Expression level of miR-155 in peripheral blood

Yu-Hui Zhang, Liang-Hua Xia, Jia-Mei Jin, Ming Zong, Ming Chen, Bo Zhang*

1Department of Ultrasound, East Hospital Affiliated to Medicine School, Tongji University, Shanghai 200120, China

2Department of Ultrasound, Shanghai Huadong Hospital Affiliated to Medicine School, Fudan University, Shanghai 200040, China

Expression level of miR-155 in peripheral blood

Yu-Hui Zhang1△, Liang-Hua Xia1△, Jia-Mei Jin2, Ming Zong1, Ming Chen1, Bo Zhang1*

1Department of Ultrasound, East Hospital Affiliated to Medicine School, Tongji University, Shanghai 200120, China

2Department of Ultrasound, Shanghai Huadong Hospital Affiliated to Medicine School, Fudan University, Shanghai 200040, China

ARTICLE INFO

Article history:

Received15 December 2014

Received in revised form 20 January 2015

Accepted 15 February 2015

Available online 20 March 2015

miR-155

Objective: To investigate the relationship between the expression level of miR-155 and the severity of coronary lesion, and explore the action mechanism. Methods: Peripheral blood mononuclear cells (PBMC) were isolated form blood simple from patients with acute myocardial infarction (AMI), unstable angina (UAP), stable angina (SAP) and chest pain syndrome (CPS). RT-PCR was performed to analysis the expression level of miR-155 in peripheral blood mononuclear cells, plasma and RAW264.7 macrophagocyte. MTT was used to analyze the cell viability of OxLDL treated RAW264.7 macrophagocyte. Results: The expression level of miR-155 in blood sample from coronary heart disease patients was much lower than in the blood sample of non-coronary heart disease (P＜0.05). The level of miR-155 in PBMCs was much higher in the blood sample from CPS group than the other three group, and the level of miR-155 in plasma was higher in the CPS group than in the UAP and the AMI group, the difference was statistically significant (P＜0.05). The expression level of miR-155 in PBMCs is positively associated with the level in the plasma (r=0.861, P=0.000). OxLDL can induce the expression of miR-155 in RAW264.7 macrophagocyte, decrease the cell viability of RAW264.7 macrophagocyte, and with the concentration and the treatment time of OxLDL increased, the effort become more obvious. The inhibition effort of OxLDL to RAW264.7 macrophagocyte with high miR-155 expression is much lower than the control group, and it is statistically significant after treated for 12, 24 and 48 h. Conclusions: miR-155 plays a protective role in the progression of atherosclerosis, and it may be achieved by reducing the apoptosis effort of OxLDL to RAW264.7 macrophagocyte.

1. Introduction

MiR-155 is widely expressed in various cells, such as T cells, B cells, mononuclear cells and endothelial cells. As a multifunction miRNA, it is extensively involved with the differentiation, proliferation and apoptosis of many cells, and the development of many tissues[1]. MiR-155 can inhibit inflammatory response and affect lipid uptake in macrophages through regulating other targets[2,3]. CAD is a kind of inflammatory disease closely related to lipid metabolism. There are no obvious symptoms during the early stage of CAD. However, as stable plaque develops into a vulnerable plaque, the fibrous cap may rupture and expose the liquid in the plaque, which could activate the fibrinolytic system in circulation. Eventually, occlusive thrombus may form, causing myocardial infarction[4-6]. Stephan Fichtlscherer et al[7] measured the miR-155 levels of patients with CAD and healthy patients; the results showed that miR-155 levels clearly declined for patients with CAD,compared with healthy patients. Huang et al[8] found a marked increase in the protein amount of the myeloid differentiation primary response gene 88 (MyD88), which can activate the NF-κB pathway. The results demonstrated that miR-155 serves as a negative feedback regulator in oxLDL-stimulated THP-1 inflammatory responses and lipid uptake. These phenomena suggest that miR-155 may be closely related to the disease. However, the relationship of miR-155 with the severity of the disease and the action mechanism is still unclear.

To investigate the clinical value of miR-155 in the diagnosis of CAD, we detected the miR-155 expression levels in plasma and mononuclear cells of peripheral blood from patients with different severities of CAD, analyzed the effort of miR-155 on the apoptosis of macrophage; the correlation of miR-155 with the severity of CAD.

