Wang Jie, Liu Lanchun, Liu Chao, Cheng Nuo, Mao Qiyuan, Chen Cong, Hu Jun, He Haoqiang, Hui Xiaoshan, Qu Peirong, Lian Wenjing, Duan Lian, Dong Yan, Liu Yongmei, Li Jun
Department of Cardiology, China Academy of Chinese Medical Sciences Guang'anmen Hospital, Beijing, China.
Department of Graduate, Beijing University of Chinese Medicine, Beijing, China.
Front Cardiovasc Med. 2023 Oct 30;10:1186297. doi: 10.3389/fcvm.2023.1186297. eCollection 2023.
This aim of this study is to screen the differential molecules of kidney deficiency and blood stasis (KDBS) syndrome in coronary heart disease by high-throughput sequencing. In addition, the study aims to verify the alterations in the expression levels of miR-4685-3p and its regulated downstream, namely, C1QC, C4, and C5, using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), and to determine whether the complement and coagulation cascade pathway is the specific pathogenic pathway.
Patients diagnosed with unstable angina pectoris with KDBS syndrome, patients with non-kidney deficiency blood stasis (NKDBS) syndrome, and a Normal group were recruited. The clinical symptoms of each group were further analyzed. Illumina's NextSeq 2000 sequencing platform and FastQC software were used for RNA sequencing and quality control. DESeq software was used for differential gene expression (DGE) analysis. qPCR and ELISA verification were performed on DGE analysis.
The DGE profiles of 77 miRNA and 331 mRNA were selected. The GO enrichment analysis comprised 43 biological processes, 49 cell components, and 42 molecular functions. The KEGG enrichment results included 40 KEGG pathways. The PCR results showed that, compared with the Normal group, the miR-4685-3p levels decreased in the CHD_KDBS group ( = 0.001), and were found to be lower than those observed in the CHD_NKDBS group. The downstream mRNA C1 regulated by miR-4685-3p showed an increasing trend in the CHD_KDBS group, which was higher than that in the Normal group ( = 0.0019). The mRNA C4 and C5 in the CHD_KDBS group showed an upward trend, but the difference was not statistically significant. ELISA was utilized for the detection of proteins associated with the complement and coagulation cascade pathway. It was found that the expression level of C1 was significantly upregulated in the CHD_KDBS group compared with the Normal group ( < 0.0001), which was seen to be higher than that in the CHD_NKDBS group ( < 0.0001). The expression levels of C4 and C5 in the CHD_KDBS group were significantly lower than the Normal group, and were lower than that in the CHD_NKDBS group ( < 0.0001).
The occurrence of CHD_KDBS might be related to the activation of the complement and coagulation cascade pathway, which is demonstrated by the observed decrease in miR-4685-3p and the subsequent upregulation of its downstream C1QC. In addition, the expression levels of complement C4 and C5 were found to be decreased, which provided a research basis for the prevention and treatment of this disease.
本研究旨在通过高通量测序筛选冠心病肾虚血瘀证的差异分子。此外，本研究旨在使用定量聚合酶链反应（qPCR）和酶联免疫吸附测定（ELISA）验证miR-4685-3p及其调控的下游分子C1QC、C4和C5的表达水平变化，并确定补体和凝血级联途径是否为特定致病途径。
招募诊断为不稳定型心绞痛且伴有肾虚血瘀证的患者、非肾虚血瘀证患者及正常组。进一步分析每组的临床症状。使用Illumina的NextSeq 2000测序平台和FastQC软件进行RNA测序及质量控制。使用DESeq软件进行差异基因表达（DGE）分析。对DGE分析结果进行qPCR和ELISA验证。
筛选出77个miRNA和331个mRNA的DGE图谱。GO富集分析包括43个生物学过程、49个细胞成分和42个分子功能。KEGG富集结果包括40条KEGG通路。PCR结果显示，与正常组相比，冠心病肾虚血瘀组中miR-4685-3p水平降低（P = 0.001），且低于冠心病非肾虚血瘀组。miR-4685-3p调控的下游mRNA C1在冠心病肾虚血瘀组呈上升趋势，高于正常组（P = 0.0019）。冠心病肾虚血瘀组中的mRNA C4和C5呈上升趋势，但差异无统计学意义。使用ELISA检测与补体和凝血级联途径相关的蛋白质。发现与正常组相比，冠心病肾虚血瘀组中C1的表达水平显著上调（P < 0.0001），高于冠心病非肾虚血瘀组（P < 0.0001）。冠心病肾虚血瘀组中C4和C5的表达水平显著低于正常组，且低于冠心病非肾虚血瘀组（P < 0.0001）。
冠心病肾虚血瘀证的发生可能与补体和凝血级联途径的激活有关，这通过观察到的miR-4685-3p降低及其下游C1QC上调得以证实。此外，发现补体C4和C5的表达水平降低，为该病的防治提供了研究依据。
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