Differential expression of miRNA in THP-1-derived foam cell model induced by drug-containing serum of Yimaijiangzhi decoction

LAI Chun-bing, GU Zhan-xin, LIU Rui

1.Guangxi University of Traditional Chinese Medicine,Nanning 530000,China

2.Rui Kang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine, Nanning 530000, China

Keywords:

ABSTRACT Objective: To investigate the differential expression of miRNA and related biological functions and signaling pathways after the intervention of the THP-1-derived foam cell model with the drug-containing serum of Yimaijiangzhi Decoction.Methods: The experiment was divided into macrophage group, foam cell model group and Yimaijiangzhi drug-containing serum group.THP-1 cells were induced into macrophages by Fopol ester, and induced differentiated macrophages were given ox-LDL to establish foam cell model, and Yimaijiangzhi decoction rat serum was used to intervene the foam cells.Total RNA was extracted from cells in each group for miRNA sequencing, differential expression of miRNA was screened, and relevant target genes were predicted for GO analysis and KEGG analysis, protein interaction network and miRNA-target gene interaction network were established, and RT-qPCR was used to verify the possible signaling pathways for improving atherosclerosis.Result: The difference miRNA between blank group and model group was hsa-miR-302c-3p, hsa-miR-302d-3p, hsamir-30d-3p, hsa-mir-3189-3p, hsa-mir-374b-5p, hsa-mir-423-5p, hsa-mir-423-5p, and hsamir-4781-3p, hsa-mir-663a; The miRNAs of model group and Yimaijiangzhi drug-containing serum group were hsa-mir-3150a-3p, hsa-mir-7704, hsa-mir-887-3p, hsa-mir-150-5p, hsamir-423-5p, hsa-mir-374c-3p, hsa-mir-374c-3p, hsa-mir-374b-5p; The difference of miRNAs prediction target genes between model group and Yimaijiangzhi drug-containing serum group showed that the miRNA prediction target genes were mainly enriched in MAPK signaling pathway, ErbB signaling pathway, Hippo signaling pathway, Wnt signaling pathway and other signaling pathways.SCN1A, PRKACA, MECP2, EIF4E, SRSF1, MBNL1, PRKCA,PPARGC1A may be the potential key targets for the effect of the drug-containing serum of Yimaijiangzhi Decoction on THP-1-derived foam cells.Conclusion: hsa-mir-374c-3p, hsamir-423-5p, and hsa-mir-374b-5p are important miRNAs that the drug-containing serum of Yimaijiangzhi Decoction acts on foam cells.The significantly differentially expressed mirnas and significantly enriched related signaling pathways may provide new ideas for the diagnosis and treatment of atherosclerosis.

1.Introduction

Atherosclerosis (AS) is a fibro-fatty lesion that forms on the walls of large and medium-sized arteries and is an important risk factor for coronary artery disease, myocardial infarction, peripheral vascular disease, and stroke[1].Various factors can damage the vascular endothelium, leading to the expression of adhesion molecules that allow monocytes to enter the intima through chemotaxis.This process results in the production of oxidized lowdensity lipoprotein (LDL) and the recruitment of macrophages,which engulf oxidized LDL particles and transform into foam cells.The contents released by dead foam cells gradually accumulate and lead to localized lipid deposition, which progresses into plaques and calcifications[2].It is evident that the foam cell formation of macrophages is a key process in the development of atherosclerosis.miRNA, an endogenous non-coding RNA, has been found to delay the progression of atherosclerosis and can also serve as a biomarker for atherosclerosis[3-4].

