| Objective:Ischemic stroke is a major cause of disability and death worldwide.At present,studies have reported that Long non-coding RNA-SNHG17(LncRNA SNHG17)is involved in colorectal cancer-related angiogenesis and whether it can participate in angiogenesis after ischemic stroke remains to be further confirmed.The present study detected the expression of LncRNA SNHG17 in the serum of patients with ischemic stroke and in vitro injury model of the oxygen-glucose deprivation/ re-oxygenation(OGD/R)of mouse brain microvascular endothelial cells(bEnd.3),aiming to provide experimental basis and theoretical basis for the diagnosis and treatment of ischemic stroke.Methods:1.A total of 79 patients with ischemic stroke due to unilateral middle cerebral artery occlusion who were treated in Qingdao University Affiliated Hospital from October 2018 to June 2020 were selected.All the enrolled patients underwent digital subtraction angiography(DSA)to evaluate collateral compensation,and were divided into two groups: the good collateral circulation group and the poor collateral circulation group.The serum was collected after centrifugation,and quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect the expression level of LncRNA SNHG17 in the serum of the patients.2.To detect the expression of LncRNA SNHG17,Vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)in the OGD/R injury model of bEnd.3cells:(1)Construct an OGD/R injury model of bEnd.3 cells and use Observe the morphology,number,and structure changes of bEnd.3 cells in the Control group and each OGD/R group with an inverted microscope.Trypan Blue assay,CCK-8 assay,and Lactate dehydrogenase(LDH)release assay kit were used to determine the survival ratio,cell viability,and LDH content of bEnd.3 cells in each culture medium,respectively,to determine the appropriate time of oxygen and glucose deprivation.(2)RT-qPCR was used to detect the expression level of LncRNA SNHG17 in bEnd.3 cells after hypoxia for 6h,12 h,18h,24 h and 30 h.(3)Three small interfering RNAs(siRNAs)targeting LncRNA SNHG17 were constructed,and the siRNAs were transfected into bEnd.3 cells,respectively.The siRNAs with the highest interfering efficiency were screened out by quantitative real-time polymerase chain reaction(RT-qPCR).(4)After siRNA knockdown,the mRNA expression changes of LncRNA SNHG17 and angiogenesis-related genes such as VEGF and bFGF were detected by RT-qPCR.Results:1.Among 79 patients with ischemic stroke,there were 39 cases in the good collateral circulation group and 40 cases in the poor collateral circulation group.The comparison of general clinical data showed that there was no significant difference in age,gender,hypertension,diabetes,coronary heart disease,smoking history,drinking history and inspection index between the two groups of patients(P>0.05).RT-qPCR results showed that the expression of LncRNA SNHG17 in the good collateral circulation group was significantly higher than that in the poor collateral circulation group(expression abundances were 2.69±0.79 and 1.01±0.02,respectively),and the difference was statistically significant(P<0.001).2.(1)The degree of morphological and structural damage of bEnd.3 cells in each OGD/R group was significantly more serious than that in the control group.With increasing time of exposure to oxygen-glucose deprivation,the damage was gradually aggravated and the cell density was also significantly reduced.The results of the Trypan Blue assay and CCK-8 assay showed that the cell survival ratio and cell activity in each OGD/R group were significantly decreased compared with the control group(P<0.05),and the cell survival ratio was only 55.93% after 24 h hypoxia.The results of LDH release showed that the content of LDH value in cell culture medium in OGD/R groups was significantly higher than that in the control group(P<0.05).In LDH release assay kit,LDH release in the culture medium of each OGD/R group was significantly higher compared with the control group(P<0.05).(2)In each OGD/R group,the expression of LncRNA SNHG17 in bEnd.3 cells increased gradually with the prolonging of hypoxia and glucose deficiency time,and the peak abundance was 11.77±0.29,which was about12 times the normal level.(3)The sequence inhibition rate of siRNA-2 was significantly higher than that of siRNA-1 and siRNA-3 groups(P<0.05),and the inhibition rate was as high as 80.11%.(4)Compared with the control group,The relative expression levels of VEGF and bFGF mRNA in bEnd.3 cells induced by OGD/R for 24 h were significantly up-regulated(P<0.05).After transfection with siRNA-2 for 24 h and induction of bEnd.3cells by OGD/R for 24 h,RT-qPCR detected VEGF and bFGF mRNA relative expression level were significantly reduced(P<0.05).Conclusion:1.Compared with the poor collateral circulation group,the relative expression level of LncRNA SNHG17 in the good collateral circulation group was up-regulated,suggesting that there may be a certain correlation between the expression level of LncRNA SNHG17 and the formation of collateral circulation after ischemic stroke.2.The relative expression level of LncRNA SNHG17 was increased in OGD/R injury model of bEnd.3 cells,suggesting that LncRNA SNHG17 may be involved in the formation of angiogenesis after ischemic stroke.3.LncRNA SNHG17 was associated with increased expression of angiogenesis-related genes VEGF and bFGF in bEnd.3 cells under OGD/R environment,suggesting that the expression level of LncRNA SNHG17 was related to angiogenesis,laying an important theoretical foundation for the diagnosis and treatment of ischemic stroke. |
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