lncRNA HOTAIR improves the cognitive ability of neonatal epileptic rats by regulating the miR-20a-5p/TGF-β1 axis
This study proposed to explore the regulation of long non-coding RNA(lncRNA)HOX transcript antisense RNA(HOTAIR)on miR-20a-5p/TGF-β1 signaling axis,and its effect on the cognitive ability of neonatal rats with epilepsy.The elbow venous blood was collected from twenty epilepsy patients and twenty healthy controls,the serum was isolated and the expression of lncRNA HOTAIR was detected by qRT-PCR.Neonatal rats were intraperitoneally injected with kainic acid to establish the epilepsy models,and were randomly divided into normal control group,model group,lncRNA HOTAIR over-expression(Lv-HOTAIR)group,negative control(Lv-NC)group,TGF-β1 inhibitor group,and Lv-HOTAIR+TGF-β1 inhibitor group,with twenty rats per group.Racine grading standard was referred to to assess the degree of epileptic seizures.Water maze test was performed to evaluate the rats cognitive function.Timm staining was used to observe the sprouting status of mossy fibers.Immunohistochemistry assay was used to detect the positive expression of M2-type microglia-specific protein marker Arginase 1(Arg-1).TUNEL staining was set out to detect the apoptosis rate of rat hippocampal neurons.qRT-PCR was used to detect the expression of lncRNA HOTAIR and miR-20a-5p in hippocampus.Western blotting was run to detect the expressions of TGF-β1,M1 type microglia polarization protein marker CD11,M2 type microglia polarization protein marker CD206,IL-6,and IL-1β in hippocampus.Dual-luciferase report gene experiment was performed to verify the targeting regulation of lncRNA HOTAIR on miR-20a-5p and that of miR-20a-5p on TGF-β1.An in vitro epilepsy cell model was established,and lncRNA HOTAIR and miR-20a-5p were simultaneously over-expressed to verify the reversal of miR-20a-5p on the anti-epileptic effect of lncRNA HOTAIR.The results showed that the expression of lncRNA HOTAIR in the serum of epilepsy patients was significantly lower than that of healthy controls(P＜0.05).lncRNA HOTAIR negatively regulates the miR-20a-5p expression,which in turn negatively regulates the TGF-β1 expression.Compared to the normal control group,rats in the epilepsy model group had higher seizure grading scores,decreased cognitive function,severe mossy fiber sprouting,and increased neuronal apoptosis.In addition,the expression of miR-20a-5p was increased,the expressions of lncRNA HOTAIR and TGF-β1 were decreased,and the anti-inflammatory phenotype activation of microglia type M2 was decreased(all with P＜0.05).Over-expression of lncRNA HOTAIR decreases the level of seizures in epileptic rats,improves cognitive function,mossy fiber sprouting and reduces neuronal apoptosis,inhibits miR-20a-5p expression,improves TGF-β1-mediated microglia M2 type anti-inflammatory phenotype activation,and exerts the anti-epileptic effect(all with P＜0.05).TGF-β1 inhibitor intervention or elevating miR-20a-5p expression can partially weaken the neuronal survival-promoting effect and anti-epileptic effect of lncRNA HOTAIR(all with P＜0.05).In conclusion,lncRNA HOTAIR may reduce the expression of miR-20a-5p and up-regulate the expression of TGF-β1 to increase the activation of M2-type anti-inflammatory phenotype of microglia and exhibit anti-epileptic function.
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