| Objective:micro RNA 30a(miR-30a)gene is a newly discovered small non-coding RNA.miR-30 a is an important gene expression regulator,which plays a significant role in regulating various biological processes such as cell proliferation,apoptosis,inflammatory reaction and angiogenesis.Studies in recent years have found that it has a prominent effect on the pathogenesis of atherosclerosis(AS).AS is an important pathological basis of Ischemic stroke(IS).However,the correlation between the polymorphism of miR-30 a gene and IS,and whether miR-30a-5p contributes the pathogenesis of IS by regulating SOCS1,a downstream target gene,have not been reported in the literature so far.Therefore,it remains to be studied and discussed.In this paper,the technical approaches including RT-q PCR,CCK8,Western blot and flow cytometry were combined with cell functional experiments to explore the molecular mechanism of miR-30a-5p/SOCS1 axis involved in IS,so as to provide foundation for the molecular diagnosis and gene therapy of IS.Method:(1)EDTA anticoagulant blood samples of a total 248 IS patients and 230 healthy subjects in Guangxi were collected and genomic DNA were extracted subsequently.The SNPscan high-throughput genotyping technology was adopted to detect the genotyping of miR-30 a rs2222722 sites in the IS group and control group before the statistic analysis of the correlation between the polymorphism of miR-30 a gene and IS.(2)The respective 33 samples from the IS group and the control group with matching age and sex were randomly collected.The expression of miR-30 a gene in peripheral blood mononuclear cells of the both group samples was detected by RT-q PCR,and the influence of the miR-30 a gene’s polymorphism on the expression level of miR-30 a gene was further analyzed.(3)HUVEC were treated with 100 μg/ml ox-LDL to construct an atherosclerosis cell models that were transfected afterwards through the up-regulation and down-regulation of miR-30a-5p;the expressions of miR-30a-5p and SOCS1 were analyzed by RT-q PCR and Western blot,cell proliferation activity was detected by CCK-8 assay and apoptosis was detected by flow cytometry.(4)Four online sites(targetscan,Pic Tar,RNA22 and PITA)were employed to predict the interaction between miR-30a-5p and SOCS1.The combination of miR-30a-5p and SOCS1 was verified by double luciferase assay.Result:(1)SNPscan genotyping showed that AA,AG and GG genotypes at rs2222722 sites of miR-30 a gene were found in both the case group and the control group.(2)rs2222722 sites are associated with genetic susceptibility to ischemic stroke.AA genotype,A allele and dominant model significantly increased the risk of IS.The expression of miR-30 a in plasma of IS group was significantly higher than that of healthy control group.(3)miR-30a-5p was found to act on the non-coding region of the 3 ’-UTR of SOCS1 based on Target Scan Human database,and the binding of SOCS1 to miR-30a-5p was confirmed by Dual luciferase experiment reports.(4)In the atherosclerotic cell model,the expression level of miR-30a-5p was increased compared with the normal control group.The up-regulation of miR-30a-5p expression level decreased HUVEC activity and increased apoptosis,and the down-regation of miR-30a-5p expression level increased HUVEC activity and decreased apoptosis.(5)q RT-PCR showed that the expression level of SOCS1 was significantly decreased after the up-regation of miR-30a-5p;or otherwise,SOCS1 expression level was significantly increased.Western blot results also revealed that the overexpression of miR-30a-5p inhibited the expression of SOCS1 protein,but conversely,it promoted the expression of SOCS1 protein.Conclusion:(1)SNPscan typing showed that there was a genetic polymorphism at rs2222722 of miR-30 a gene,which was correlated with the genetic susceptibility of IS.(2)The study confirmed that miR-30 a expression was significantly increased in peripheral blood mononuclear cells of IS patients and was involved in the pathogenesis of IS by regulating the target gene SOCS1. |
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