Traditional Chinese medicine does not have a specific term for atherosclerosis.Based on its clinical manifestations, it can be classified into the categories of “chest obstruction,” “pulse obstruction,” “true heart pain,”“phlegm turbidity,” and “adipose”[5].From the perspective of modern biology, the primary pathological factor of atherosclerosis is “phlegm-dampness.” The spleen governs the transformation of water and grains.If the spleen’s transforming function is impaired, it leads to the formation of phlegm due to water retention.Phlegm obstruction causes stagnation of qi, which further leads to blood stasis and the intersection of phlegm and blood,resulting in arterial blockage[6].Therefore, the appropriate treatment should focus on invigorating the spleen, resolving phlegm, activating blood circulation, and nourishing the liver and kidneys[7].The Yimaijiangzhi Decoction is a prescription formulated by the Geriatric Department of Rui Kang Hospital affiliated with Guangxi University of Chinese Medicine.The decoction has effects of invigorating the spleen, resolving dampness, activating blood circulation, and remove blood stasis.Clinical experiments have shown that the Yimaikangzhi Decoction can effectively reduce the levels of total cholesterol and low-density lipoprotein cholesterol in patients with hyperlipidemia[8].

This study aimed to establish a foam cell model using human myeloid leukemia mononuclear cells (THP-1) and investigate the effects of Yimaijiangzhi Decoction-containing serum on the expression of miRNAs in THP-1-derived foam cells.The study also conducted bioinformatics analysis and preliminary validation of key miRNA target genes.

2.Materials and methods

2.1 General materials

2.1.1 Experimental animals

Thirty SD rats, SPF grade, female, weighing 180 g-200 g, were purchased from Hunan Slake Jingda Experimental Animal Co.Ltd.[SCXK (Hunan)2018-0002].The rats were raised in the SPF animal feeding center of the Guangxi University of Chinese Medicine Science Experimental Center.The temperature was 20-25 ℃, the relative humidity was 50%-65%.

2.1.2 Experimental cells

Human monocytic leukemia cell line THP-1, derived from ATCC cell bank, was purchased from Guangxi Zhuoyi Biotechnology Co.Ltd.

2.1.3 Experimental drugs

The experimental drug is Yimaijiangzhi decoction, the composition is as follows: Huangjing 20 g, Poria 10 g, Hawthorn 20 g,atractylodes macrocephala 10 g, Salvia miltiorrhiza 10 g, alisma orientalis 10 g, Gynostemma Pentaphyllum 10 g, Polygonum multiflorum 10 g, astragalus membranaceus 10 g, Qi Zi 10 g,Chrysanthemum 10 g, Cassia obtusifolia 10 g, provided by Guangxi University of Chinese Medicine Hospital.

2.1.4 Preparation of Yimai Jiangzhi decoction

The preparation of Yimaijiangzhi decoction and its concentration were carried out by reference[9].The Yimaijiangzhi decoction was soaked in ultra-pure water for 30 min, then boiled twice with ultrapure water.After filtering, the medicine was mixed and boiled twice.The medicine was concentrated to 2.84 g/mL containing crude drugs in a cyclone and stored in a 4 ℃ refrigerator.

2.1.5 Main laboratory reagents and instruments

RPMI 1640 medium (Gibco) , Australian imported fetal bovine serum (GIBCO) , oxidized Low-density lipoprotein (OX-LDL)(Beijing Solarbio) , RNA analysis kit (American Aati ) , Tatal RNA extraction kit (Shanghai Progmega) , Biosafety cabinet (Qingdao Haier) , real-time fluorescent quantitative PCR (ABI, QuantStudio 6), cell culture box (Japanese Sanyo Motor) , BGISEQ-500 sequencing platform (Shenzhen Bgi) , RT-qPCR Kit (Vazyme)

2.2 Methods

2.2.1 Prepare drug-containing serum of Yimaijiangzhi decoction

Thirty SD rats were randomly divided into blank group and Yimaijiangzhi decoction group with 15 rats in each group.Rats were given Yimaijiangzhi decoction once a day for 7 d.After 1 hour of the last administration, the rats in each group were anesthetized,and the blank rat serum and the yimai jiangzhi-containing serum were extracted from the abdominal aorta.The whole blood was left at room temperature for 3 h, centrifuged at 3000 rpm for 10 min,inactivated serum, filtered and sterilized, and then frozen in -80 ℃refrigerator.

2.2.2 Establishment and identification of foam cell model

The foam cell model was established by reference[9].Thp-1 cells were incubated with 320 mmol/L phorbol ester in a thermostatic cell incubator in complete medium.After 24 h, it was identified that THP-1 cells could differentiate into phagocytic macrophages.The foam cell model was established by adding 80mg/l ox-LDL to complete medium for 48 h.PBS was washed, fixed with 4%paraformaldehyde, differentiated with 60% isopropanol, and stained with oil red o.The cells were removed after washing and added glycerol gelatin.The foam cell model was observed under the microscope.

