The regulated profile of noncoding RNAs associated with inflammation by tanshinone IIA on atherosclerosis

Atherosclerosis (AS) is the principal cause of heart attack, sudden cardiac death, stroke, and necrosis of the extremities, in which significant changes in gene expression associated with inflammation are found. However, the molecular mechanisms of AS are not clearly elucidated. In this study, ApoE−/− mice were fed with a high fat diet for 12 weeks to induce atherosclerosis and half of the mice were treated with tanshinone IIA (TAN). Then sequencing analysis was performed to investigate the expression patterns of ncRNAs in AS plaques obtained from mice treated with TAN and AS Model mice. A total of 22 long noncoding RNAs (lncRNAs), 74 microRNAs (miRNAs), 13 circular RNAs (circRNAs), and 1359 mRNAs in AS plaque were more significantly regulated from TAN mice, compared with model mice. Bioinformatics tools and databases were employed to investigate the potential ncRNA functions and their interaction. Our data showed that the most significantly pathways regulated by TAN were associated with inflammation, and involved in the signaling pathways of Ras, Rap1, MAPK, cAMP, T cell receptor, and so on. In addition, the competitive endogenous RNA (ceRNA) network had been constructed and the core nodes included circ‐Tns3/let‐7d‐5p/Ctsl, circ‐Wdr91/miR‐378a‐5p/Msr1, and circ‐Cd84/ miR‐30c/ Tlr2. The DERNAs were validated by quantitative RT‐PCR and dual luminescence reporter assay in RAW264.7 cells in vitro. This study identified ceRNAs network regulated by TAN and elucidated the ncRNAs profile and signal pathways to attenuate AS comprehensively. This research would contribute to further research on the pathogenesis of AS, and facilitate the development of novel therapeutics targeting ncRNAs.

the major component extracted from the root of Salvia miltiorrhiza.
Research found TNA has several pharmacological functions in clinical, such as anti-bacteria, anti-inflammation, and obvious antitumor.
TAN has been used in the treatment of cardiovascular diseases in China for decades. It has been reported that TNA can act as a natural anti-oxidant to reduce myocardial oxygen consumption and antiatherosclerosis. However, the therapeutic molecular mechanisms of TNA are not fully elucidated.
Noncoding RNAs (ncRNAs) comprise a category of RNA molecules that typically cannot encode proteins but functionally regulate protein expression. 2 ncRNA genes include certain ample and functional RNAs, such as microRNAs, siRNAs, long ncRNAs, and so on. 3 ncRNAs are involved in many biological processes, and the mutations or imbalances of the repertoire could cause multifarious of diseases. Although ncR-NAs have been confirmed to play a role in AS pathogenesis, the exact effects of ncRNAs in AS still remain unknown. In this study, sequencing analysis was performed to investigate the expression patterns of ncR-NAs in AS from mice treated with TAN and the AS Model mice fed with a high fat diet with background of ApoE gene knockout, and then the comparison between them was made.
Our study is aimed to identify the dynamic network of inflammation, and optimally balance the levels of genes and protein activation regulated by TAN to attenuate atherosclerosis. We try to explore the mechanism of TAN on regulating the genes encoding and develop new diagnostic and therapeutic agents of AS.

H&E and oil red O staining of aorta
For H&E staining, paraffin sections were deparaffinized with alcohol, and dyed with hematoxylin for 10 min, then color separation with 10% hydrochloric acid in alcohol for 10 s, backed to blue by 0.5% ammo-nia, and dyed with eosin for 5 min, gradient dehydrated with alcohol.
Finally, the slides were observed under a microscope.  PRJNA604080) and will be released upon publication.

GO annotations and KEGG pathways analysis
GO analysis was applied to elucidate genetic regulatory networks by forming hierarchical categories according to the differentially expressed genes (http://www.geneontology.org). Pathway analysis was performed to explore the significant pathways of the differentially expressed genes in terms of KEGG (http://www.genome.jp/kegg/).

Construction of the ceRNA network
Competitive endogenous RNAs can bind to miRNAs through miRNA response elements (MREs), which affects the expression of genes regulated by miRNAs. 6 As known, lncRNAs/circRNAs can compete with each other to interact with miRNAs. 7 F, forward; R, reverse.
For measurement of luciferase activity, a procedure based on the protocol of Dual-Glo luciferase reporter assay (Promega, WI, USA) was used and detected by GloMax R 20/20 Luminometer (Promega) 48 h after co-transfection.

Statistical analysis
Statistical analyses in 2 groups were performed using a 2-tailed Student't-test. For multiple comparisons, P-value was determined by

RNA name Primer
2-way ANOVA followed by a Bonferroni posttest. All analyses were performed with GraphPad Prism 5 software. A value of P < 0.05 was considered to be statistically significant. Data are shown as mean ± SD.

