Abstract:

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.

Key words: Circular RNA_0114428, MiR-139-5p, Transforming growth factor β receptor 2, Lipopolysaccharide, Human renal tubular epithelial cells