2.2.3 Experimental grouping and treatment

The experiment was divided into macrophage group, foam cell model group, Yimai lipid-lowering drug-containing serum group.The macrophage group and the foam cell model group were added to the complete culture medium containing 10% of the blank rat serum,and the foam cells were added to the complete culture medium containing 10% of the rat serum of Yimai Jiangzhi decoction,namely the Yimaijiangzhi containing drug serum group, the cells of each group were treated in 37 ℃ and 5% CO2incubator for 48 h.

2.2.4 Total RNA was extracted

The cells of each group were seeded in 6-well plates at a concentration of 1× 106/mL.After the intervention, RNA lysate was added, transferred to EP tube, diluted solution was added and centrifuged, and the supernatant was obtained, add in anhydrous ethanol, mix and centrifuge.The centrifuge column was added to the RNA wash, and the filtrate was discarded after further centrifugation.The centrifuge column was added to the dnase I incubation solution.Transfer centrifuge column to elution tube, add non-ribozyme water,static centrifugation, the extracted liquid is Total RNA.

2.2.5 miRNA sequencing

The integrity and concentration of Total RNA were detected by Standard Sensitivity RNA Analysis Kit (DNF-471) , and the library was constructed by Agilent bioanalyzer 2 100, small RNA was sequenced by bgiseq -500 sequencing platform of BGI, and the results were filtered and compared by AASRA software.

2.2.6 Bioinformatics analysis of miRNA

The target genes were predicted by software such as RNAhybrid,Miranda and Targetscan.The predicted target genes were analyzed by cluster analysis, GO analysis and KEGG analysis, to construct PPI protein network interaction map and miRNA-target gene regulatory network.

2.2.7 RT-qPCR Verification improves arteriosclerosis possible signaling pathways

The cells in each group were lysed with Trizol, centrifuged with chloroform, the supernatant was added with isopropanol and centrifuged with absolute ethanol, trace photometer to determine the purity and concentration of RNA.According to the requirements of the kit, the reagent was added for reverse transcription and qPCR detection.The internal parameter was β-actin.The target gene primers were designed and synthesized by Enko Biotechnology Co.Ltd.

Tab 1 Primer sequences for real-time fluorescence quantitative PCR

2.2.8 Statistics and analysis

The results of real-time quantitative PCR were Quantitative analysis by 2-△△t.GraphPad Prism 9.0(GraphPad software, USA) was used for statistical analysis and graph generation, and P ＜ 0.05 considered the difference to be significant.

3.Results

3.1 Establishment of foam cell model

After 24 h of induction by Phorbol Ester, the THP-1 cells were adherent to the wall, and the shape of the cells was spindle, round and irregular, which showed the extension of pseudopodia.The THP-1-derived macrophages were incubated with 80 mg/L ox-LDL.The cells showed red and orange lipid droplets after oil red O staining.However, the cells without ox-LDL intervention had few or no red and orange lipid droplets, which confirmed the successful establishment of foam cell model, as shown in Figure 1.

Fig 1 lipid accumulation of Non-ox-LDL Group and ox-LDL group after oil red O staining(20×)

3.2 Screening mirnas and clustering analysis

miRNA sequencing results suggested that 55 miRNAs were differentially expressed in the blank group and the model group,with 28 miRNAs up-regulated and 67 mirnas down-regulated.After intervention by Yimai lipid-lowering serum, 55 miRNAs were differentially expressed in Yimai lipid-lowering serum group and model group,39 miRNAs were up-regulated and 16 miRNAs were down-regulated.To exclude false-positive differences and further narrow the range of screening, the screening condition was upregulated to log2 2, qvalue＜ 0.01, with expression levels ＞ 1 in either or both groups, eight miRNAs with distinct expression were screened out in the Blank Group and model group: hsa-mir-302c-3p,hsa-mir-302d-3p, hsa-mir-30d-3p, hsa-mir-3189-3p, hsa-mir-374b-5p, hsa-mir-423-5p, hsa-mir-4781-3p, hsa-mir-663a.The model group and Yimaijiangzhi-containing serum group screened out seven mirnas with significant difference: hsa-mir-3150a-3p, hsa-mir-7704,hsa-mir-887-3p, hsa-mir-150-5p, hsa-mir-423-5p, hsa-mir-374c-3p,hsa-mir-374b-5p.The mirnas with the highest expression level were hsa-mir-374c-3p and hsa-mir-423-5p and hsa-mir-374b-5p.

