Comparative transcriptomic analysis reveals genes related to the rapid accumulation of oleic acid in Camellia chekiangoleosa, an oil tea plant with early maturity and large fruit.

Camellia chekiangoleosa has a higher oleic acid content and a shorter reproductive cycle than typical oil tea plants. It was intensively sampled over six C. chekiangoleosa seed development stages. The content of fatty acids determined by GC showed that the accumulation of fatty acids gradually increased from the S1 to S5 stages, and the maximum concentration was reached in S5. Then, fatty acids declined slightly in S6. The main fatty acid component showed the same accumulation trend as the total fatty acids, except linolenic acid, which remained at a low level throughout seed developmental stages. Changes in the expression of fatty acid accumulation-related genes were monitored using second-generation and SMRT full-length transcriptome sequencing. Finally, 18.92 G accurate and reliable data were obtained. Differential expression analysis and weighted coexpression analysis revealed two "gene modules" significantly associated with oleic acid and linoleic acid contents, and the high expression of ENR, KAS I, and KAS II, which accumulate substrates for oleic acid synthesis, was thought to be responsible for the rapid accumulation of fatty acids in the early stage. The rapid increase in fatty acids in the second stage may be closely related to the synergy between the high expression of SAD and low expression of FAD2. In addition, many transcription factors, such as ERF, GRAS, GRF, MADS, MYB and WRKY, may be involved in the fatty acid synthesis. Our data provide a rich resource for further studies on the regulation of fatty acid synthesis in C. chekiangoleosa.

Corrigendum to "miR-221 Alleviates the Ox-LDL-Induced Macrophage Inflammatory Response via the Inhibition of DNMT3b-Mediated NCoR Promoter Methylation".

[This corrects the article DOI: 10.1155/2019/4530534.].

miR-221 Alleviates the Ox-LDL-Induced Macrophage Inflammatory Response via the Inhibition of DNMT3b-Mediated NCoR Promoter Methylation.

Atherosclerosis (AS) is a chronic inflammatory disease, and macrophages play a key role in all phases of AS. Recent studies have shown that miR-221 is a biomarker for AS and stroke; however, the role and mechanism of miR-221 in AS are unclear. Herein, we found that miR-221 and NCoR levels were decreased in ox-LDL-treated THP-1-derived macrophages. In contrast, DNMT3b, IL-6, and TNF-α expression levels were increased under these conditions. Upregulation of miR-221 or NCoR could partially inhibit ox-LDL-induced IL-6 and TNF-α expression. Further studies showed that DNMT3b was a target of miR-221. DNMT3b inhibition also suppressed IL-6 and TNF-α expression and increased NCoR expression in the presence of ox-LDL. Moreover, DNMT3b was involved in ox-LDL-induced DNA methylation in the promoter region of NCoR. These findings suggest that miR-221 suppresses ox-LDL-induced inflammatory responses via suppressing DNMT3b-mediated DNA methylation in the promoter region of NCoR. These results provide a rationale for using intracellular miR-211 as a possible antiatherosclerotic target.

TRIF Regulates BIC/miR-155 via the ERK Signaling Pathway to Control the ox-LDL-Induced Macrophage Inflammatory Response.

Toll/IL-1R-domain-containing adaptor-inducing IFN-ß (TRIF) is an important adaptor for TLR3- and TLR4-mediated inflammatory signaling pathways. Recent studies have shown that TRIF plays a key role in vessel inflammation and atherosclerosis; however, the precise mechanisms are unclear. We investigated the mechanisms of the TRIF-regulated inflammatory response in RAW264.7 macrophages under oxidized low-density lipoprotein (ox-LDL) stimulation. Our data show that ox-LDL induces TRIF, miR-155, and BIC expression, activates the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways, and elevates the levels of IL-6 and TNF-α in RAW264.7 cells. Knockdown of TRIF using TRIF siRNA suppressed BIC, miR-155, IL-6, and TNF-α expression and inhibited the ERK1/2 and SOCS1-STAT3-NF-κB signaling pathways. Inhibition of ERK1/2 signaling also suppressed BIC and miR-155 expression. These findings suggest that TRIF plays an important role in regulating the ox-LDL-induced macrophage inflammatory response and that TRIF modulates the expression of BIC/miR-155 and the downstream SOCS1-STAT3-NF-κB signaling pathway via ERK1/2. Therefore, TRIF might be a novel therapeutic target for atherosclerosis.

