KCNQ1OT1 promotes autophagy by regulating miR-200a/FOXO3/ATG7 pathway in cerebral ischemic stroke

Abstract

Dysregulation of long noncoding RNAs (lncRNAs) is associated with abnormal development and pathophysiology in the brain. Increasing evidence has indicated that ischemic stroke is becoming the most common cerebral disease in aging populations. The treatment of ischemic stroke is challenging, due in part to ischemia and reperfusion (I/R) injury. In this study, we revealed that potassium voltage-gated channel subfamily Q member 1 opposite strand 1 (KCNQ1OT1) was significantly upregulated in ischemic stroke. Knockdown of KCNQ1OT1 remarkably reduced the infarct volume and neurological impairments in transient middle cerebral artery occlusion (tMCAO) mice. Mechanistically, KCNQ1OT1 acted as a competing endogenous RNA of miR-200a to regulate downstream forkhead box O3 (FOXO3) expression, which is a transcriptional regulator of ATG7. Knockdown of KCNQ1OT1 might inhibit I/R-induced autophagy and increase cell viability via the miR-200a/FOXO3/ATG7 pathway. This finding offers a potential novel strategy for ischemic stroke therapy.

Keywords: ATG7; FOXO3; autophagy; lncRNA KCNQ1OT1; miR-200a; stroke.

Figure 1

KCNQ1OT1 was upregulated in focal ischemia (a) Expression of KCNQ1OT1 in plasma of AIS patients (n = 42) and healthy controls (n = 40) detected by real‐time qPCR. Data are presented as the mean ±SD. ***p < 0.001 vs. health control group. (b) Linear regression analysis was conducted to each individual about KCNQ1OT1 expression and National Institute of Health Stroke Scale (NIHSS) score, **p < 0.01. (c,d) Expression of KCNQ1OT1 in plasma (c) and brain tissue (d) of tMCAO and sham mice detected by qRT‐PCR. Data are presented as the mean ± SD (n = 10 in each group). ***p < 0.001 vs. sham group. (e) Linear regression analysis was conducted to each individual KCNQ1OT1 expression in plasma and brain tissue in tMCAO group (n = 10), **p < 0.01. (f) Protein levels of ATG7, SQSTM1, and LC3B II in tMCAO and sham mice with GAPDH as an endogenous control. Data are presented as the mean ± SD (n = 6 in each group). *p < 0.05 vs. sham group. (g) Infarct region was visualized by triphenyltetrazolium chloride (TTC) staining. (h) Infarct size was measured using Image J software. Data are presented as the mean ± SD (n = 6 in each group). *p < 0.05 vs. tMCAO + sh‐NC group

Figure 2

Knockdown of KCNQ1OT1 restrained autophagy in vitro. (a) Expression of KCNQ1OT1 in cells after transfected with sh‐KCNQ1OT1 plasmids and scrambled vectors (NC), respectively. **p < 0.01 vs. sh‐NC group. (b) Western blot analysis of ATG7, SQSTM1, and LC3B II altered expression in OGD/R cells with GAPDH as an endogenous control. *p < 0.05 vs. control group. (c) CCK‐8 assay was performed to assess the influences of KCNQ1OT1, 3‐MA, and rapamycin (RAPA) on cell viability. (d) Western blot analysis of ATG7, SQSTM1, and LC3B II expression in OGD/R cells with the treatment of sh‐KCNQ1OT1 and RAPA. GAPDH was regarded as an endogenous control. (e) Flow cytometry analysis of cells after the intervention of sh‐KCNQ1OT1, 3‐MA, or RAPA. The apoptosis rates equated to the sum percent of right lower quadrant (representing early apoptosis) and right upper quadrant (representing late apoptosis). Statistical analysis was applied with nonparametric Mann–Whitney test. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + sh‐NC group. ^▲ p < 0.05 vs. OGD/R group. ^▼ p < 0.05 vs. OGD/R + sh‐KCNQ1OT1 group. ^◆ p < 0.05 vs. OGD/R + 3‐MA + sh‐NC group. ^◇ p < 0.05 vs. OGD/R + RAPA + sh‐NC group. (f) Transmission electron microscopy was applied to observe the ultrastructural features in OGD/R cells with altered KCNQ1OT1 expression. Arrows show autophagic vacuoles (AVs) with double membranes. Scale bars represent 1 µm. (g) The number of AVs was calculated statistically, at least 40 cells were counted in each experiment. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R group. For (a‐f), data are presented as the mean ± SD (n = 3 in each group)