3.3 differential miRNA target gene bioinformatics analysis between blank group and model group

3.3.1 GO enrichment Analysis

Eight differentially expressed mirnas (hsa-miR-302c-3p, hsa-miR-302d-3p, hsa-mir-30d-3p, hsa-mir-3189-3p, hsa-mir-374b-5p, hsamir-423-5p, hsa-mir-4781-3p, hsa-mir-663a) were used for target gene prediction, GO enrichment analysis was performed on the cross-linked target genes, the results showed that the target genes were mainly involved in the positive regulation of RNA polymerase II promoter transcription, the negative regulation of RNA polymerase II promoter transcription, aging, and the negative regulation of ERK1 and ERK2 Cascades The transcription factor activity, sequencespecific DNA binding and RNA polymerase II transcription factor activity were displayed It is involved in chromatin, transcription factor complex, nuclear plasma, actin cytoskeleton and Cul4-RING E3 ubiquitin ligase complex.

Tab 2 miRNA expression in different groups

Fig 2 heat map of differential gene cluster

Tab 3 The difference of miRNA expression between model group and Yimai lipid-lowering group

3.3.2 KEGG Pathway analysis

KEGG enrichment analysis of miRNA target genes between the blank group and the model group was carried out, and the miRNA target gene regulatory pathway with significantly different expression was obtained.KEGG results showed that predicted miRNA target genes collectively participated in the enrichment of 18 signaling pathways, there were significant differences in Neurotrophin signaling pathway, Relaxin signaling pathway, MAPK signaling pathway, c-type lectin signaling pathway and estrogen signaling pathway.

Fig 3 GO enrichment analysis of target genes predicted in control group VS model group

Tab 4 Analysis of intersection gene signaling pathway

3.4 Analysis of miRNA target gene bioinformatics between model group and Yimaijiangzhi-containing serum group

3.4.1 GO enrichment analysis

The main point of this study is to discuss the effect of Yimai lipidlowering serum on the miRNA of THP-1 derived foam cells.Seven mirnas (hsa-mir-3150a-3p, hsa-mir-7704, hsa-mir-887-3p, hsa-mir-150-5p, hsa-mir-423-5p, hsa-mir-374c-3p, hsa-mir-374b-5p) with the most significant differential expression were predicted as target genes.Go analysis showed that these target genes were involved in the regulation of RNA polymerase II promoter transcription,chondrocyte differentiation, cholinergic synaptic transmission, lipid metabolism and Glutathione metabolism It showed the molecular functions of heparin binding, ornithine cyclase activity and Glutathione transferase activity, and participated in cell components such as postsynaptic membrane, medium density lipoprotein granule and Low-density lipoprotein granule.

3.4.2 Differential miRNA target gene KEGG analysis

KEGG enrichment analysis of miRNA target genes between model group and Yimai lipid-lowering drug-containing serum group showed that 22 signaling pathways were enriched by predicted miRNA target genes, MAPK signaling pathway, ErbB signaling pathway, Hippo signaling pathway, Wnt signaling pathway, Ras signaling pathway were significantly different.

Tab 5 Analysis of intersection gene signaling pathway

3.4.3 Construct the network of predicted target gene protein interactions

The predicted target genes were imported into the String database(https://cn.String-db.org/) to construct protein-protein interaction networks.The results were analyzed by Cytoscape software according to degree value, mediator, and shortest path, indicates that the degree value is greater.Among them, SCN1A, PRKACA,MECP2, EIF4E, SRSF1, MBNL1, PRKCA, PPARGC1A have the darkest color, which indicates that the degree value of the target gene is in the front rank, suggesting that the target gene may be the core potential target of Yimaijiangzhi decoction-containing serum.

