miR-34b Alleviates High Glucose-Induced Inflammation and Apoptosis in Human HK-2 Cells via IL-6R/JAK2/STAT3 Signaling Pathway

Abstract

BACKGROUND It is well established that inflammation and apoptosis of renal tubular epithelial cells caused by hyperglycemia contribute to the development of diabetic nephropathy (DN). Although microRNAs (miRNAs) are known to have roles in inflammation-related disorders, the exact role of miR-34b in DN has not been defined, and the regulatory mechanism has been unclear. This study aimed to clarify the role of miR-34b in DN pathogenesis. MATERIAL AND METHODS Expression of miR-34b, IL-6R, and other key factors of inflammation, apoptosis (TNF-alpha, IL-1ß, IL-6, caspase-3) in high glucose (HG)-induced HK-2 cells were measured by real-time PCR, Western blot, and flow cytometric cell apoptosis assays. We used luciferase reporter assay to detect the target of miR-34b. Moreover, the targeting gene of miR-34b and its downstream JAK2/STAT3 signaling pathway were explored. RESULTS It was demonstrated that miR-34b overexpression inhibited apoptosis and expression levels of TNF-alpha, IL-1ß, IL-6, and caspase-3 in HG-treated HK-2 cells. We also found that IL-6R is a direct target of miR-34b, which could rescue inflammation and apoptosis in HG-treated HK-2 cells transfected with miR-34b mimic. Furthermore, we showed that overexpression of miR-34b inhibited the IL-6R/JAK2/STAT3 signaling pathway in HG-treated HK-2 cells. CONCLUSIONS Our data suggest that overexpression of miR-34b improves inflammation and ameliorates apoptosis in HG-induced HK-2 cells via the IL-6R/JAK2/STAT3 pathway, indicating that miR-34b could be a promising therapeutic target in DN.

Figure 1

miR-34b was downregulated in HG-treated HK-2 cells. The HK-2 cells were incubated with 5 mM (NG group) or 25 mM (HG group) at different time points (12 h, 24 h, 48 h, 72 h). The expression of miR-34b was measured by qRT-PCR. Data are presented as mean ±SD and shown as fold change relative to the control group. Data were assessed using one-way ANOVA. * p<0.05 and ** p<0.01. HG – high glucose; NG – normal glucose.

Figure 2

miR-34b attenuates inflammation in HG-treated HK-2 cells. (A, B) The expression of miR-34b was measured by qRT-PCR. (C) qRT-PCR detection of TNF-α, IL-1β, and IL-6 mRNA expression in HG-treated HK-2 cells in each group. (D, E) Western Blot detection of TNF-α, IL-1β, and IL-6 protein expression in HG-treated HK-2 cells in each group. Data are presented as mean ±SD and shown as fold change relative to the control group. Data were assessed using one-way ANOVA. * p<0.05 and ** p<0.01. HG – high glucose; NG – normal glucose.

Figure 3

miR-34b attenuates apoptosis in HG-treated HK-2 cells. (A, B) Percentage appoptosis in HG-treated HK-2 cells transfected with miR-34b mimic or mimic-NC by using flow analysis. (C) qRT-PCR detection of caspase-3 mRNA expression in HG-treated HK-2 cells transfected with miR-34b mimic or mimic-NC. (D, E) Western blot detection of cleaved caspase-3 protein expression in HG-treated HK-2 cells transfected with miR-34b mimic or mimic-NC. Data are presented as mean ±SD and shown as fold change relative to the control group. Data were assessed uding one-way ANOVA. * p<0.05 and ** p<0.01. HG – high glucose; NG – normal glucose.

Figure 4

IL-6R is a direct target of miR-34b. (A) The predicted binding sequences of the 3′-UTR of IL-6R. (B) Relative luciferase activity was analyzed by luciferase analysis (C–E) The mRNA and protein expression of IL-6R in HG-treated HK-2 cells transfected with miR-34b mimic or miR-34b inhibitors. Data are presented as mean ±SD and shown as fold change relative to the control group. Data were assessed using one-way ANOVA. * p<0.05 and ** p<0.01. NC – negative control.

Figure 5

miR-34b attenuates inflammation and apoptosis by targeting IL-6R in HG-treated HK-2 cells. (A) The mRNA expression of TNF-α, IL-1β, IL-6, and caspase-3 in HG-induced HK-2 cells co-transfected with pcDNA-IL-6R, empty vector, miR-34b mimic, or mimic-NC. (B, C) The protein expression of TNF-α, IL-1β, IL-6, and cleaved caspase-3 in HG-induced HK-2 cells co-transfected with pcDNA-IL-6R, empty vector, miR-34b mimic, or mimic-NC. Data are presented as mean ±SD and shown as fold change relative to the control group. Data were assessed with one-way ANOVA. * p<0.05 and ** p<0.01.

Figure 6

miR-34b suppresses activation of the JAK2/STAT3 signaling pathway. (A, B) The p-STAT3, t-STAT3, p-JAK2, and t- JAK2 expression in HG-treated HK-2 cells tranfected with miR-34b mimic or controls. (C, D) The p-STAT3, t-STAT3, p-JAK2, and t-JAK2 expression in HG-treated HK-2 cells co-transfected with pcDNA-IL-6R, empty vector, miR-34b mimic, or mimic-NC. Data are presented as mean ±SD and shown as fold change relative to the control group. Data were assessed with one-way ANOVA. * p<0.05, ** p<0.01, and *** p<0.001. p-STAT3 – phosphorylated STAT3; p-JAK2 – phosphorylated JAK2; t-STAT3 – total STAT3; t-JAK2 – total JAK2; HG – high glucose; NG – normal glucose.