Advances in Research on the circRNA-miRNA-mRNA Network in Coronary Heart Disease Treated with Traditional Chinese Medicine

There has been an increase in morbidity and mortality related to coronary heart disease (CHD) in China in recent years. Numerous clinical experiences and studies have shown that traditional Chinese medicine (TCM) plays an important role in the prevention, treatment, and prognosis of CHD. However, the mechanism of TCM in the treatment of CHD has not yet been elucidated. The circRNA-miRNA-mRNA network consists of miRNA that is competitively bound by circRNA, and miRNA regulates the transcription level of mRNA. Through literature review, we found that the circRNA-miRNA-mRNA network acts to contribute to certain effects to CHD such as myocardial hypertrophy, myocardial fibrosis, and heart failure. TCM contains constituents that act against CHD by antiatherosclerosis and apoptosis inhibition action, cardiac and cardiomyocyte protection, and these components also promote cell growth and protection of the vascular system by regulating miRNAs. Therefore, we consider that the circRNA-miRNA-mRNA network may be a new regulatory mechanism for the effective treatment of CHD by TCM.


Introduction
e morbidity and mortality caused by coronary heart disease (CHD) are steadily increasing in China. According to a 2018 report on cardiovascular disease in China, in 2013, 11,396,104 people died from CHD, with CHD mortality in 2016 of 113.46/100 thousand for urban residents and 118.78/ 100 thousand for rural residents. erefore, it is urgent to find effective methods for the prevention and treatment of CHD events [1]. e etiology of CHD is cavity vascular stenosis, coronary insufficiency, acute or transient ischemia, and hypoxia of myocardial cells caused by coronary atherosclerosis (AS). CHD belongs to the XiongBi or heartache category in traditional Chinese medicine (TCM). With a history of thousands of years, TCM is unique in theory and rich in clinical practice. Syndrome differentiation and treatment and holistic concept are two basic characteristics of TCM. A syndrome reflects the pathological essence of a certain stage in the development of disease. Phlegm and blood syndrome (PBSS) is one of the common syndrome types in CHD, and its formation and development are closely related to blood stasis, obesity, hyperlipidemia, hyperglycemia, body deficiency, old age, and environment. For PBSS, a satisfactory clinical outcome is achieved by promoting blood circulation and dispersing phlegm.
ere are three types of Chinese medicine used for the prevention and treatment of CHD, namely, a single Chinese medicine (e.g., redroot salvia, Rhodiola, and pseudoginseng), effective ingredients (e.g., tanshinone IIA, salvianolic acid, and ligustrazine), and prescriptions (e.g., Xuefu-Zhuyu decoction, Chaihu-Shugan powder, Gualou-Xiebai-Banxia decoction, and Ditan decoction). However, the mechanisms of these Chinese herbal medicines (CHMs) have not been fully elucidated. Circular RNAs (circRNA) and micro-RNAs (miRNA) are noncoding RNAs and have been confirmed to contribute to the pathogenesis and progression of CHD [2,3]. CircRNA and miRNA interact with each other and regulate the mRNA expression of critical genes in CHD genesis and progression. erefore, we speculated that there is a connection between the circRNA-miRNA-mRNA network and TCM and gathered supporting data through reviewing and summarizing the literature.

The Therapeutic Effect of TCM in CHD
TCM aims to treat CHD based on holistic regulation and syndrome differentiation and treatment. According to the model theory of gene constitution syndrome pattern, the occurrence and development of TCM syndromes are mainly based on the genomics of gene expression, which is a powerful approach to gain insight into the prevention and treatment of diseases [4].
In TCM, CHD is also described as XiongBi, which belongs to the category of chest pain, thoracic obstruction, heartache, or angina pectoris as a consequence of cold invasion, improper diet, mood disorder, fatigue, bodily internal injury, or body deficiency due to old age. CHD entails mainly obstruction of the heart channel with injury to other organs including the lungs, liver, spleen, and kidneys [5]. e syndromes of CHD in Chinese clinical medicine manifest as intermingled deficiency in origin (mostly Qi, blood, but also Qi-Yin and Yang) and excess in superficiality (mainly blood stasis and phlegm turbidity, but also cold coagulation and Qi stagnation) [6]. Qi in the TCM is a kind of substance with strong vitality and constant movement and extremely subtle, which is the unity of life substance and physiological function. Yin and Yang represent two opposite aspects of every object and their implicit conflict and interdependence. Generally, anything that is moving, ascending, bright, progressing, and hyperactive, including functional disease of the body, pertains to yang. e characteristics of stillness, descending, darkness, degeneration, and hypoactivity, including organic disease, pertain to Yin.
