The role of lncRNA MALAT1 and NEAT1 in neuroprotection of hypoxia preconditioning mouse hippocampus cell
10.3969/j.issn.1672-5921.2024.08.003
- VernacularTitle:长链非编码核糖核酸肺腺癌转移相关转录本1和核旁斑组装转录本1在低氧预适应小鼠海马神经保护中的作用研究
- Author:
Haidong HOU
1
;
Lei YAN
;
Liping WANG
;
Jing YANG
;
Yucheng GUI
;
Yongqiang DU
;
Guo SHAO
Author Information
1. 523660 广东省东莞市清溪医院神经外科
- Keywords:
Hypoxic preconditioning;
Long non-coding RNA;
MALAT1;
NEAT1;
NR2B
- From:
Chinese Journal of Cerebrovascular Diseases
2024;21(8):525-536
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the expression of long non-coding ribonucleic acid(lncRNA)metastasis-associated lung adenocarcinoma transcript 1(MALAT1)and nuclear paraspeckle assembly transcript 1(NEAT1)in the hippocampus and HT22 cells of hypoxia pre-acclimated(HPC)mice and their relationship with neuroprotection.Methods(1)Thirty-six male Institute of Cancer Research(ICR)mice were randomly divided into three groups according to the random number table method of complete randomization:the control group,the hypoxia group and the hypoxia preconditioning group,with 12mice in each group.Mice in the control group were not exposed to hypoxia,mice in the hypoxia group were exposed to hypoxia once,and mice in the hypoxia preconditioning group were exposed to hypoxia four times.Immediately after the end of hypoxia treatment,all mice were decapitated and killed and hippocampal tissues were isolated and preserved in groups.(2)HT22 cells were cultured in medium containing 10％foetal bovine serum and 100 U/ml penicillin-streptomycin.When cell confluence was greater than 90％,they were transferred to 24-well plates for culture and then processed in 2 batches.6 pmol disordered small interfering RNA(siRNA),MALAT1 siRNA(siMALAT1),NEAT1 siRNA(siNEAT1),siMALAT1+siNEAT1 were transfected into the negative control group,siMALAT1 group,siNEAT1 group,and siMALAT1+siNEAT1 group of the first batch of HT22 cells one by one by transfection reagent,and the blank group did not have any treatment;then they were cultured under normal conditions(5％CO2 and 95％air)for 48 h.In the second batch of HT22 cells,6 pmol of disordered siRNA,disordered siRNA,siMALAT1,siMALAT1,siNEAT1 and siNEAT1 were transfected one by one correspondingly to the negative control group and the negative control+oxygen-glucose deprived/reoxygen(OGD/R)group,siMALAT1 group,siMALAT1+OGD/R,siNEAT1 group,siNEAT1+OGD/R group.48 h after transfection,HT22 cells of negative control group,siMALAT1 group and siNEAT1 group were cultured under normal conditions(5％CO2 and 95％air),and the cells of negative control+OGD/R group,siMALAT1+OGD/R group and siNEAT1+OGD/R group were treated with OGD/R.That is,under low oxygen conditions(1％O2+5％CO2+94％N2)exposure for 8 h,and then culture under normal conditions for 16 h.(3)The real-time fluorescence quantitative polymerase chain reaction(PCR)and Western blot was used to determine the expression of MALAT1,NEAT1,N-methyl-D-aspartate receptor subunit 2B(NR2B)messenger RNA(mRNA)and NR2B protein in the hippocampus of mice,the relative expression levels of NR2B mRNA and NR2B protein after transfection of HT22 cells in each group,and the relative expression levels of haemoglobin breakdown products and activated cysteine protease protein 3 after transfection and OGD/R of HT22 cells in each group.The survival rate of HT22 cells in each group was calculated.Results(1)The differences in relative expression of MALAT1(F=43.92),NEAT1(F=506.4),NR2B mRNA(F=50.64)and NR2B protein(F=41.24)in the hippocampus of mice in the three groups were statistically significant(all P＜0.05).The relative expression of MALAT1([1.68±0.06]vs.