Figures & data
Figure 1. Expression characteristics of OIP5-AS1 and miR-128-3p in the sepsis model. CLP surgery was used to induce septic rat model. 48 hours after the construction of the mode, the lung tissues were isolated and subjected to further experiments. NR8383 and RLE-6TN cells were treated with LPS (25, 50, 100 ng/mL) for 6 hours. A-E. qRT-PCR was taken to determine OIP5-AS1 and miR-128-3p relative expression in septic rats (a-b) and LPS-treated NR8383 and RLE-6TN cells (c-f). G-I. The relative expression of SIRT1, p-NF-κB and iNOS in septic rats (g) or was LPS-treated NR8383 and RLE-6TN cells (h-i) was tested applying Western blot. *P < 0.05, ** P < 0.01, *** P < 0.001 vs. sham group or control group. N = 3
Figure 2. Overexpressing OIP5-AS1 attenuated lung epithelial cell damage and macrophage inflammation. RLE-6TN cells and NR8383 cells were transfected with OIP5-AS1 overexpression plasmids or the negative control (NC), and then both were treated with 100 ng/mL LPS for 6 hours. A-D RLE-6TN cell and NR8383 cell viability and apoptosis were examined adopting CCK8 assay (a-b) and flow cytometry (c-d), respectively. E-F. The test of the relative expression of SIRT1, p-NF-κB and iNOS in RLE-6TN cells and NR8383 cells was carried out using Western blot. (g, h) We employed the ELISA method for detecting the levels of related inflammatory factors IL-6, IL-1β, and TNFα in RLE-6TN cell and NR8383 cell. * P < 0.05, ** P < 0.01, *** P < 0.001. N = 3
Figure 3. OIP5-AS1 overexpression weakened lung injury in vivo. Ad-GFP or Ad-OIP5-AS1 was injected into the SD rats via tail vein. CLP surgery was used to induce septic rat model. 48 hours after the construction of the mode, the lung tissues were isolated and subjected to further experiments. (a) The examination of the pathological changes in rat lung tissue of each group was conducted by HE staining method. (b) The dry/wet method was adopted for testing pulmonary edema. (c) ICH was used to evaluate MPO-labeled polymorphonuclear neutrophils (PMN). (d) The relative expression of apoptosis-related proteins Caspase3, Bax and Bcl2 related by apoptosis in each group was examined applying the Western blot method. (e) Western blot method was performed for the examination of the relative expression of SIRT1, p-NF-κB and iNOS. (f) SIRT1 expression in lung tissue was measured by immunohistochemical staining. (g) ELISA was applied for measuring the levels of related inflammatory factors IL-1β, TNFα and IL-6 in the lung homogenate supernatant. ** P < 0.01, *** P < 0.001. N = 5
Figure 4. Overexpressing miR-128-3p accelerated lung macrophage inflammation and epithelial cell damage. RLE-6TN cells and NR8383 cells were transfected with miR-128-3p mimics or the negative control (miR-NC), and then both were treated with 100 ng/mL LPS for 6 hours. A-D. CCK8 assay and flow cytometry were conducted for the measurement of RLE-6TN cells and NR8383 cells cell viability (a-b) and apoptosis (c-d), respectively. (e) WB detected the expression of SIRT1, p-NF-κB and iNOS in RLE-6TN cells and NR8383 cells. F-G. We took ELISA to test the levels of IL-1β, TNFα and IL-6 in RLE-6TN cells and NR8383 cells. * P < 0.05, ** P < 0.01, *** P < 0.001. N = 3
Figure 5. OIP5-AS1 overexpression attenuated miR-128-3p-mediated effects. RLE-6TN cells and NR8383 cells were transfected with miR-128-5p mimics, OIP5-AS1 overexpression plasmids, or treated with SIRT1 inhibitor (EX527, 100 nM), LPS (100 ng/mL) for 6 hours. A-B. We applied the qRT-PCR method for determining the relative expression of OIP5-AS1 and miR-128-3p in RLE-6TN and NR8383 cells. C-D. CCK8 method and flow cytometry were implemented for detecting cell viability and apoptosis of RLE-6TN and NR8383 cells. E. The relative expression of SIRT1, p-NF-κB and iNOS in RLE-6TN and NR8383 cells was examined employing Western blot. F. ELISA was adopted for the detection of the levels of IL-1β, TNFα and IL-6 in RLE-6TN and NR8383 cells. * P < 0.05, ** P < 0.01, *** P < 0.001. N = 3
Figure 6. MiR-128-3p targeted OIP5-AS1 and SIRT1. A. The underlying binding sites between OIP5-AS1 and miR-128-3p, as well as miR-128-3p and SIRT1 predicted by bioinformatics were as shown. B. The dual-luciferase gene reporter method verified the targeting interrelationship between OIP5-AS1 and miR-128-3p, and miR-128-3p and SIRT1. C. The RIP method was utilized for validation of the targeting interrelationship between OIP5-AS1 and miR-128-3p, as well as miR-128-3p and SIRT1. nsP> 0.05, ** P < 0.01, *** P < 0.001. N = 3
Availability of data and materials
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.