EPO activates PI3K-IKKα-CDK1 signaling pathway to promote the proliferation of Glial Cells under hypoxia environment

Abstract

Erythropoietin (EPO), supports the function and survival of neurons through astrocytes and has a protective role in neonatal asphyxia brain injury; yet, its mechanism of action remains unclear. As a neuroprotective factor, EPO is also used in the treatment of various diseases, such as neurodegenerative diseases, Parkinson's disease, traumatic brain injury, by decreasing inflammatory reaction, resisting apoptosis, and lowering oxidative stress. The aim of this study was to examine the effect and mechanism of EPO on promoting human brain glial cell proliferation under hypoxia in vitro. Under CoC12-induced hypoxia, after adding EPO, high-throughput sequencing was used to screen out meaningful up-regulated and significant differentially expressed genes PI3K, IKKα CDK1 related to proliferation, and make further verification by qPCR and western blotting. Under hypoxia, EPO promoted cell proliferation and the expression of PI3K while this effect was inhibited (along with a decrease of downstream genes IKKα and CDK1 decreased) after adding PI3K inhibitor to cell culture. EPO can promote cell proliferation and CDK1 expression, while after inhibiting CDK1 expression, the promotion of EPO on cell proliferation was eliminated. These data proved that EPO promotes the proliferation of U251 cells by activating the PI3K-IKKα-CDK1 signaling pathway under CoC12-induced hypoxia.

Figure 1 -. Establishment and evaluation of hypoxia model of U251 cells using CoCl₂. (A) CCK-8 assay to detect the effect of CoCl₂ on cell proliferation; * P<0.05 vs. the blank control group, # P<0.05 vs. the MgCl₂ group, Newman-Keuls multiple comparisons test. (B) qPCR was used to determine the expression of HIF-1α mRNA in U251 cells of each group after adding CoC1₂; *P<0.05 vs. the blank control group, T-test. (C) WB method to detect the expression of HIF-1α protein after adding CoC1_2. (D) *P<0.05 vs. the blank control group, T-test.

Figure 2 -. The effect of EPO on cell transcriptome under hypoxia by high throughput sequencing. (A) The heat map,50 genes of up-regulation and 50 genes of down-regulation were selected for visualization, and the genes were sorted according to p value significance. (B) The volcano plot is mapped according to log2FC>0.5, p<0.05, marking three target genes. (C-E) mRNA transcription levels of PIK3, IKKα and CDK1 were verified by qPCR method. * P<0.05 vs. the CoC1₂ group, t-test.

Figure 3 -. Effect of EPO on U251 cells under hypoxia. (A) The effect of EPO on the proliferation of U251 cells under hypoxia was determined by CCK-8 assay. ^#P<0.05 vs. the blank control group.* P<0.05 vs. the CoC1₂ group. (B-H) Expression of PIK3, p-PIK3,IKKα and CDK1 verified by WB method. ^# P<0.05 vs. the blank control group.* P<0.05 vs. the CoC1₂ group, Newman-Keuls multiple comparisons test.

Figure 4 -. The intervention effect of EPO on U251 cells under hypoxia through PI3K signaling pathway. (A) The IC50 concentration of the inhibitor LY294002. (B) The cell viability of effect of EPO on U251 cells. ^#P<0.05 vs. the CoC1₂+DMSO group.* P<0.05 vs. the CoC1₂ group and &P<0.05 vs. the CoC1₂+EPO group. (C, D) The effect of inhibition of PI3K expression on IKK α and CDK1 transcription level under hypoxia.#P<0.05 vs. the CoC1₂ group.* P<0.05 vs. the CoC1₂+DMSO group, Newman-Keuls multiple comparisons test.

Figure 5 -. Intervention of EPO on U251 cells under hypoxia through CDK1. (A) Partial sequencing of Sh-CDK1-2 (The part marked by black line is target sequence). (B) The expression of green fluorescent protein in each group: B-1, NC group; B-2, Sh-CDK1-1 group; B-3, Sh-CDK1-2 group; B-4, Sh-CDK1-3 group, 400*. (C) The interference effect of CDK1 interfering RNA eukaryotic expression vector under hypoxia environment.* P<0.05 vs. the NC group. (D) CCK-8 assay was used to detect the intervention effect of EPO on cells under hypoxia through CDK1.* P<0.05 vs. the CoC1₂ group; ^# P<0.05 vs. the CoC1₂+NC group and & P<0.05 vs. the CoC1₂+EPO group, Newman-Keuls multiple comparisons test.