Yunnanterpene G, a spiro-triterpene from the roots of Cimicifuga foetida, downregulates the expression of CD147 and MMPs in PMA differentiated THP-1 cells.

A new cycloartane triterpene, yunnanterpene G (1), containing an oxaspiro[5.4]decane moiety, was purified from the roots of Cimicifuga foetida. The new structure was determined from spectroscopic data and the X-ray diffraction method. Biological evaluations revealed that compound 1 significantly inhibited the mRNA expression of the atherosclerosis-related adhesion molecule CD147 (extracellular matrix metalloproteinase inducer, EMMPRIN), and proteolytic enzymes matrix metalloproteinase 2 (MMP-2), MMP-9 and MMP-14, in a dose-dependent manner in phorbol-12-myristate-13-acetate-induced human monocytic THP-1 cells by quantitative real-time PCR method. At the same time, the migration ability of the induced THP-1 cells was potently inhibited. Furthermore, western blot experiments showed that compound 1 at 25 µM strongly suppressed phosphorylation of NF-κB p65 and p38 MAPK in the differentiated THP-1 cells.

Ox-LDL-Induced MicroRNA-155 Promotes Autophagy in Human Endothelial Cells via Repressing the Rheb/ mTOR Pathway.

BACKGROUND/AIMS: Autophagy, an evolutionary conserved biological process, is activated in cells to cope with various types of stress. MicroRNAs control several activities related to autophagy. However, the role of autophagy-related microRNAs during atherosclerosis is far from known. MicroRNA-155 was identified to be a crucial regulator of atherosclerosis. The objectives of the study were to analyze the effect of microRNA-155 on autophagic signaling and explore its mechanism in human endothelial cells under ox-LDL stress. METHODS: The study included human endothelial cells surrogate EA.hy926 lines (EA.hy926 cells). The expression of microRNA-155 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effect of microRNA-155 on endothelial autophagy was observed along with the expression levels of Rheb, LC3B, Beclin1, and P62/SQSTM1 by western blotting (WB) and immunofluorescence through microRNA-155 overexpression or inhibition. Bioinformatics analysis and Luciferase reporter assay were used to explore the target gene of microRNA-155. Cell viability and apoptosis were examined by 3-[4,5-dimethylthiazol-2-yl]-5- [3-carboxy-methoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium inner salt (MTS) assay and TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis assay. RESULTS: MicroRNA-155 expression was significantly increased under ox-LDL stress. MicroRNA-155 increased autophagic activity, while inhibition of it alleviated ox-LDL-induced autophagy in EA.hy926 endothelial cells. In addition, dual-luciferase reporter assays showed that microRNA-155 suppressed Rheb transcription. MicroRNA-155 increased autophagic activity in EA.hy926 cells via inhibition of Rheb-mediated mTOR/P70S6kinase/4EBP signaling pathway. Furthermore, we demonstrated that microRNA-155 could regulate not only autophagy but also apoptosis in EA.hy926 cells. CONCLUSIONS: MicroRNA-155 works as a regulator of endothelial function under ox-LDL stress, making it a potential candidate for the novel therapeutic strategies against atherosclerotic diseases.

[Effect of microRNA-155 on inflammatory response and lipid uptake of macrophages and its mechanism].

