of TGFBR2 mRNA and protein， and expression level of Bax protein， the apoptotic rate， and levels of IL-6， TNF-α and IL-1β in cell supernatant of the HK-2 cells in LPS group were significantly increased （P<0.05），and the expression levels of miR-139-5p and Bcl-2 protein were significantly decreased （P<0.05）. Compared with LPS+si-NC group， the expression levels of circ_0114428， expression levels of TGFBR2 mRNA and protein， and expression level of Bax protein， the apoptotic rate， and the levels of IL-6， TNF-α and IL-1β in cell supernatant of the HK-2 cells in LPS+si-circ_0114428 group were significantly decreased （P<0.05），and the expression levels of miR-139-5p and Bcl-2 protein were significantly increased （P<0.05）. Compared with LPS+si-circ_0114428+in-NC group， the expression levels of circ_0114428， expression levels of TGFBR2 mRNA and protein， and expression level of Bax protein， the apoptotic rate， and the levels of IL-6， TNF-α and IL-1β in cell supernatant of the HK-2 cells in LPS+si-circ_0114428+in-miR-139-5p group were significantly increased （P<0.05）， and the expression levels of miR-139-5p and Bcl-2 protein were significantly decreased （ P<0.05）. Conclusion Circ_0114428 can alleviate the LPS-induced HK-2 cell injury， and its mechanism may be related to the regulation of the miR-139-5p/TGFBR2 axis.