3.4.4 Construct miRNA-target gene regulatory network

Cytoscape was used to construct a miRNA-target gene regulatory network for the seven target genes predicted by different mirnas.The mirnas with the largest number of target genes were hsa-mir-3150a-3p, hsa-mir-887-3p, hsa-mir-423-5p, the significantly regulated genes were SP1, AGAP1, Bach2, BTBD9, GRIN2B, LARP1,MDM4 and so on.See Figure 6.

Fig 4 GO enrichment analysis of different miRNA prediction target genes in model group VS Yimaijiangzhi drug-containing serum group

Fig 5 network diagram of predicted target gene protein interaction

Fig 6 miRNA-target genes control network diagram

3.5 Follow-up verification of Yimaijiangzhi decoctioncontaining serum on differential expression of miRNA target genes in THP-1 derived foam cells

In order to verify the reliability and accuracy of target gene prediction, Wnt signaling pathway and upstream and downstream genes of MAPK signaling pathway were selected to validate.On the WNT signal pathway, the expression level of Wnt mRNA in the model group was significantly higher than that in the blank group (P＜ 0.01) , and the expression level of Wnt mRNA was down-regulated in the Yimaijiangzhi-containing serum group compared with that in the model group, the difference was statistically significant (P ＜ 0.05).Compared with the blank group,the expression level of β-catenin mRNA in the model group was significantly higher (P ＜ 0.01) , and the expression level of β-catenin mRNA in the Yimai lipid-lowering drug-containing serum group was significantly lower than that in the model group(P ＜ 0.01) , the difference was statistically significant (P ＜ 0.01) , as shown in Figure 7.

In MAPK signal pathway, the expression level of MKK4 mRNA in the model group was significantly higher than that in the blank group (P ＜ 0.05) , and the expression level of MKK4 mRNA in the Yimaijiangzhi-containing serum group was significantly lower than that in the model group (P＜0.05) , the difference was statistically significant (P ＜ 0.05).Compared with the blank group, the expression level of ELK1 mRNA in the model group was significantly higher (P ＜ 0.01) , and the expression level of ELK1 mRNA in the YIMAI jiangzhi-containing serum group was significantly lower than that in the model group (P ＜ 0.01) , the difference was statistically significant (P ＜ 0.01) , as shown in Figure 7.

Fig 7 Real-time fluorescent quantitative PCR was used to verify genes related to MAPK signaling pathway and WNT signaling pathway

4.Discussion

The arteriosclerosis is due to dysfunction of the Viscera organs,which leads to blood dysfunction, blood stasis and toxin, and deficiency of the body.Yimaijiangzhi decoction in Atractylodes Macrocephala, Tuckahoe, Alisma, cassia, Astragalus spleen phlegm,qi and turbidity, Salvia miltiorrhiza, Alisma huoxue Huayu Tongmai;Wolfberry fruit, Polygonum multiflorum, polygonatum tonify the liver and kidney, gynostemma pentaphyllum, hawthorn coke for disease differentiation, pharmacological studies showed that it has the role of lipid[11,12].The combined use of all the medicines can play the role of invigorating the spleen, resolving phlegm and lowering turbidity, promoting blood circulation, removing blood stasis and dredging the arteries, nourishing the liver and kidney.Modern pharmacological studies have shown that danshen diol C of Danshen compounds is able to inhibit macrophage foaming by activating the Nrf2/SIRT1 signaling pathway[13]; Astragaloside, a astragalus compound, can delay the process of arteriosclerosis by inhibiting the formation of foam cells, while astragaloside can also reduce lipid arteriosclerosis by down-regulating lipid synthesis[14].More recently, it has been shown that gypenosides in Gynostemma pentaphyllum enhance SIRT1/FOXO1-mediated restoration of autophagic flux, effectively inhibiting ox-LDL uptake as well as foam cell formation[15].Therefore, many effective compounds of Yimaijiangzhi decoction have the potential to inhibit the foam formation of macrophages.