Objective To investigate the effect of microRNA-155 on inflammatory response and lipid uptake of macrophages after the cells are stimulated by ox-LDL and its potential mechanism. Methods Macrophage RAW264.7 cells were treated with 0, 25, 50 and 100 µg/mL ox-LDL for 24 hours or with 50 µg/mL ox-LDL for 0, 6, 12, 24 hours. The level of miR-155 was evaluated in all above samples through real-time quantitative PCR. In our research, RAW264.7 cells were divided into six groups: control group, ox-LDL group, ox-LDL/negative control group, ox-LDL/anti-miR-155 group, ox-LDL/shRNA negative control group and ox-LDL/PPARγ-shRNA group. Oil red O staining was used to observe lipid uptake in the cells. Filipin staining was used to evaluate the cellular uptake of ox-LDL. Cholesterol testing was performed to examine the levels of total cholesterol (TC) and free cholesterol (FC). Real-time quantitative PCR was done to detect the expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 mRNAs. According to study purpose, we explored the potential mechanisms of miR-155 inhibitor (including control group, ox-LDL group, ox-LDL/negative control group and ox-LDL/miR-155 inhibitor group), miR-155 mimic (including negative control group and miR-155 mimic group), and PPARγ shRNA (including control group, ox-LDL group, ox-LDL/shRNA negative control group and ox-LDL/PPARγ shRNA group) in ox-LDL-treated RAW264.7 cells through evaluating the expressions of p-STAT3, PPARγ, CD36 and NF-κBp65 using Western blotting. Results Ox-LDL stimulation increased the relative expression of miR-155 in a dose- and time-dependent manner. Through oil red O staining, Filipin staining, cholesterol testing and real-time PCR experiment, we found the relative absorbance, levels of TC and FC, filipin fluorescence intensity, and levels of TNF-α, IL-1ß and IL-6 mRNAs were significantly lower in ox-LDL/anti-miR-155 group than in ox-LDL and ox-LDL/negative control group. Similarly, the relative absorbance, levels of TC and FC, filipin fluorescence intensity and levels of TNF-α, IL-1ß and IL-6 mRNAs were significantly lower in ox-LDL/ PPARγ shRNA group than in ox-LDL group and ox-LDL/shRNA negative control group. The expressions of p-STAT3, PPARγ, CD36 and NF-κBp65 proteins were suppressed in ox-LDL/anti-miR-155 group as compared with ox-LDL group and ox-LDL/negative control group. Similarly, p-STAT3, PPARγ, CD36 and NF-κBp65 protein levels decreased in ox-LDL/PPARγ shRNA as compared with ox-LDL/vector group. Moreover, p-STAT3, PPARγ, CD36 and NF-κBp65 protein levels were higher in miR-155 mimic group than in negative control group. Conclusion Mediated by PPARγ, miR-155 induced inflammation response and lipid uptake of macrophages via STAT3/NF-κB signal pathway and CD36.

MicroRNA-99a inhibits insulin-induced proliferation, migration, dedifferentiation, and rapamycin resistance of vascular smooth muscle cells by inhibiting insulin-like growth factor-1 receptor and mammalian target of rapamycin.

Patients with type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and are subsequently at high risk for atherosclerosis. Hyperinsulinemia has been associated with proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) during the pathogenesis of atherosclerosis. Moreover, insulin-like growth factor-1 receptor (IGF-1R) and mammalian target of rapamycin (mTOR) have been demonstrated to be the underlying signaling pathways. Recently, microRNA-99a (miR-99a) has been suggested to regulate the phenotypic changes of VSMCs in cancer cells. However, whether it is involved in insulin-induced changes of VSCMs has not been determined. In this study, we found that insulin induced proliferation, migration, and dedifferentiation of mouse VSMCs in a dose-dependent manner. Furthermore, the stimulating effects of high-dose insulin on proliferation, migration, and dedifferentiation of mouse VSMCs were found to be associated with the attenuation of the inhibitory effects of miR-99a on IGF-1R and mTOR signaling activities. Finally, we found that the inducing effect of high-dose insulin on proliferation, migration, and dedifferentiation of VSMCs was partially inhibited by an active mimic of miR-99a. Taken together, these results suggest that miR-99a plays a key regulatory role in the pathogenesis of insulin-induced proliferation, migration, and phenotype conversion of VSMCs at least partly via inhibition of IGF-1R and mTOR signaling. Our results provide evidence that miR-99a may be a novel target for the treatment of hyperinsulinemia-induced atherosclerosis.

miR-155 Regulated Inflammation Response by the SOCS1-STAT3-PDCD4 Axis in Atherogenesis.