[1.00±0.08]),NR2B mRNA([1.26±0.06]vs.[1.00±0.01]),and NR2B protein([1.47±0.05]vs.[1.00±0.01])was increased in the hypoxia group as compared to the control group(all P＜0.05),whereas the relative expression of NEAT1([1.02±0.10]vs.[1.00±0.03])were not statistically significant(P＞0.05),and the relative expression of MALAT1([1.12±0.13]vs.[1.00±0.08])and NEAT1([2.88±0.10]vs.[1.00±0.03])were increased in hypoxic preconditioned group.Compared with hypoxia group,the relative expression of NR2B mRNA([0.54±0.07]vs.[1.26±0.06])and NR2B protein([1.17±0.07]vs.[1.47±0.05])were decreased(both P＜0.05).(2)The differences in the relative expression of NR2B mRNA(F=36.92)and NR2B protein(F=56.98)after transfection of HT22 cells in the five groups were statistically significant(both P＜0.05).Compared with the negative control group,siMALAT1 group(NR2B mRNA:[2.04±0.08]vs.[0.94±0.04],NR2B protein:[1.72±0.13]vs.[0.93±0.02]),siNEAT1 group(NR2B mRNA:[2.15±0.13]vs.[0.94±0.04],NR2B protein:[1.87±0.46]vs.[0.93±0.02]),siMALAT1+siNEAT1 group(NR2BmRNA:[2.09±0.16]vs.[0.94±0.04],NR2B protein:[2.07±0.30]vs.[0.93±0.02])showed the relative NR2B mRNA and NR2B protein expression were increased(all P＜0.05).(3)Differences in relative expression of haematopoietin breakdown product(145/150 kDa)protein(F=12.43),haematopoietin breakdown product(120 kDa)protein(F=7.15),and activated cysteamine protease protein 3 protein(F=6.61)were statistically significant in the 6 groups of HT22 cells transfected and treated with OGD/R(all P＜0.05).Compared with the siMALAT1 group,the siMALAT1+OGD/R group had 145/150kDa([1.42±0.48]vs.[0.85±0.34]),120 kDa([1.33±0.37]vs.[0.52±0.19])haematopoietin catabolism products and activated cysteamine protease protein 3([2.43±0.35]vs.[1.15±0.24])relative expression increased(all P＜0.05);compared with the negative control+OGD/R group,the siMALAT1+OGD/R group showed an increase in 145/150kDa([1.42±0.48]vs.[1.23±0.17]),120 kDa([1.33±0.37]vs.[0.80±0.21])relative expression of haematopoietin breakdown products and activated cysteamine protease protein 3([2.43±0.35]vs.[1.46±0.39])increased(all P＜0.05);compared with the siNEAT1 group,the siNEAT1+OGD/R group had a higher expression of 145/150 kDa([1.28±0.44]vs.[0.87±0.32]),120 kDa([0.81±0.36]vs.[0.63±0.16])relative expression of haematopoietic proteolytic products and activated cysteamine protease protein 3([1.51±0.45]vs.[1.01±0.27])increased(all P＜0.05).(4)The difference in HT22 cell survival rate among the 6 groups was statistically significant(F=5.54,P＜0.05).Compared with the negative control group,HT22 cell survival was decreased in the siMALAT1,siNEAT1,siMALAT1+OGD/R and siNEAT1+OGD/R groups([0.65±0.40],[0.76±0.35],[0.24±0.17],[0.23±0.16]vs.[0.84±0.04],all P＜0.05);cell viability was reduced in the siMALAT1+OGD/R group compared with the siMALAT1 group([0.24±0.17]vs.[0.65±0.40],P＜0.05);and cell viability was reduced in the siNEAT1+OGD/R group compared with the siNEAT1 group([0.23±0.16]vs.[0.76±0.35],P＜0.05).Conclusion HPC increased the expression of MALAT1 and NEAT1 in the hippocampus of mice,and MALAT1 and NEAT1 may participate in the neuroprotective effect of mice after ischemia and hypoxia by affecting the expression of NR2B.