Traditional Chinese syndromes in XiongBi involve (a) heart-blood stasis syndrome (Xin Xue Yu Zu), (b) cold coagulation in heart-vessel syndrome (Han Ning Xin Mai), (c) Qi stagnation blood stasis syndrome (Qi Zhi Xin Xiong), (d) phlegm stasis in heart-vessel syndrome (Tan Zhuo Bi Zu), (e) deficiency of both Qi and Yin syndrome (Qi Yin Liang Xu), (f ) Yin deficiency of heart and kidney syndrome (Xin Shen Yin Xu), and (g) Yang deficiency of heart and kidney syndrome (Xin Shen Yang Xu).
ese syndrome types are shown in Table 1 [6].
Based on the principle of holistic regulation and syndrome differentiation and treatment, CHMs have been widely used in disease prevention and treatment in China and have been proven to have great clinical effectiveness on cardiovascular system disease. A previous study that summarized and introduced the application of plant products in CHD, AS, dyslipidemia, and hypertension also sorted formulae and herbs according to specific cardiovascular diseases [7]. e plant products applied in AS and dyslipidemia Over the last decades, some TCM decoctions have been confirmed to be effective in the treatment of CHD. For instance, combination therapy with Xuefu Zhuyu decoction and traditional antianginal medications (TAMs) was more effective in treating angina pectoris than TAMs alone. Evidence has indicated that Xuefu Zhuyu decoction combined with TAMs was more effective in improving RAS (RR � 1.29; 95% CI � (1.20, 1.38)), ECG (RR � 1.37; 95% CI � (1.22, 1.54)), and blood HDL-C level (MD � 0.29 mmol/L; 95% CI � (0.23, 0.35)) compared with TAMs alone from a meta-analysis and systematic review of 14 randomized controlled trials [11]. ere was benefit in the use of Tongxinluo (TXL) capsule for secondary prevention after acute myocardial infarction (AMI). A systematic review and meta-analysis of randomized controlled trials indicated that TXL improves cardiac function; regulates blood lipid total cholesterol (TC), triglycerides (TG), and low-density lipoprotein-C (LDL-C); and decreases the level of hs-C-reactive protein (hs-CRP) [12]. A meta-analysis indicated that Huoxue Huayu therapy (HXHY) is an effective and safe therapy for patients after percutaneous coronary intervention (PCI). HXHY had a greater beneficial effect on reducing the instent restenosis (ISR) rate and the degree of restenosis, improving Seattle Angina Questionnaire (SAQ) scores and increasing the revascularization rate compared with placebo. However, the rate of death and myocardial infarction (MI) of patients treated with HXHY was no different from that of those treated with placebo (P > 0.05) [13].
According to new studies, the pharmacological mechanisms of CHM are relevant to the effects on vascular smooth muscle cells (VSMCs), endothelial cells, cardiomyocytes, macrophages, and monocytes. Details are shown in Table 2 [7].

Regulation of miRNAs in CHD
With the improvement in modern living standards, phlegm and blood stasis commonly occur and then aggravate each other during CHD progression [14,15]. Phlegm is a pathological substance caused by the disturbance of body fluid. Blood stasis is a form of pathology caused by the disturbance of blood circulation. Research on CHD with PBSS has mainly focused on lipid metabolism, inflammatory factors, hemorheology, coagulation function, endothelial cell injury, endoplasmic reticulum autophagy, insulin resistance, genomics, proteomics, and metabolomics [16].