Figure 3

miR‐200a promoted cells survival and was targeted by KCNQ1OT1 in OGD/R‐induced autophagy. (a) qRT‐PCR analysis of miR‐200a expression in tMCAO (left panel, n = 10) and OGD/R cells (right panel, n = 5). Data are presented as the mean ± SD. **p < 0.01 vs. sham group. ***p < 0.001 vs. control group. (b) CCK‐8 assay was conducted to explore the influence of changing miR‐200a on cell viability in OGD/R. (c) Western blot analysis of ATG7, SQSTM1, and LC3B II with altered miR‐200a using GAPDH as an endogenous control. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + pre‐NC group. ^▲ p < 0.05 vs. OGD/R + anti‐NC group. (d) Presentation of the putative binding site of miR‐200a and KCNQ1OT1 (KCNQ1OT1‐wt), and the designed mutant sequence (KCNQ1OT1‐mut). (e) The relative luciferase activities of N2a cells co‐transfected either KCNQ1OT1‐wt or KCNQ1OT1‐mut with either pre‐miR‐200a or pre‐NC. *p < 0.05 vs. KCNQ1OT1‐wt + pre‐NC. ^# p < 0.05 vs. KCNQ1OT1‐mut + pre‐miR‐200a. (f) CCK‐8 assay was performed to evaluate the effects of KCNQ1OT1 and miR‐200a on cell viability. (g) Western blot analysis of ATG7, SQSTM1, and LC3B II affected by KCNQ1OT1 accompanied with miR‐200a alteration using GAPDH as an endogenous control. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + sh‐NC + pre‐NC group. For (b,e,f,g), data are presented as the mean ± SD (n = 3 in each group)

Figure 4

FOXO3 was upregulated and attenuated cell viability in ischemia and reperfusion (I/R). (a) Western blot analysis of FOXO3 in brain tissue of tMCAO mice (n = 6 in each group). *p < 0.05 vs. sham group. (b) Western blot analysis of FOXO3 in OGD/R cells. *p < 0.05 vs. control group. (c) Altered protein levels of FOXO3 after transfection of FOXO3(+), FOXO3(−) plasmids or their scrambled vectors (NC), respectively. *p < 0.05 vs. FOXO3(+)‐NC group. ^# p < 0.05 vs. FOXO3(−)‐NC group. (d) CCK‐8 assay was applied to estimate the impacts of FOXO3 expressing alteration on OGD/R cell viability. (e) Western blot analysis of ATG7, LC3B II and SQSTM1 expression influenced by changing FOXO3 protein levels with GAPDH as an endogenous control. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + FOXO3(+)‐NC group. ^▲ p < 0.05 vs. OGD/R + FOXO3(−)‐NC. For (b–e), data are presented as the mean ± SD (n = 3 in each group)

Figure 5

FOXO3 was a target gene of miR‐200a and modulated by both KCNQ1OT1 and miR‐200a. (a) Western blot analysis of FOXO3 affected by KCNQ1OT1 knockdown in OGD/R. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + sh‐NC group. (b) Linear regression analysis was conducted to each individual expression of miR‐200a and FOXO3 in tMCAO mice (n = 10). **p < 0.01. (c) Western blot analysis of FOXO3 expression affected by altered miR‐200a. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + pre‐NC group. ^▲ p < 0.05 vs. OGD/R + anti‐NC group. (d) Western blot analysis of FOXO3 expression affected by altered KCNQ1OT1 and miR‐200a with GAPDH as an endogenous control. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + sh‐NC + pre‐NC group. (e) Presentation of the putative binding site of miR‐200a and FOXO3 (FOXO3‐wt), and the designed mutant sequence (FOXO3‐mut). (f)The relative luciferase activities of N2a cells co‐transfected FOXO3‐wt or FOXO3‐mut with pre‐miR‐200a or pre‐NC. *p < 0.05 vs. FOXO3‐wt + pre‐NC. ^# p < 0.05 vs. FOXO3‐mut + pre‐miR‐200a. (g) CCK‐8 assay was used to investigate the effect of altered miR‐200a and FOXO3 expression on OGD/R cells viability. (h) Western blot analysis of ATG7, SQSTM1, and LC3B II expression impacted by altered miR‐200a and FOXO3. *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + pre‐NC + FOXO3(+)‐NC group. ^▲ p < 0.05 vs. OGD/R + pre‐miR‐200a + FOXO3(+)‐NC group. ^▼ p < 0.05 vs. OGD/R + pre‐NC + FOXO3(+) group. For (a,c,d,f–h), data are presented as the mean ± SD (n = 3 in each group).

Figure 6

ATG7 was a downstream gene of KCNQ1OT1/miR‐200a/FOXO3 axis. (a) Linear regression analysis was conducted to each individual expression of FOXO3 and ATG7 in tMCAO mice (n = 10). **p < 0.01. (b) CCK‐8 assay was conducted to investigate the effect of altered FOXO3 and ATG7 expression on OGD/R cells viability. Data are presented as the mean ± SD (n = 3 in each group). *p < 0.05 vs. control group. ^# p < 0.05 vs. OGD/R + FOXO3(+)‐NC + ATG7(−)‐NC group. ^▲ p < 0.05 vs. OGD/R + FOXO3(+) + ATG7(−)‐NC group. ^▼ p < 0.05 vs. OGD/R + FOXO3(+)‐NC + ATG7(−) group. (c) Different reporter constructs with schematic depiction was applied, and the luciferase activity is detected. The Y‐bar shows the deletions on the DNA fragments. X‐bar shows the plasmid activity with normalized to the co‐transfection of reference vector, and relative activity to pEX3 empty vector, which activity was set to 1. Data are presented as the mean ± SD (n = 3, each). (d) Presentation of the predicted binding site for FOXO3 and ATG7 promoter region 3,000 bp upstream of the transcription start site (TSS) which designated as +1. Immunoprecipitated DNA was amplified by PCR. Normal rabbit IgG was used as a negative control. (e) The schematic description of the mechanism of KCNQ1OT1/miR‐200a/FOXO3/ATG7 axis in regulating autophagy during ischemia and reperfusion (I/R) injury