Inflammation response plays a critical role in all phases of atherosclerosis (AS). Increased evidence has demonstrated that miR-155 mediates inflammatory mediators in macrophages to promote plaque formation and rupture. However, the precise mechanism of miR-155 remains unclear in AS. Here, we also found that miR-155 and PDCD4 were elevated in the aortic tissue of atherosclerotic mice and ox-LDL treated RAW264.7 cells. Further studies showed that miR-155 not only directly inhibited SOCS1 expression, but also increased the expression of p-STAT and PDCD4, as well as the production of proinflammation mediators IL-6 and TNF-α. Downregulation of miR-155 and PDCD4 and upregulation of SOCS1 obviously decreased the IL-6 and TNF-α expression. In addition, inhibition of miR-155 levels in atherosclerotic mice could notably reduce the IL-6 and TNF-α level in plasma and aortic tissue, accompanied with increased p-STAT3 and PDCD4 and decreased SOCS1. Thus, miR-155 might mediate the inflammation in AS via the SOCS1-STAT3-PDCD4 axis. These results provide a rationale for intervention of intracellular miR-155 as possible antiatherosclerotic targets.

[Relationship between atherosclerotic plaque characteristics and extracellular matrix metalloproteinase inducer and urokinase-type plasminogen activator in patients with coronary artery disease].

OBJECTIVE: To explore the association between extracellular matrix metalloproteinase inducer (EMMPRIN) and urokinase-type plasminogen activator (uPA) and the severity of coronary artery lesions in coronary heart disease (CHD) patients. METHODS: This study enrolled 88 patients with acute coronary syndrome (ACS) and 46 patients with stable angina pectoris (SAP). The mean fluorescence intensity (MFI) of EMMPRIN on monocytes of peripheral blood (PBMCs) were examined by flow cytometry. uPA in serum was measured with ELISA . 64-slice spiral computed tomography coronary artery imaging was performed in 108 CHD patients. Coronary artery plaques were divided into type I (33 patients), type II (59 patients) and type III (44 patients) through plaque morphology characteristics according to coronary angiography. Coronary artery plaques were divided into soft (42 patients), fibrous (34 patients) and calcified plaque (32 patients) according to CT characteristics. RESULTS: (1) Type II plaque (48 patients) and soft plaque (35 patients) were the major plaque types in the ACS patients, while type Iplaque (20 patients) and type III plaque (17 patients) and fibrous plaque (16 patients) and calcified plaque (22 patients) were the major plaque types in the SAP patients. (2) The EMMPRIN expression and uPA levels were significantly higher in typeII plaque group (EMMPRIN MFI: 11.61 ± 0.81, uPA: (0.89 ± 0.17) mg/L) than those in the typeIplaque group (EMMPRIN MFI: 6.65 ± 1.32, uPA: (0.53 ± 0.06) mg/L) and in the type III plaque group (EMMPRIN MFI: 9.47 ± 1.16, uPA:(0.56 ± 0.04) mg/L, all P < 0.05). The EMMPRIN expression was higher in the typeIII plaque group (MFI: 9.47 ± 1.16) than in the typeIplaque group (MFI:6.65 ± 1.32, P < 0.05), but uPA levels were similar between the 2 groups ((0.56 ± 0.04) mg/L vs. (0.53 ± 0.06) mg/L). (3) The EMMPRIN expression and uPA levels in the soft plaque group (EMMPRIN MFI:11.37 ± 0.76, uPA: (0.97 ± 0.12)mg/L) were significantly higher than those in the fibrous plaque group (EMMPRIN MFI: 8.93 ± 1.21), uPA:(0.52 ± 0.09) mg/L) and calcified plaque group (EMMPRIN MFI: 6.94 ± 1.19, uPA:(0.49 ± 0.12) mg/L, P < 0.05). The EMMPRIN expression in the fibrous plaque group (MFI:8.93 ± 1.21) was higher than in the calcified plaque group (MFI:6.94 ± 1.19, P < 0.05), but uPA levels were similar between the two groups. CONCLUSION: Higher EMMPRIN expression and uPA levels were associated with plaque instability, which might be used to evaluate plaque stability in CHD patients.

microRNA-155 is inversely associated with severity of coronary stenotic lesions calculated by the Gensini score.