CircRNA-miRNA-mRNA in CHD
CircRNA is an endogenous noncoding RNA. It has no 5′end cap or 3′-end poly (A) tail as a covalently closed loop from precursor mRNA back-splicing by the spliceosome machinery. Some circRNAs have been shown to act as miRNA sponges, to interact with RNA-binding proteins, to regulate transcription, or to be translated into proteins. CircRNAs have been widely explored in antiaging strategies, diabetes, cardiovascular diseases, and malignant tumors [44][45][46][47][48][49][50]. In various cardiovascular tissues and organs, circRNA participates in physiological and pathological processes such as myocardial repair regulation and fibrosis of vascular smooth muscle cells and myocardium, without e circRNA-miRNA network may regulate mRNA participating in the development of CHD through competitive endogenous RNA (ceRNA). A new regulation model of gene expression and a complex network system has been developed that involves ceRNA, in which coding RNA and noncoding RNA interact with each other. is regulatory network is constructed to affect the mutual regulation, restriction, and interaction among lncRNA, circRNA, miRNA, and mRNA, thus affecting the target genes participating in CHD [51]. e circRNAs that have been reported to be differentially expressed in Qi stagnation and blood stasis syndrome include circRNA 0 9849, circRNA 1 1523, circRNA 1 8046, and circRNA 2 4450, with circRNA 11523 being related to hsa-circ-0005860 in circBase [52].
rough the KEGG pathway enrichment analysis of differential circRNA in Qi stagnation and blood stasis syndrome, five enrichment pathways were found, namely, path:hsa05144 (malaria), path:hsa04914 (progesterone-mediated oocyte maturation), path:hsa04650 (natural killer cell-mediated cytotoxicity), path:hsa04110 (cell cycle), and path:hsa05203 (viral carcinogenesis). ese circRNAs also participate in the regulation of natural killer cell-mediated cytotoxicity and cell cycle. Previous studies showed that a total of 1670 circRNAs and 13 miRNAs were differentially expressed in AMI, and 110 circRNAs and 11 miRNAs were differentially expressed in CHD. Gene ontology (GO) and pathway analyses for differentially expressed circRNAs showed that many pathways, diseases, and functions participate in the development of AMI [53]. One study also showed that there were nine circRNAs, namely, hsa_circ_0089378, hsa_circ_0083357, hsa_circ_0082824, hsa_circ_0068942, hsa_circ_0057576, hsa_circ_0054537, hsa_circ_0051172, hsa_circ_0032970, and hsa_circ_0006323, that could negatively regulate miR-130A-3p, resulting in the upregulation of transient receptor potential cation channel subfamily M member 3 (TRPM3) in patients with CHD, with nine circRNAs promoting TRPM3 expression by inhibiting hsa-miR-130a-3p in CHD patients [54]. e effects of the circRNA-microNA-mRNA network on CHD include myocardial hypertrophy, myocardial fibrosis, and heart failure, which are shown in Table 5.
CircRNAs with a covalently closed continuous loop are an abundant class of endogenous RNAs that are formed during the maturation of precursor mRNA, and they have been widely researched with respect to cardiovascular diseases. When CircANRIL binds to pescadillo homologue 1 (PES1), it impairs exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages and induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation for conferring atheroprotection [55]. Cdr1as also functioned as a powerful miR-7a sponge in  Evidence-Based Complementary and Alternative Medicine myocardial cells, involving the function of miR-7a targets PARP and SP1 in MI injury [56]. CircRNA HRCR can sequester and inhibit miR-223 activity and was then used in the treatment of cardiac hypertrophy and heart failure through increasing the ARC expression [57]. MFACR regulates mitochondrial dynamics and apoptosis through miR-652-3p and MTP18 [58]. CircRNA_010567 promotes myocardial fibrosis via suppressing miR-141 by targeting TGF-β1 [25]. CircNCX1 promotes cardiomyocyte apoptosis via the miR-133a-3p/CDIP1 [59]. Circ-SATB2 can regulate vascular smooth muscle cell phenotypic differentiation, proliferation, apoptosis, and migration by promoting the expression of STIM1 (a target gene of miR-939) [60]. CircCHFR facilitates the proliferation and migration of vascular smooth muscle via the miR-370/FOXO1/cyclin D1 pathway [61]. CircSlc8a1 can function as an endogenous sponge for miR-133a in cardiomyocytes, and it can regulate the serum response factor, connective tissue growth factor, adrenoceptor beta 1, and adenylate cyclase 6 through directly intervening the knockdown and overexpression in heart failure [62]. CircNfix can suppress Ybx1 ubiquitindependent degradation and increase miR-214 activity. It also can promote cardiac regenerative repair and functional recovery after MI [63]. Overexpression of circHIPK3 reverses miR-29b-3p-induced inhibition of cardiac fibrosis proliferation and migration, and then alters the expression levels of miR-29b-3p-targeting genes (a-SMA, COL1A1, COL3A1) in vitro [64].

