微小RNA-150及靶基因SRC激酶信号抑制剂1在下肢深静脉血栓中的表达及作用机制

Expression and action mechanism of microRNA-150 and target gene SRC kinase signaling inhibitor 1 in lower extremity deep vein extremity

目的探讨微小RNA(miRNA)-150及靶基因SRC激酶信号抑制剂1(SRCIN1)在下肢深静脉血栓(LEDVT)中的表达及作用机制。方法收集2022年1月至2023年1月柳州市人民医院收治的60例LEDVT患者的临床资料作为LEDVT组,另纳入同期进行体检的60例健康者作为对照组。采集两组受试者的外周静脉血,并分离内皮祖细胞。通过荧光定量逆转录聚合酶链反应(qRT-PCR)检测miRNA-150的表达水平,利用生物信息学分析和双荧光素酶报告基因实验验证miRNA-150的靶基因,采用蛋白质印迹法(Western blot)检测SRCIN1蛋白的表达,采用四甲基偶氮唑蓝(MTT)实验检测内皮祖细胞增殖情况。结果与对照组相比,LEDVT组内皮祖细胞中miRNA-150的相对表达量明显降低(P﹤0.01)。生物信息学分析和双荧光素酶报告基因实验证实SRCIN1是miRNA-150的靶基因;在野生型SRCIN1中,与转染miRNA-NC的细胞相比,转染miRNA-150 agomir细胞的荧光素酶活性明显降低(P﹤0.01)。在突变型SRCIN1中,miRNA-150对SRCIN1的负向调控作用消失。Western blot检测结果显示,与miRNA-150antagomir组内皮祖细胞相比,miRNA-150 agomir组内皮祖细胞中SRCIN1蛋白的表达水平降低(P﹤0.05);与对照组相比,LDVT组内皮祖细胞中SRCIN1蛋白的表达水平升高(P﹤0.05)。MTT实验检测结果显示,与miRNA-150antagomir组相比,miRNA-150 agomir组的吸光度(OD)值在48、72 h时均明显升高(P﹤0.01)。结论miRNA-150在内皮祖细胞中的表达水平降低可能与LEDVT的发生、发展有关。高表达的miRNA-150可能通过靶向SRCIN1促进内皮祖细胞的增殖,从而达到治疗LEDVT的目的。

Objective To investigate the expression and action mechanism of microRNA(miRNA-150)and target gene SRC kinase signaling inhibitor 1(SRCIN1)in lower extremity deep vein extremity(LEDVT).Method The clinical data of 60 LEDVT patients admitted to Liuzhou People's Hospital from January 2022 to January 2023 was collected,and include them as the LEDVT group,the 60 healthy people who underwent physical examinations during the same period were included as the control group.The peripheral venous blood from two groups of subjects were collected and endothelial progenitor cells were isolated.The level of miRNA-150 was detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR),the target gene of miRNA-150 was predicted by bioinformatics analysis and double luciferase reporter gene experiment,the expression of SRCIN1 protein was detected by Western blot assay,and the proliferation of endothelial progenitor cells were detected by methyl thiazolyl terazolium(MTT).Result The relative expression level of miRNA-150 in endothelial progenitor cells in the LEDVT group were significantly lower than that in the control group(P<0.01).Bioinformatics analysis and double luciferase reporter gene experiment confirmed that SRCIN1 was the target gene of miRNA-150.In wild-type SRCIN1,compared with cells transfected with miRNA-NC,the luciferase activity of cells transfected with miRNA-150 agomir was significantly reduced(P<0.01).In mutant SRCIN1,the negative regulatory effect of miRNA-150 on SRCIN1 disappears.Western blot assay showed that compared with endothelial progenitor cells in the miRNA-150 antagomir group,the expression level of SRCIN1 protein in endothelial progenitor cells in the miRNA-150 agomir group was reduced;compared with the control group,the expression level of SRCIN1 protein in endothelial progenitor cells in the LDVT group was increased.The methyl thiazolyl terazolium(MTT)experimental results showed that compared with the miRNA-150 antagomir group,the optical density(OD)values of the miRNA-150 agomir group were significantly increased at 48 and 72 hours(P<0.01).Conclusion The decreased expression of miRNA-150 in endothelial progenitor cells may be one of the mechanisms for the occurrence and development of LEDVT.Highly expressed miRNA-150 may promote the proliferation of endothelial progenitor cells by targeting SRCIN,thereby achieving the goal of treating LEDVT.