Previous studies confirmed that Yimaijiangzhi decoction contained serum can reduce the foam accumulation of macrophages[9].In this study, the foam cell model was taken as the research object, and the Yimaijiangzhi decoction contained serum interfered with the foam cell, to explore the regulatory mechanism of Yimaijiangzhi oral liquid on improving macrophage foaming based on miRNA level.The results showed that 55 miRNAs differentially expressed in the model group were compared with those in the Yimai lipid-lowering drug-containing serum group.When the absolute value of difference fold | log2(MODEL/DRUG) | 2, qvalue ＜ 0.01 and the expression quantity ＞ 1 in any one or two groups of two groups, seven differential mirnas were screened.The expression quantity of hsamir-423-5p, hsa-mir-374c-3p, hsa-mir-374b-5p were abundant, these results suggest that these three miRNAs may play an important role in the regulation of lipid metabolism of foam cells by Yimaijiangzhi decoction-containing serum.The most significantly upregulated miRNA was hsa-mir-374c-3p, which was found to be associated with autoimmune encephalomyelitis and pulmonary hypertension in the current literature[16.17].hsa-mir-423-5p and hsa-mir-374b-5p were the most down-regulated 16 mirnas.The study showed that mir-423-5p could be used as a biomarker to judge the prognosis of acute myocardial infarction and predict adverse cardiovascular events[18] , most studies of hsa-mir-374b-5p have been associated with cancer, sepsis, thrombosis, and cystic echinococcosis[19-22].Among these three mirnas, only mir-423-5p has been reported to regulate cardiovascular disease, hsa-mir-374b-5p, mir-374c-3p or other differentially expressed miRNAs have not been reported to be directly or potentially associated with arteriosclerosis, and their role in arteriosclerosis remains to be verified.

According to KEGG, the target genes predicted by differential miRNAs were enriched in MAPK, ErbB, Hippo, Wnt and Ras arteriosclerosis.In this study, we selected the WNT signal pathway and the key target genes of MAPK signal pathway for PCR verification.The mRNA expressions of Wnt, β-catenin, MKK4 and Elk1 in the model group were higher than those in the blank group, the mRNA expressions of Wnt, β-catenin, MKK4 and Elk1 were down-regulated compared with the model group, indicating that Yimai lipid-lowering serum may regulate foam cells through Wnt signal pathway and MAPK signal pathway, to improve the arteriosclerosis.

SCN1A, PRKACA, MECP2, EIF4E, SRSF1, MBNL1, PRKCA,PPARGC1A were found to be related to macrophage foaming.The miRNA-target gene regulatory network showed that the mirnas regulating the most target genes were hsa-mir-3150a-3p, hsa-mir-887-3p, hsa-mir-423-5p.The significantly regulated genes were SP1, AGAP1, BACH2, BTBD9, GRIN2B, LARP1 and MDM4.The molecular mechanism of Yimaijiangzhi decoction in modulating macrophage foaming in the previous study has not been fully clarified, and the miRNA regulation mechanism is complex, further studies are needed to determine the specific mechanisms involved in the regulation of THP-1-derived foam cells by differentially expressed miRNAs and to improve the arteriosclerosis of THP-1-derived foam cells.

In this study, we used miRNA sequencing to predict the target genes of yimajiangzhi decoction serum interfering with the differential expression of miRNA in THP-1-derived foam cell model,the protein interaction network and miRNA-target gene network were constructed by analyzing the differentially expressed miRNA target genes by GO and KEGG, the main prediction results were verified by RT-qPCR according to the correlation and importance,and the biological structure and molecular function of Yimaijiangzhi decoction-containing serum in foam cell model were preliminarily elucidated, to provide experimental basis and thinking for the treatment of arteriosclerosis by Yimaijiangzhi decoction.

Author’s contribution

Lai Chunbing was in charge of bioinformatics analysis, RT-qPCR and thesis writing, Gu Zhanxin was in charge of experimental operation and data processing, and Liu Rui was in charge of experimental design, experimental guidance and thesis revision.

All authors state that there is no conflict of interest.