关键词: 长链非编码RNA, 肺腺癌转移相关转录本1, 血管生成, 不均一核糖核蛋白K, 血管内皮生长因子A

Abstract:

Objective To discuss the effect of long non-coding RNA （lncRNA） lung cancer metastasis-associated transcript 1 （MALAT1） on the angiogenesis of the human brain microvascular endothelial cells （HBMECs） induced by oxygen-glucose deprivatioin/reoxygenation（OGD/R）， and to clarify its potential molecular mechanism. Methods The binding sites of MALAT1， heterogeneous nuclear ribonucleoprotein K （hnRNPK）， and vascular endothelial growth factor A （VEGFA） were predicted by bioinformatics. The HBMECs were treated with OGD/R to establish the cerebral ischemia cell model. The HBMECs were divided into control group， OGD/R model group， OGD/R+siNC group， OGD/R+ silencing MALAT1 （OGD/R+siMALAT1） group and OGD/R+siMALAT1+over-expression of VEGFA （OGD/R+siMALAT1+VEGFA） group.Small interference RNA（siRNA） was used to silence the expression of MALAT1， and pcDNA vector was used to construct the VEGFA over-expression vector. The constructed siMALAT1 and pcDNA VEGFA vectos were transfected into the HBMECs separately or simultaneously. The angiogenesis abilities of cells in various groups were detected by tube formation assay； the expression levels of VEGFA protein in the cells in various groups were detected by Western blotting method. Twenty 6-week-old healthy male C57BL/6 J mice were randomly divided into sham operation group， middle cerebral artery occlusion （MCAO） model group， MCAO+NC blank vector（MCAO+NC） group， and MCAO+ over-expression of MALAT1 （MCAO+MATAL1） group，and there were 5 mice in each group. Except for sham operation group， the mice in the other groups were used to construct the MCAO mouse models by suture method， and the NC empty vector and MALAT1 over-expression vector were injected into the right ventricle of the mice in MCAO+NC group and MCAO+MATAL1 group， respectively. The percentage of cerebral infarction area of mice in various groups were detected by 2，3， 5-triphenyltetrazole chloride （TTC） staining，and the expression levels of VEGFA protein in brain tissue of mice in various groups were detected by Western blotting method. Results The bioinformatics results showed that there were binding sites among MALAT1 and hnRNPK， hnRNPK and VEGFA. The results of tube formation experiment and Western blotting showed that compared with control group， the number of the tubes in OGD/R model group was significantly increased （P<0.01）， and the expression level of VEGFA protein in the cells in OGD/R model group was significantly increased （P<0.01）； compared with OGD/R model group， the number of the tubes in OGD/R+siMALAT1 group was significantly decreased （P<0.01）， and the expression level of VEGFA protein in the cells in OGD/R+siMALAT1 group were significantly decreased （P<0.01）； compared with OGD/R+siMALAT1 group， the number of the tubes in OGD/R+siMALAT1+VEGFA group was significantly increased （P<0.01）， and the expression level of VEGFA protein in the cells in OGD/R+siMALAT1+VEGFA group was significantly increased （P<0.01）. In the MCAO model mouse experiment， the results of TTC staining and Western blotting showed that compared with sham operation group， the percentage of cerebral infarction area and the expression level of VEGFA protein in brain tissue of the mice in MCAO model group were significantly increased （P<0.01）； compared with MCAO model group， the percentage of cerebral infarction area of the mice in MCAO+MALAT1 group was significantly decreased （P<0.01）， and the expression level of VEGFA protein in brain tissue of the mice in MCAO+MALAT1 group was significantly increased （P<0.01）. Conclusion LncRNA MALAT1 can enhance the stability of the target gene VEGFA and upregulate its expression， and promote the angiogenesis of the cerebral ischemic stroke mice， which provides a new direction for the treatment of cerebral ischemic stroke.

Key words: Long non-coding RNA, Metastasis associated lung adenocarcinoma transcript 1, Angiogenesis, Heterogeneous nuclear ribonucleoprotein K, Vascular endothelial growth factor A