2. Materials and methods

2.1. Materials

miR-155 mimics and miR-155 inhibitor were purchased from the Gene Pharma; OxLDL was purchased from the Beijing Xiesheng biological technology; DMEM culture medium and fetal calf serum were provided by HyClone; trypsin and MTT were from Sigma; total RNA isolation kit and Fist Strand cDNA Synthesis kit were from TaKaRa Bio Inc. Real-time PCR was purchased from Thermo; microplate reader was from Molecular Devices.

2.2. Cell isolation

The blood sample was provided by Shanghai East hospital. There were four kind of blood samples, namely blood form patients with acute myocardial infarction (AMI group), patients with tunstable angina pectoris (UAP group), patients with stable angina pectoris (SAP group), and patients with chest pain syndrome (CPS group). PBMCs were isolated from the blood by density gradient centrifugation method using lymphocyte isolation liquid. After centrifugation, the liquid divided into 3 layers; cells on the top of the forth layer were collected into a centrifuge tube, and was resuspended by PBS. The liquid was centrifuged in 1 500 rpm for 15 minutes, then the supernatant was abandoned. The process was repeated for three times.

2.3. Transfect macrophage with miR-155 OligoRNA

RAW 264.7 was seeded in the 24 plate (2×105/well), and cultured to 80% confluence for later use. Lipofectamin 2000 (1 μL/well) and FAM-siRNA (2 μL/well) were diluted by 50 μL Opti-MEMI Reduced Serum Medium respectively, and cultured for 5 min; Mix the solution thoroughly and let it stand for about 20 min before using it. The FAM-siRNA-transfection reagent was added into the plate with culture medium, and after cultured in an incubator at 37 ℃ for another 6 h, the liquid was replaced with DMEM culture medium containing 10% FBS. The cell was then collected and the expression level of miR-155 was analyzed by quantitative real time PCR.

2.4. Quantitative real-time PCR analysis

Certain amount of cell was re-suspended by PSB in a centrifuge tube, and an appropriate amount of Trizol was added into the centrifuge tube with the PBMCs. After incubating for 15 h the tube was centrifuged at 12 000 rpm at 4 ℃ for 5 min, and the supernatant was removed. Total mRNA was isolated using an Eastep Universal RNA Extraction Kit (Promega). The RNA sample were treated with DNase Ⅰ(Sigma), then quantified and reverse-transcribed into cDNA using a Fist Strand cDNA Synthesis kit (TAKARA). Quantitative real-time PCR was conducted using a RealPlex4 realtime PCR detection system (Eppendrof) with SYBR Green Real time PCR Master MIS (TOYOBO). The reaction system: SYBR Premix Ex Taq (2×)12.5 μL, PCR Forward Primer (10 μM) 1 μL, PCR Reverse Primer (10 μM) 1 μL, DNA template 2 μL, dH2O 8.5 μL. Reaction parameters: pre denaturation at 94 ℃ for 30 s, denaturation at 95 ℃ for 15 s, anneal at 60 ℃ for 20 s, and extend at 72 ℃ for 10 s. The application was performed for 40 cycles. A comparative threshold cycle (Ct) was used to determine the relative gene expression normalized to 18S rRNA for each sample, and the relative expression level of each sample was calculated using the formula, 2-△△Ct. All primers used in our study were designed according to the sequence published online (GenBank), and synthesized by Invitrogen.

2.5. MTT detect the viability of the cell

The RAW264.7 macrophage and miR-155 overexpressed RAW264.7 macrophage were digested by trypsin after they achieved 80% confluence. Adjust the concentration of the cell suspension to 2 ×105/mL, and 200 μL/well was added into the 96 well plate. After cultured for 24 h, OxLDL was added to one group of the cell with the final concentration of 0, 20, 40, 80, 160 μg/mL. the cell was cultured in an incubator at 37 ℃ for another 4 h, and then the culture medium with MTT was abandoned, 150 μL dimethyl sulfoxide (DMSO) was added to the plate to dissolve the formazan crystalsfor 30 min. Then the absorbance was measured at 490nm using a Microplate reader. Corrected absorbance values were obtained by subtracting the blank absorbance from the absorbance obtained. OxLDL was added to the other group with the final concentration of 80 μg/mL, after cultured for 1, 3, 6, 12, 24, 48 h, detect the absorbance as we described above.