OBJECTIVES: This study aimed to investigate the association between microRNA-155 (miR-155) and the severity and extent of coronary stenotic lesions. PATIENTS AND METHODS: We measured the miR-155 expression by real-time PCR in 110 consecutive patients undergoing coronary angiography for suspected coronary artery disease. The severity and extent of coronary stenotic lesions were evaluated on the basis of coronary angiography findings by the Gensini score. RESULTS: The miR-155 expression was significantly lower in 56 patients with coronary heart disease than those in 54 controls (P<0.01). The level of miR-155 in peripheral blood mononuclear cells or plasma was lower in patients with unstable angina pectoris and acute myocardial infarction than in patients with chest pain syndrome, whereas no statistically significant differences were observed between patients with stable angina pectoris and chest pain syndrome. Spearman's correlation analysis showed that the expression of miR-155 in plasma correlated positively with the expression in peripheral blood mononuclear cells. The levels of miR-155 in the patients with diseased vessels of two and three or more were significantly lower than in those with diseased vessel of zero and one. The levels of miR-155 were not significantly different among groups with diseased vessels of zero and one. miR-155 were associated negatively with Gensini scores (r = -0.663, P<0.001). The miR-155 expression was correlated significantly to age (r = -0.227), hypertension (r = -0.440), total cholesterol (r = 0.239), high-density lipoprotein cholesterol (r = 0.280), low-density lipoprotein cholesterol (r = -0.315), tobacco use (r = -0.363), angiotensin-converting enzyme inhibitor (r = -0.250), statins (r = -0.368), and high-sensitivity C-reactive protein (r = -0.515). CONCLUSION: miR-155 expression is associated inversely with complicated proatherogenic metabolic risk factors, and the severity of coronary stenotic lesions calculated by Gensini scores.

miR-155 inhibits oxidized low-density lipoprotein-induced apoptosis of RAW264.7 cells.

Macrophage apoptosis is a prominent feature of advanced atherosclerotic plaques. Here, we examined the hypothesis that the apoptotic machinery is regulated by microRNA-155 (miR-155). Constitutive expression of miR-155 was detected in RAW264.7 cells, which was increased following stimulation with oxidized low-density lipoprotein (OxLDL) in a dose- and time-dependent manner. OxLDL-treated RAW264.7 cells showed a marked time- and dose-dependent increase in apoptosis, which was suppressed in the presence of mimics and increased with antagonists of miR-155. Bioinformatics analysis revealed Fas-associated death domain-containing protein (FADD) as a putative target of miR-155. Luciferase reporter assay and Western blot further disclosed that miR-155 inhibits FADD expression by directly targeting the 3'-UTR region. We propose that miR-155 attenuates the macrophage apoptosis, at least in part, through FADD regulation, since forced expression of FADD blocked the ability of miR-155 to inhibit apoptosis. Our results collectively suggest that miR-155 attenuates apoptosis of OxLDL-mediated RAW264.7 cells by targeting FADD, supporting a possible therapeutic role in atherosclerosis.

Functional blockage of EMMPRIN ameliorates atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Extracellular matrix metalloproteinase inducer (EMMPRIN), a 58-kDa cell surface glycoprotein, has been identified as a key receptor for transmitting cellular signals mediating metalloproteinase activities, as well as inflammation and oxidative stress. Clinical evidence has revealed that EMMPRIN is expressed in human atherosclerotic plaque; however, the relationship between EMMPRIN and atherosclerosis is unclear. To evaluate the functional role of EMMPRIN in atherosclerosis, we treated apolipoprotein E-deficient (ApoE(-/-)) mice with an EMMPRIN function-blocking antibody. METHODS AND RESULTS: EMMPRIN was found to be up-regulated in ApoE(-/-) mice fed a 12-week high-fat diet in contrast to 12 weeks of normal diet. Administration of a function-blocking EMMPRIN antibody (100 µg, twice per week for 4 weeks) to ApoE(-/-) mice, starting after 12 weeks of high-fat diet feeding caused attenuated and more stable atherosclerotic lesions, less reactive oxygen stress generation on plaque, as well as down-regulation of circulating interleukin-6 and monocyte chemotactic protein-1 in ApoE(-/-) mice. The benefit of EMMPRIN functional blockage was associated with reduced metalloproteinases proteolytic activity, which delayed the circulating monocyte transmigrating into atherosclerotic lesions. CONCLUSION: EMMPRIN antibody intervention ameliorated atherosclerosis in ApoE(-/-) mice by the down-regulation of metalloproteinase activity, suggesting that EMMPRIN may be a viable therapeutic target in atherosclerosis.

