Long non-coding RNA MEG3 promotes tumor necrosis factor-alpha induced oxidative stress and apoptosis in interstitial cells of cajal via targeting the microRNA-21 /I-kappa-B-kinase beta axis

Abstract

Interstitial Cells of Cajal (ICC) plays a critical role in the peristaltic contractions of the gastrointestinal and urinary tract. The dysfunction and loss of ICC contributes to hypokinetic disease, such as gallstoneand ureteropelvic junction obstruction . In the present study, we identified the underlying driving molecular signals of oxidative stress and apoptosis in ICC. ICC was isolated from small intestine of Balb/c mice, and stimulated with tumor necrosis factor-alpha (TNF-α). MTT and flow cytometry were performed to assess cell viability, apoptosis, and the level of reactive oxygen species in ICC, respectively. The level of malondialdehyde, superoxide dismutase, and glutathione peroxidase in cells were measured to assess oxidative stress. The expression of inflammatory factors (interleukin, IL-1 and IL-6) and apoptosis-related proteins were detected by western blot. We observed that TNF-αinduced inflammation, oxidative stress and cell apoptosis in ICC. By using quantitative real-time PCR , we verified that the expression of long non-coding RNAMEG3 was elevated by TNF-α in ICC. Silencing MEG3 reversed inflammation, oxidative stress, and cell apoptosisin TNF-α-treated ICC. Subsequently, we confirmed that MEG3 sponged cytoprotective miR-21 to upregulate the expression of I-kappa-B-kinase beta (IKKB) and activate the nuclear factor kappa-B (NF-κB) pathway. Both miR-21 overexpression and IKKB knockdown reduced TNF-α-induced above symptoms in ICC. Taken together, we can conclude that MEG3 mediates inflammation, oxidative stress and apoptosis in TNF-α-treated ICC via the miR-21/IKKB-NF-κB axis. The study improves our understanding of the molecular mechanism of ICC reduction related diseases.

Keywords: Interstitial Cells of Cajal; MEG3; apoptosis; nuclear factor kappa-B; oxidative stress.

Graphical abstract

Figure 1.

TNF-α induced oxidative stress, inflammation and apoptosis in ICC. a:Cell viability in ICC treated with different concentrations of TNF-α(0, 10, 20, 30 ng/mL), ‘*’ means P < 0.05 compared with ICC treated with 0 ng/mL of TNF-α. ICC was treated with or without 20 ng/mL of TNF-α. b-c: Flow cytometry was performed to detect cell apoptosis rate (b) and ROS level (c). d: Oxidative stress reaction markers were detected by ELISA method with a microplate reader. e:Western blot was used to determine the expression of IL-1, IL-6, Bax, Bcl2 and cleaved caspase3.‘*’ means P < 0.05 compared with control group.

Figure 2.

MEG3 silence reduced inflammation and apoptosis induced by TNF-α in ICC. a: MEG3 expression was detected by qRT-PCR in ICC treated with or without 20 ng/mL of TNF-α. ICC was transfected with siRNA-MEG3 and treated with 20 ng/mL of TNF-α. b: Cell viability was detected by MTT method, c-d: Flow cytometry was performed to detect cell apoptosis rate (c) and ROS level (d). e: Oxidative stress reaction markers were detected by ELISA method with a microplate reader. f:Western blot was used to determine the expression of IL-1, IL-6, Bax, Bcl2 and cleaved caspase3.‘*’ means P < 0.05 compared with control group. ‘#’ means P < 0.05 compared with TNF-α group.

Figure 3.

MiR-21 was a target of MEG3 and IKKB was directly targeted by miR-21. a: Luciferase reporter gene assay was conducted to verify the targeting relationship of miR-21 and MEG3. ‘*’ means P < 0.05 compared with vector group. b: The regulatory effect of MEG3 on the expression of miR-21 was measured using western blot. ‘*’ means P < 0.05 compared with siRNA-NC group. ‘#’ means P < 0.05 compared with pcDNA-NC group. c: The expression of miR-21 in ICC transfected with siRNA-MEG3 and treated with TNF-α. ‘*’ means P < 0.05 compared with control group. ‘#’ means P < 0.05 compared with TNF-α group. d: The binding site between miR-21 and IKKB was predicted on the miRwalk website, and then luciferase reporter gene assay was conducted to verify the targeting relationship. ‘*’ means P < 0.05 compared with vector group. e: The regulatory effect of miR-21 on the expression of IKKB was measured using qRT-PCR and western blot. ‘*’ means P < 0.05 compared with NC group. f: The expression of IKKB mRNA and protein in ICC transfected with siRNA-MEG3 and treated with TNF-α. ‘*’ means P < 0.05 compared with control group. ‘#’ means P < 0.05 compared with TNF-α group.

Figure 4.

MEG3 activated NF-κB pathway by miR-21 in ICC. ICC was transfected with pcDNA-MEG3 and miR-21 mimic. a: IKKB mRNA was detected by qRT-PCR. b:The expression of IKKB and NF-kB pathway related protein was detected by western blot. ‘*’ means P < 0.05 compared with NC group. ‘#’ means P < 0.05 compared with OE-MEG3 group.

Figure 5.

MiR-21/IKKB axis reversed TNF-α-induced oxidative stress, inflammation and apoptosis in ICC. ICC was transfected with miR-21 mimic and siRNA-IKKB, and then treated with TNF-α. a: MEG3 expression was detected by qRT-PCR in ICC treated with or without 20 ng/mL of TNF-α. ICC was transfected with siRNA-MEG3 and treated with 20 ng/mL of TNF-α. b: Cell viability was detected by MTT method, c-d: Flow cytometry was performed to detect cell apoptosis rate (c) and ROS level (d). e: Oxidative stress reaction markers were detected by ELISA method with a microplate reader. f: Western blot was used to determine the expression of IL-1, IL-6, Bax, Bcl2 and cleaved caspase3. ‘*’ means P < 0.05 compared with control group. ‘#’ means P < 0.05 compared with TNF-α group.