LINC00963 targeting miR-128-3p promotes acute kidney injury process by activating JAK2/STAT1 pathway

Abstract

The role of long non-coding RNAs (lncRNAs) in kidney diseases has been gradually discovered in recent years. LINC00963, as an lncRNA, was found to be involved in chronic renal failure. However, the role and molecular mechanisms of LINC00963 engaged in acute kidney injury (AKI) were still unclear. In this study, we established rat AKI models by ischaemia and reperfusion (I/R) treatment. Urea and creatinine levels were determined, and histological features of kidney tissues were examined following HE staining. CCK8 assay was chosen to assess the viability of hypoxia-induced HK-2 cells. Dual-luciferase reporter gene assays were performed to verify the target relationship between LINC00963 and microRNA. The mRNA and protein levels were assayed by RT-qPCR and Western blot, respectively. Annexin V-FITC/PI and TUNEL staining were used to evaluate apoptosis. LINC00963 was highly expressed in the cell and rat models, and miR-128-3p was predicted and then verified as a target gene of LINC00963. Knockdown of LINC00963 reduced acute renal injury both in vitro and in vivo. LINC00963 activated the JAK2/STAT1 pathway to aggravate renal I/R injury. LINC00963 could target miR-128-3p to reduce G1 arrest and apoptosis through JAK2/STAT1 pathway to promote the progression of AKI.

Keywords: LINC00963; STAT1 pathway; acute kidney injury; apoptosis; miR-128-3p.

FIGURE 1

Expression of LINC00963 in hypoxia‐induced HK‐2 cells and AKI rat models. A, Representative morphology of HK‐2 cell models. B, HE staining of renal tissues in AKI rat models. C, The viability of HK‐2 cells analysed by CCK‐8. D, Relative LINC00963 mRNA expression in HK‐2 cell models at 4, 6 and 8 h of reoxygenation. (E) BUN and (F) Scr levels of AKI model rats after reperfusion at various time‐points. G, Relative LINC00963 mRNA expression of LINC00963 in renal tissue. H, RNA FISH analysis of LINC00963 in hypoxia‐induced HK‐2 cells after reoxygenation for 8 h. Data were expressed as mean ± SEM, and different letters represented significant differences (P < .05) between groups

FIGURE 2

Targeting relationship between miR‐128‐3p and LINC00963. A, Potential binding sites of LINC00963 and miR‐128‐3p via RegRNA 2.0. B, Potential binding sites between LINC00963 and miR‐128‐3p. C, Binding relationship of LINC00963 and miR‐128‐3p was confirmed by dual‐luciferase reporter gene assay. D, Relative mRNA expression of LINC00963 in HK‐2 cells after transfected with miR‐128‐3p mimics/inhibitor. E, RNA pull‐down assay and qPCR detection of LINC00963 binding to miR‐128‐3p. Data are expressed as mean ± SEM, and different letters represented significant differences (P < .05) between groups

FIGURE 3

The impacts of LINC00963 and miR‐128‐3p on the apoptosis and cell cycle in hypoxia‐induced HK‐2 cells. A, Cell transfection efficiency of si‐LINC00963 and miR‐128‐3p‐ASO. B, Apoptosis analysis examined by flow cytometric using Annexin V/PI assay. C, Expression of apoptosis‐related proteins by Western blot. D, The distribution of cell cycle analysed by flow cytometry. E, Expression of cell cycle‐related proteins by Western blot. Data are expressed as mean ± SEM, and different letters represent significant differences (P < .05) between groups

FIGURE 4

Effect of si‐LINC00963 on renal I/R‐induced AKI rat model. A, Transfection efficiency of si‐LINC00963 and miR‐128‐3p‐ASO in renal tissues by qRT‐PCR. B, HE staining of I/R renal after reperfusion for 24 h among groups. C, Expression of apoptosis‐related proteins by Western blot. Data are expressed as mean ± SEM, and different letters represent significant differences (P < .05) between groups

FIGURE 5

The relationship between LINC00963 and JAK2/STAT1 pathways in hypoxia‐induced HK‐2 cell model. A, The protein levels of p‐JAK2 and p‐STAT1 in different groups. B, Apoptosis analysed by flow cytometry. C, Expression of apoptosis‐related proteins by Western blot. Data are expressed as mean ± SEM, and different letters represent significant differences (P < .05) between groups

FIGURE 6

The relationship between LINC00963 and JAK2/STAT1 pathways on I/R‐induced AKI rat model. A, Expression of p‐JAK2 and p‐STAT1 under different conditions. B, TUNEL staining and cell apoptosis proportion of different groups. C, The protein levels of Bax and Bcl‐2. Data are expressed as mean ± SEM, and different letters represent significant differences (P < .05) between groups