2.6. Statistical analysis

In this study, SPSS 13.0 software was used for statistical analysis. The measured data were expressed as mean±SD, and t-test was applied to the results for statistical analysis. The chi-square test was applied to the count data for statistical analysis. P＜0.05 was considered significant.

3. Results

3.1. Levels of miR-155 in PBMCs and miR-155

Figure 1 shows the same trend of miR-155 expression levels between plasma and PBMCs, in the four kind of blood sample. The miR-155 levels declined as the disease become more severe: the highest miR-155 level came from the CPS group, followed by the SAP and UAP groups, and the AMI group had the lowest expression level. In PBMCs, the miR-155 levels were much higher than the other three groups, and the difference was statistically significant (P＜0.05). In plasma, there were no significant differences between the miR-155 levels of the CPS and SAP groups (P＞0.05), however, the miR-155 levels of the CPS group were obviously higher than the UAP and AMI groups (P＜0.05).

In PBMCs, the miR-155 level in the CPS group was higher than in the SAP, UAP, and AMI groups; the differences were statistically significant (CPS vs. SAP t=28.891, P=0.000; CPS vs. UAP t=20.739, P=0.000; CPS vs. AMI t=26.118, P=0.000). In plasma, the miR-155 level in CPS was higher than in the UAP and AMI groups; the differences were statistically significant (CPS vs. SAP t=10.065, P=0.167; CPS vs. UAP t=10.774, P=0.005; CPS vs. AMI t=15.036, P=0.000).

3.2. miR-155 levels in PBMCs and plasma

Spearman correlation coefficients were used in evaluating and analyzing the correlation of miR-155 expression levels in PBMCs and plasma; and the results revealed that miR-155 levels has a positive correlation with PBMCs and plasma (r=0.861, P=0.000 (Figure 2).

3.3. miR-155 levels between CAD blood sample and non-CAD blood sample

Patients were diagnosed with CAD when the degree of coronary stenosis was higher than 50%. As shown in figure 3, the miR-155 level in PBMCs was higher in the non-CAD group than in the CAD group (2.705±0.310 and 0.983±0.220, respectively); and the miR-155 level in plasma was also higher in the non-CAD group than in the CAD group (2.060±0.410 and 1.128±0.250, respectively).

In PBMCs and plasma, the miR-155 levels in CAD patients were lower than non-CAD patients, and the difference was statistically significant (In PBMCs: non-CAD vs. CAD t=11.550, P=0.000; in plasma: non-CAD vs. CAD t=14.003, P=0.002).

3.4. Expression of miR-155 was induced by OxLDL by RAW264.7 macrophage

The level of miR-155 was detected by Real-Time PCR. We found that, after treated with OxLDL, the expression level of miR-155 in RAW264.7 macrophage raised with the improvement of the OxLDL concentration (Figure 3).

3.5. OxLDL reduce the viability of RAW264.7 macrophage

After treated with OxLDL of different concentration for 24 h, the viability of RAW264.7 macrophage declined with the concentration improved. Compared with the control group, the viability of the cell treated with the concentration of 20, 40, 80 and 160 μg/mL declined obviously, and the difference was statistically significant (P＜0.05). When treated with OxLDL at the concentration of 80 μ g/mL, the viability of the cell also declined with the time prolonged. Compared with the control group, the viability of the cell at 24 and 48 hours improved significantly (P＜0.05) (Figure 4).

3.6. Effort of OxLDL on the miR-155 overexpressed RAW264.7 macrophage

After treated with OxLDL, the viability of the cell in the control group, miR-155 overexpression group, and the miR-155 overexpression + miR-155 inhibitor group declined with the treatment time prolonged. And the viability of the cell in miR-155 overexpression group was much higher than the control group and the inhibitor group in the 12, 24 and 48 h, the difference is statistically significant (P＜0.05). There was no statistical difference between the control group and the inhibitor group (P＞0.05) (Figure 5).