The levels of plasma cholesterol and triglycerides
The plasma triglycerides (TG), total cholesterol (TC), HDL-C (cholesterol), VLDL-C, and LDL-C were measured in mice. Compared with

Histology of atherosclerotic lesions
Atherosclerosis assessment was done via intimal lesion areas of atherosclerotic lesions by H&E staining and lipid accumulation by Oil

The expression profiling changes of genes in proximal aortas
Volcano Plot indicates 605 up-regulated and 754 down-regulated genes (mRNAs) in proximal aortas from TAN group compared with Model group, as shown in Fig. 1C; heat map showed the hierarchical clustering of changed genes. In clustering analysis, up-and down-regulated genes are colored in red and blue, respectively ( Fig. 1D).

The expression profiling changes of miRNAs in proximal aortas from ApoE −/− mice and WT mice
Forty-three up-regulated miRNAs and 31 down-regulated miRNAs in proximal aortas from TAN group compared with Model group are shown in the volcano plot (Fig. 1E); In clustering analysis, up-and down-regulated genes are colored in red and green, respectively. Heat map of miRNAs showed the hierarchical clustering of changed miR-NAs of in proximal aortas of TAN group compared with Model group (Fig. 1F).  Fig. 1G and 1H, respectively.

GO analysis of miRNAs target genes
We used Gene Ontology database (http://www.geneontology.org) to perform GO analysis on the miRNA target genes. The 508 up-regulated and 449 down-regulated genes were analyzed individually. P-value and FDR was calculated by Fisher's exact test and multiple comparisons test respectively (P < 0.05). Around 197 differentially expressed genes were classified in the light of GO term, including biological process, molecular function, and cellular component ( Fig. 2A).

Construct of ceRNAs network
Since MREs are the mediators of lncRNA-miRNA interaction, we  With the significant difference of miRNAs between TAN mice and Model mice, we selected miR-146b-5p, miR-378c, miR-30-2-3p, miR-378a-5p, and let-7d-5p to validate the expression of the miRNAs.

circRNAs directly interacts with miRNAs in RAW264.7 cells by dual luminescence reporter assay
We found that circ-Cd84 (SLAMF5), circ-Wdr91, and circ-Tns3 were mainly located in the cytoplasm. The level of lnc-Pirb was low in both cytoplasm and nucleus of cells (Fig. 3D). Given that cytoplasmic cir-cRNA mainly functions by acting as an effective molecular sponge for miRNAs, we thus searched for miRNAs that might be adsorbed by these 3 circRNAs via using miRWalk online. Only 4 miRNAs (miR-378a-5p, miR-378c, let-7d-5p, and miR-30c) were predicted to harbor 3 cir-cRNAs binding site (Fig. 3E). The 2D construction of miRNAs and the predicted binding site with relative mRNAs are shown in Fig. 3F.

The expression of RNAs in RAW264.7 cells treated with TNA in vitro
To find if the mechanism was different between in vivo and in vitro, we

DISCUSSION
To date, the impact of ncRNAs in regulating immune response during atherosclerosis has not been evaluated in detail. Evidence has revealed that inflammation is the early trigger in the formation of atheroscle-  [15][16][17] In this study, we specifically focused on the miRNAs regulated by TAN controlling innate and inflammatory responses during F I G U R E 5 The expression of selected RNAs in RAW264.7 cells treated with TNA in vitro. RAW264.7 cells were treated with TNA or miRNAs inhibitors or circRNAs mimics in vitro and the quantitation of miRNAs (A-C) and mRNA (D-F) were detected by qRT-PCR. n = 3. ** P < 0.01 and * P < 0.05 versus control group, ## P < 0.01 and # P < 0.05 versus ox-LDL treated group atherosclerosis. We found that miR-378a-5p, miR-30c and let-7d-5p were the key nodes in the network. In the co-expression network, the inflammation-related miRNAs are connected with lncRNAs, circRNAs, and mRNAs, including positively and negatively regulated genes. Three ceRNAs (circ-Tns3/let-7d-5p/Ctsl, circ-Wdr91/miR-378a-5p/Msr1, and circ-Cd84/miR-30c/Tlr2) were interacted in the network.
The key mRNAs in the ceRNAs network include Msr1, Tlr2, the mechanism in this study, the complex inflammatory microenvironment in atherosclerotic plaque should be further considered. We will fully consider these limitations and carry out in-depth research in the future.
Since manipulating the activation of immune response is essential to promote innate host defenses, the accumulation of evidence on miR-NAs as inflammatory regulators is likely to make the new progress in the treatment of atherosclerosis. Future studies will contribute to develop novel and effective RNA interference therapeutics.