微小RNA在高血压相关性脑出血患者中表达的初步分析

Preliminary study of microRNAs expression in patients with hypertension related intracerebral hemorrhage

目的探讨微小RNA对高血压相关性脑出血(HRICH)患者脑血管组织的影响。方法回顾性连续纳入2018年9月至2019年12月四川绵阳四〇四医院神经外科自发性高血压脑出血住院患者9例为观察组,回顾性连续纳入同期四川绵阳四〇四医院神经外科因颅脑外伤需行颅内减压术且伴高血压病史的住院患者9例为对照组。术中取两组患者术区皮质造瘘口的少量脑血管标本,通过Trizol法提取标本总RNA,筛选差异表达的微小RNA。从微小RNA差异表达谱中随机选择3种微小RNA,用实时荧光定量逆转录聚合酶链反应(PCR)技术验证结果的可靠性。采用TargetScan、miRDB、miRanda三种在线分析工具预测差异表达微小RNA的靶基因,并对靶基因进行京都基因与基因组百科全书(KEGG)分析,富集程度越高,相应的P值越小。结果(1)与对照组标本比较,观察组患者脑血管组织细胞中有35种差异表达的微小RNA,其中15种微小RNA表达下调,即hsa-let-7g-3p、hsa-miR-103a-2-5p、hsa-miR-126-5p、hsa-miR-130a-3p、hsa-miR-210-3p、hsa-miR-214-3p、hsa-miR-28-5p、hsa-miR-34a-5p、hsa-miR-378a-3p、hsa-miR-145-5p、hsa-miR-4785、hsa-miR-549a-5p、hsa-miR-574-3p、hsa-miR-590-3p、hsa-miR-660-3pg表达降低(均P<0.05);20种微小RNA表达上调,即hsa-miR-1180-3p、hsa-miR-1224-3p、hsa-miR-128-2-5p、hsa-miR-1298-3p、hsa-miR-137-5p、hsa-miR-139-3p、hsa-miR-139-5p、hsa-miR-2682-5p、hsa-miR-370-3p、hsa-miR-433-3p、hsa-miR-485-5p、hsa-miR-541-3p、hsa-miR-543、hsa-miR-598-5p、hsa-miR-6505-5p、hsa-miR-668-3p、hsa-miR-7-5p、hsa-miR-744-5p、hsa-miR-873-3p、hsa-miR-942-3p表达升高(均P<0.05)。(2)对已知的差异表达微小RNA采用TargetScan、miRDB、miRanda三种在线分析工具进行基因预测,结果显示,共同交集预测得到的靶基因有87个交集。(3)随机抽取3种差异表达的微小RNA进行荧光定量逆转录PCR验证,结果显示,与对照组(颅脑外伤伴高血压病史者)比较,观察组(自发性高血压脑出血者)hsa-miR-145-5p、hsa-miR-28-5p表达降低(0.22±0.17比1.00±0.29,t=0.153;0.13±0.03比1.00±0.62,t=0.421),hsa-miR-139-3p表达升高(1.95±0.29比1.00±0.52,t=0.492),组间差异均有统计学意义(均P<0.05)。(4)交集靶基因的KEGG富集分析结果显示,主要涉及Ras相关蛋白1信号通路、黏附斑激酶、肿瘤微小RNA、环磷酸鸟苷-环磷酸鸟苷依赖性蛋白激酶信号通路、磷脂酰肌醇3激酶-蛋白激酶B信号通路、丝裂原活化蛋白激酶(MAPK)信号通路、人乳头状瘤病毒、肿瘤蛋白聚糖、肿瘤通道蛋白,其中MAPK信号通路的富集程度较高(P<0.01)。结论微小RNA可能在调控HRICH的相关生物学功能中具有重要作用。