4. Discussion

miR-155 is a key factor that regulates inflammatory reactions and participates in inflammatory related diseases, while CAD is a kind of inflammatory disease; To elucidate the relationship of miR-155 with CAD, we measured the PBMCs and plasma miR-155 levels of blood

sample form patients with CPS, SAP, UAP, and AMI; and analyzedthe relationship of miR-155 with the severity of CAD and plaque stability; detected the effort of OxLDL on RAW264.7 macrophage and miR-155 overexpression RAW264.7 macrophage, analyzed the effort of miR-155 on the apoptosis of RAW264.7 macrophage, these results further confirmed that miR-155 levels declines as CAD progresses and the overexpression of miR-155 can inhibit the apoptosis of . RAW264.7 macrophage induced by OxLDL.

The miR-155 gene is located in chromosome 21q21, a highly conserved coding region within the third extron of the B-cell integration cluster gene[9,10]. Research revealed that miR-155 has been found up-regulated in several activated immune cells; which could modulate the immune response by regulating the differentiation of the immune cells, such as Th1 cell and T cell, and the secretion of cytokines[11,12]. Apart from inflammatory stimulation, the Oxidized LDL, which plays an important role in atherosclerosis, can also stimulate the THP-1 macrophage, and upregulate the miR-155 level, which suggests that miR-155 plays an important role in CAD[13,14]. Fichtlscherer et al[15] found that miR-155 levels declines, as CAD progresses; while Menno Hoekstra et al[16] reported that there was no significant difference between the patient's miR-155 levels and the different degrees of CAD.

Our results showed that the miR-155 levels in plasma and PBMCs gradually declined from SAP to AMI blood sample. According to the correlation analysis, miR-155 levels in plasma were positively related to the levels in PBMCs. Although, the process on how miR-155 is released in blood circulation is still unclear, it can still be detected in serum, at a steady form[17]. This phenomenon revealed that miR-155 can resist RNA enzyme dependent degradation. During cell culture, miR-155 secretion can be stimulated by serum elimination[18,19]. Moreover, miR-155 can also be detected in endothelial cell-derived apoptotic bodies; and the RNA, incorporated into apoptotic bodies or microvesicles, can be delivered into recipient cells[20]. Combined with the results of our study, wherein, miR-155 can be detected in peripheral blood and that miR-155 levels in plasma has the same trend as the levels in PBMCs, we can deduce that miR-155 in plasma may have originated from PBMCs.

There was an over expression of miR-155 in the plaque and macrophage of patients with atherosclerosis[21,22], which suggesting that miR-155 could participate in the development of atherosclerosis. Although, miR-155 could accelerate inflammatory reactions and cell apoptosis at the same time[23], its function in atherosclerosis is still controversial. In our study, we found that the miR-155 levels in blood sample from patients with CAD were lower than blood sample from patients without CAD. This suggests that the miR-155 levels were negatively related to the severity of coronary lesions. Combined with the research of Li et al[24,25], we can speculate that miR-155 has a vascular protective effect; miR-155 can control inflammation and reduce tissue damage through its negative feedback effects on inflammatory factors. Moreover, miR-155 can also directly act on angiotensin Ⅱ-1 receptors, preventing it to bond with angiotensinⅡ; which may block endothelial cell migration, repress angiotensinⅡ-1 transcription, and inhibit the occurrence and development of atherosclerosis[26,27].

The improvement of apoptosis in vivo is a major cause of CAD, but the action mechanism is still unclear. The opoptosis of macrophage has been confirmed to be an outstanding factor in advanced atherosclerotic plaque. In this study, we use miR-155 mimics as intervention factor, induce the apoptosis of macrophage by OxLDL to stimulate the apoptosis process of macrophage in vivo. We found that OxLDL can induce the apoptosis of macrophage in an time and dosage dependent way. We also found, OxLDL can induce the expression of miR-155 improved slightly. The overexpression of miR-155 can improve the viability of macrophage, inhibit the apoptosis induced by OxLDL and the phenomena can be inhibited by miR-155 inhibitor. These results suggest that the regulation effort of OxLDL and miR-155 on the apoptosis of macrophage is achieved by a negative feedback pathway. Zhu et al[28] reported, miR-155 suppress the apoptosis of macrophage by regulate FADD.