Potential circRNA-miRNA-mRNA Network in CHD Treated with TCM
TCM may play a positive role in the treatment of cardiovascular diseases through the circRNA/miRNA/mRNA regulatory network (Figure 1). In myocardial ischemiareperfusion injury, increasing amounts of circNCX1 competitively bind to the miRNA-133a family, decreasing miRNA-133a-3p levels and increasing the activity of CDIP1, leading to cardiomyocyte deterioration [59]. Tongxinluo can increase the expression of the miRNA-133a family, weaken myocardial fibrosis, and reduce the damage caused by myocardial ischemia [65]. erefore, Tongxinluo may play a regulatory role through the network of circNCX1/miRNA-133a/mRNA. Modulation of circHRCR/miR-223/ARC levels provides an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure [57]. TCM that tonifies Qi and activates blood (Shenmai injection, Shenqi Fuzheng injection, Danhong injection, and Qishen Yiqi pills) can significantly reduce the expression of miR-223-3p in the plasma of patients with AMI. Studies have also found that activating blood and dissolving phlegm can significantly reduce the expression level of miR-223 in the serum of hyperlipidemia rats in the myocardial ischemiareperfusion model and play an anti-inflammatory role through the miR-223/PPRs pathway and inhibit apoptosis [66]. Ligustrazine, which is tetramethylpyrazine (TMP), can inhibit the proliferation, migration, apoptosis, and vascular remodeling of smooth muscle cells by downregulating the gene expression of miR-223 and then inhibiting the expression of CaMKIIδ [67]. Ginsenoside Rg1 upregulates the expression of eNOS by targeting miR-214, which is beneficial for promoting angiogenesis [68]. Electroacupuncture preconditioning can upregulate the expression of miR-214 and inhibit the increase in Ca 2+ , sodium/calcium exchanger 1 (NCX1), BCL2-like 11 (BIM), calmodulin-dependent protein kinase IIδ (CaMKIIδ), cyclophilin D (CypD), and other related proteins that have accumulated in hypoxia injury in H9c2 myocardial cells, and play a protective role in ischemia/reperfusion (I/R) [69]. e Fuzheng Huayu capsule has the effect of alleviating myocardial fibrosis after MI in rats. is mechanism may be related to upregulation of the expression levels in the miRNA-29 family and direct or indirect regulation of the ratio of matrix metalloproteinase 2/ tissue inhibitor of metalloproteinases 2 (MMP2/TIMP2) and MMP9/TIMP1, improving the metabolic balance in the extracellular matrix [70].
Single herb medicine, effective components, formulae, and Chinese patent medicines are widely used in the prevention and treatment of CHD. e functions of TCM in clinical treatment mainly include tonifying and regulating Qi, activating blood circulation, reducing phlegm, and relieving pain and consumptive disease. e treatment principles of PBSS in CHD are activating blood circulation and reducing phlegm, which are potential regulators of lipid metabolism and antiplatelet aggregation. However, due to the complex chemical components in CHM, its efficacy is actually a combination of various factors. erefore, the biggest challenge for the modernization of TCM is to reduce the ambiguity of effective substances and mechanisms. e circRNA-miRNA-mRNA network may be a new regulatory mechanism. CircRNAs and miRNAs interact with each other to regulate the mRNA expression of some critical genes in CHD genesis and progression. Previous studies showed that TCM contains active components that protect cardiomyocytes, act against atherosclerosis, inhibit apoptosis, promote cell growth, and protect the cardiovascular system by regulating miRNAs. Because there is a very tight connection among circRNAs, miRNAs, and mRNAs, it is likely that TCM may play a positive role in the treatment of CHD through the circRNA-miRNA-mRNA network. In conclusion, the circRNA-miRNA-mRNA network may be an important mechanism that can be affected by Chinese medicine, resulting in the amelioration of CHD. TCM can also be used to affect the circRNA-miRNA-mRNA network to assist with the diagnosis of CHD and the evaluation of the clinical effect of Chinese medicine on CHD.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Fei Lin and Heng-Wen Chen contributed equally to this work.
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