Angiotensin II induces extracellular matrix metalloproteinase inducer expression via an AT1R dependent pathway in aortic atherosclerotic plaque in apolipoprotein E knockout mice.

UNLABELLED: The pathogenesis of acute coronary syndrome is rupture of vulnerable plaque. Extracellular matrix metalloproteinase inducer (EMMPRIN) is reported to have a important role in the destabilization of atheroma. OBJECTIVES: this investigation examined the effect of angiotensin II (Ang II) on EMMPRIN expression in atherosclerotic plaques in ApoE knockout mice. METHODS: ApoE knockout mice were fed a high fat diet to establish an atherosclerosis model then intervention was made with Ang II and valsartan. EMMPRIN gene and its protein expression were measured by real-time PCR immunohistochemistry, and Western blot. RESULTS: EMMPRIN gene and protein expression intervened with Ang II were significantly increased; valsartan could inhibit the effect of Ang II. CONCLUSION: Ang II up-regulated EMMPRIN expression in atherosclerotic plaque via AT1R, and valsartan could inhibit the effect of Ang II.

The effect of the expression of angiotensin II on extracellular matrix metalloproteinase inducer (EMMPRIN) in macrophages is mediated via the AT1/COX-2/PGE2 pathway.

AIM: To explore the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in THP-1 macrophages induced by angiotensin II (Ang II) and the mechanism of EMMPRIN expression. METHODS: THP-1 cells were cultured and induced into macrophages, then stimulated with 10(-6) mol/L Ang II. Levels of EMMPRIN gene and its protein were measured by real-time polymerase chain reaction and western blotting. Prostaglandin E(2) (PGE(2)) expression was assayed by enzyme-linked immunosorbent assay. Antagonists of the angiotensin type-1 receptor (AT(1)R) and angiotensin type-2 receptor (AT(2)R) were used to inhibit the effect of Ang II, and PGE(2) added to detail the mechanism of Ang II-induced EMMPRIN expression. RESULTS: Ang II clearly induced the expression of EMMPRIN mRNA and protein in macrophages; this expression peaked at 12 h and declined after 24 h. The tendency of enhancement of the levels of cyclooxygenase-2 (COX-2) and PGE(2) was coincident with EMMPRIN expression. AT(1)-receptor antagonists and COX-2 inhibitors inhibited the effect of Ang II, but AT(2)-receptor antagonists did not. CONCLUSION: Ang II can up-regulate EMMPRIN expression in THP-1 macrophages via the AT(1)/COX-2/PGE(2) signal transduction pathway, and the effect can be inhibited by losartan and NS-398.

Angiotensin II induces EMMPRIN expression in THP-1 macrophages via the NF-kappaB pathway.

BACKGROUND: Recent studies on atherosclerosis showed that an inducer of MMPs, EMMPRIN, is highly expressed in human atheromas. This suggested the important role of EMMPRIN in the stability of atherosclerotic plaques. Angiotensin II, one of the main functional peptides in the renin-angiotensin system, is involved in the advancement of atherosclerosis. We evaluated the effect of angiotensin II on EMMPRIN expression in THP-1 macrophages, and postulated the potential mechanisms underlying its effects. METHODS AND RESULTS: We established THP-1 macrophages using PMA. The effect of angII on EMMPRIN expression in THP-1 macrophages was then investigated. Results from analyses of RT-PCR and western blotting showed that angII could upregulate EMMPRIN expression. This was mediated via the AT1R, but not the AT2R. The NF-kappaB inhibitor PDTC and P65 RNAi treatment could suppress the effect of angII on EMMPRIN, suggesting the involvement of the NF-kappaB pathway. A gelatin zymography assay showed that MMP-9 activity was related to EMMPRIN expression. CONCLUSION: AngII upregulates the expression of EMMPRIN. NF-kappaB is the critical factor involved in the upregulation of EMMPRIN induced by angII.