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. 2022 Dec 6:2022:2789900.
doi: 10.1155/2022/2789900. eCollection 2022.

KCNQ1OT1 Influences HK-2 Apoptosis and Inflammation in LPS-Induced Acute Renal Injury via Modulating miR-30a-5p/NLRP3 Axis

Jing Hou  1 , Jun-Ming Fan  1   2
Affiliations

KCNQ1OT1 Influences HK-2 Apoptosis and Inflammation in LPS-Induced Acute Renal Injury via Modulating miR-30a-5p/NLRP3 Axis

Jing Hou et al. Evid Based Complement Alternat Med. .

Retraction in

Abstract

Objective: To investigate the influence of KCNQ1OT1 on HK-2 apoptosis and inflammation in ARI and its molecular mechanism.

Methods: Normal cultivated HK-2 cells were used as negative control (NC) group. Three different concentrations of lipopolysaccharide (LPS) were used to treat the cells (5 μg/mL, 10 μg/mL, and 20 μg/mL). The groups included si-KCN1OT1+ LPS, si-NC + LPS, miR-30a-5p + LPS, pcDNA-NLRP3+si-KCNQ1OT1 + LPS group, miR-NC + LPS group, and pcDNA + si-KCNQ1OT1 + LPS group. CCK-8 and flow cytometry are used to measure cell viability and apoptosis, while RT-qPCR and Western blotting are used to detect KCNQ1OT1, miR-30a-5p, and NLRP3 mRNA. ELISA was used to detect the levels of TNF-α, IL-6, and IL-1β in HK-2 cells. The targeting relationship among KCNQ1OT1, miR-30a-5p, and NLRP3 was verified.

Results: After the intervention of LPS, the viability of HK-2 cells was decreased, while the apoptosis rates were increased. The mRNA and protein expressions of NLRP3 and KCNQ1OT1 were increased, while the mRNA and protein levels of miR-30a-5p were decreased (P < 0.05). The expressions of Bax and Cleaved-caspase-3 were downregulated after silencing KCNQ1OT1 and overexpressed miR-30a-5p. In addition, the viability of HK-2 cells was improved, and the apoptosis was reduced by inhibiting KCNQ1OT1 and overexpressed miR-30a-5p. Thus, KCNQ1OT1 modulated NLRP3 via targeting miR-30a-5p. Overexpression of NLRP3 reverses KCNQ1OT1 inhibition of LPS-induced apoptosis, activity, and inflammation in HK-2 cells.

Conclusions: Through modulating the miR-30a-5p/NLRP3 axis, inhibition of KCNQ1OT1 may reduce HK-2 apoptosis and inflammation in LPS-induced ARI.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Effects of different concentrations of LPS on HK-2 viability and apoptosis. (a) Cell proliferation detected by CCK-8. (b) Histogram of apoptosis rate. (c) Apoptosis detected by flow cytometry. Note: ∗P < 0.05 vs NC.
Figure 2
Figure 2
Effects of LPS at different concentrations on the expression of KCNQ1OT1, miR-30a-5p, and NLRP3 in HK-2 cells. (a) KCNQ1OT1 expression in HK-2 cells detected by RT-qPCR. (b) miR-30a-5p expression in HK-2 cells detected by RT-qPCR. (c) NLRP3 mRNA expression in HK-2 cells detected by RT-qPCR. (d) NLRP3 protein expression in HK-2 cells detected by Western blot. Note: ∗P < 0.05 vs NC.
Figure 3
Figure 3
Effects of si-KCNQ1OT1 on LPS-induced HK-2 cell viability, apoptosis, and inflammation. (a) KCNQ1OT1 expression in HK-2 cells detected by RT-qPCR. (b) Cell proliferation determined by CCK-8. (c) Changes in the expression of inflammatory cytokines. (d) Apoptosis measured by flow cytometry. (e) Bax and Cleaved-caspase-3 protein expression detected by Western blot. Note: ∗P < 0.05 vs NC; #P < 0.05 vs si-NC + LPS.
Figure 4
Figure 4
Effects of overexpression of miR-30a-5p on LPS-induced HK-2 cell viability, apoptosis, and inflammatory response. (a) miR-30a-5p expression in HK-2 cells detected by RT-qPCR. (b) Cell proliferation determined by CCK-8. (c) Changes in the expression of inflammatory cytokines. (d) Cell apoptosis determined by flow cytometry. (e) Bax and Cleaved-caspase-3 protein expression detected by Western blot. Note: ∗P < 0.05 vs miR-NC + LPS.
Figure 5
Figure 5
KCNQ1OT1 regulates the expression of NLRP3 by targeting miR-30a-5p. (a) The binding sites of KCNQ1OT1 and miR-30a-5p predicted by LncBase Predicted v.2. (b) The binding sites of miR-30a-5p and NLRP3 predicted by TargetScan. (c) Detection of luciferase activity after co-transfection of miR-NC or miR-30a-5p and KCNQ1OT1 reporter plasmid into HK-2 cells. (d) Detection of luciferase activity after co-transfection of miR-NC or miR-30a-5p and NLRP3 reporter plasmid into HK-2 cells. (e) miR-30a-5p expression in HK-2 cells detected by RT-qPCR. (f) NLRP3 expression detected by Western blot.
Figure 6
Figure 6
NLRP3 overexpression can reverse the effect of si-KCNQ1OT1 on LPS-induced HK-2 cell viability, apoptosis, and inflammatory reaction. (a) Cell proliferation detected by CCK-8. (b) Changes in the expression of inflammatory cytokines. (c) Apoptosis determined by flow cytometry. (d) NLRP3, Bax, and Cleaved-caspase-3 protein expression detected by Western blot. Note: ∗P < 0.05 vs pcDNA + si-KCNQ1OT1+LPS group.
Figure 7
Figure 7
The mechanism of KCNQ1OT1 influences HK-2 apoptosis and inflammation in LPS-induced acute renal injury.
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