Objective To investigate the effect of microRNAs on cerebrovascular tissue in patients with hypertension related intracerebral hemorrhage(HRICH).Methods From September 2018 to December 2019,9 hospitalized patients with spontaneous hypertensive intracerebral hemorrhage in the Neurosurgery Department of Sichuan Mianyang 404 Hospital were included retrospectively as the observation group;9 hospitalized patients requiring intracranial decompression due to craniocerebral trauma and with a history of hypertension in the Neurosurgery Department of Mianyang 404 Hospital during the same period were included retrospectively as the control group.During the operation,a small number of cerebrovascular specimens from the cortical ostomy in the operative area of two groups of patients were taken.The total RNA specimens were extracted by Trizol method to screen differentially expressed microRNAs.Three kinds of microRNAs were randomly selected from the microRNA differential expression spectrum;and reliability of the results was verified by real-time fluorescence-based quantitative reverse transcriptional polymerase chain reaction(PCR).TargetScan,miRDB and miRanda were used to predict the target genes of differentially expressed microRNAs.Target genes were analyzed by The Kyoto Encyclopedia of Genes and Genomics(KEGG).The higher the enrichment degree,the smaller the corresponding P value.Results(1)Compared with the control group,35 kinds of microRNAs were differentially expressed in the cerebrovascular tissue cells of patients in the observation group,15 of which were down-regulated.The expressions of hsa-let-7g-3p,hsa-miR-103a-2-5p,hsa-miR-126-5p,hsa-miR-130a-3p,hsa-miR-210-3p,hsa-miR-214-3p,hsa-miR-28-5p,hsa-miR-34a-5p,hsa-miR-378a-3p,hsa-miR-145-5p,hsa-miR-4785,hsa-miR-549a-5p,hsa-miR-574-3p,hsa-miR-590-3p,and hsa-miR-660-3p were significantly decreased(all P<0.05).The expressions of 20 microRNAs were up-regulated,including hsa-miR-1180-3p,hsa-miR-1224-3p,hsa-miR-128-2-5p,hsa-miR-1298-3p,hsa-miR-137-5p,hsa-miR-139-3p,hsa-miR-139-5p,hsa-miR-2682-5p,hsa-miR-370-3p,hsa-miR-433-3p,hsa-miR-485-5p,hsa-miR-541-3p,hsa-miR-543,hsa-miR-598-5p,hsa-miR-6505-5p,hsa-miR-668-3p,hsa-miR-7-5p,hsa-miR-744-5p,hsa-miR-873-3p,and hsa-miR-942-3p were significantly increased(all P<0.05).(2)The known differentially expressed microRNAs were predicted by using three online analysis tools of TargetScan,miRDB and miRanda.The results showed that there were 87 intersection genes predicted by common intersection.(3)Three kinds of differentially expressed microRNAs were randomly selected for real-time fluorescence-based quantitative reverse transcriptional PCR verification.The results showed that compared with patients with craniocerebral trauma and a history of hypertension,the expressions of hsa-miR-145-5p and hsa-miR-28-5p in the observation group(patients with spontaneous hypertensive cerebral hemorrhage)were decreased compared with those in the control group(0.22±0.17 vs.1.00±0.29,t=0.153;0.13±0.03 vs.1.00±0.62,t=0.421);the expression of hsa-miR-139-3p was increased(1.95±0.29 vs.1.00±0.52,t=0.492);the differences between groups were statistically significant(all P<0.05).(4)KEGG enrichment analysis was performed on target genes predicted by different miRNAs.Target genes were mainly significantly enriched in Rap1 signaling pathway,adhesion plaque kinase,microRNAs in cancer,cyclic guanosine monophosphate(cGMP)-protein kinase G(PKG)signaling pathway,phosphatidylinositol 3-kinase(PI3K)-protein kinase B(Akt)signaling pathway,mitogen-activated protein kinase(MAPK)signaling pathway,human papillomavirus infection,proteoglycans in cancer and channel protein in cancer.Among them,the enrichment degree of MAPK signaling pathway was higher(P<0.01).Conclusion MicroRNAs may play an important role in regulating the related biological functions of HRICH.