In conclusion, the miR-155 levels of CAD patients were higher than non-CAD patients, and the miR-155 levels declined as the disease developed. The over expression of miR-155 can inhibit the apoptosis of macrophage induced by OxLDL. Therefore, the miR-155 in circulation may potentially serve as a new marker for cardiovascular disease. However, our study still has its deficiencies. For inflammatory related small miRNAs, it is still unclear whether other inflammatory diseases would affect the expression level of miR-155, and further affect the sensitivity and specificity of CAD diagnosis. Further study should be done to confirm the possibility of clinically displacing the tradition marker.

Conflict of interest statement

We declare that me have no conflict of interest.

[1] Tili E, Michaille JJ, Wernicke D, Hansjuerg A, Stefan C, Stefano V, et al. Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer. P Natl Acad Sci 2011; 108(12): 4908-4913.

[2] Quinn SR, Mangan NE, Caffrey BE, Michael PG, Williams BRG, Hertzog PJ, et al. The role of Ets2 transcription factor in the induction of MicroRNA-155 (miR-155) by lipopolysaccharide and its targeting by interleukin-10. J Biol Chem 2014; 289(7): 4316-4325.

[3] Chen Y, Siegel F, Kipschull S, Bodo Haas, Holger Fröhlich, Gunter Meister, et al. miR-155 regulates differentiation of brown and beige adipocytes via a bistable circuit. Nat Commun 2013; 4: 1769.

[4] Thygesen K, Alpert JS, Jaffe AS, Jaffe AS, Harvey DW, Maarten LS, et al. Third universal definition of myocardial infarction. J Am Coll Cardiol 2012; 60(16): 1581-1598.

[5] Polonsky TS, McClelland RL, Jorgensen NW, Diane EB, Gregory LB, Alan DG, et al. Coronary artery calcium score and risk classification for coronary heart disease prediction. JAMA 2010; 303(16): 1610-1616.

[6] Hanson MA, Fareed MT, Argenio SL, William B. Coronary artery disease. Primary Care: Clin Office Pract 2013; 40(1): 1-16.

[7] Fichtlscherer S, De Rosa S, Fox H, Thomas S, Ariane F, Christoph L, et al. Circulating microRNAs in patients with coronary artery disease. Circ Res 2010; 107(5): 677-684.

[8] Huang R, Hu GQ, Lin B, Lin Z, Sun C. MicroRNA-155 silencing enhances inflammatory response and lipid uptake in oxidized low-density lipoprotein-stimulated human THP-1 macrophages. J Invest Med 2010; 58(8): 961-967.

[9] Vigorito E, Kohlhaas S, Lu D, Leyland R. miR-155: an ancient regulator of the immune system. Immunol Rev 2013; 253(1): 146-157.

[10] Gracias DT, Stelekati E, Hope JL, Boesteanu AC, Doering TA, Norton J, et al. The microRNA miR-155 controls CD8+T cell responses by regulating interferon signaling. Nat Immunol 2013; 14(6): 593-602.

[11] Singh UP, Murphy AE, Enos RT, Haidar AS, Narendra PS, Honbing G, et al. miR-155 deficiency protects mice from experimental colitis by reducing Th1/Th17 responses. Immunology 2014; 143(3): 478-489.

[12] Zhang J, Cheng Y, Cui W, Li MX, Li B, Li G. MicroRNA-155 modulates Th1 and Th17 cell differentiation and is associated with multiple sclerosis and experimental autoimmune encephalomyelitis. J Neuroimmunol 2014, 266(1): 56-63.

[13] Wagner J, Riwanto M, Besler C, Andrea K, Stephan F, Tino R, et al. Characterization of levels and cellular transfer of circulating lipoproteinbound microRNAs. Arterioscl Throm Vas 2013; 33(6): 1392-1400.

[14] Voloshyna I, Littlefield MJ, Reiss AB. Atherosclerosis and interferon-γ: New insights and therapeutic targets. Trends Cardiovas Med 2014; 24(1): 45-51.

[15] Fichtlscherer S, Zeiher AM, Dimmeler S. Circulating MicroRNAs biomarkers or mediators of cardiovascular diseases? Arteriosclerosis, Thrombosis, Vascular Biol 2011; 31(11): 2383-2390.

[16] Donners MM, Wolfs IM, Stöger LJ, Miel PC, Chantal CH, Stephane H, et al. Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice. PloS one 2012; 7(4): e35877.

[17] Hoekstra M, van der Lans CA, Halvorsen B, Lars G, Johan K, Pål A, et al. The peripheral blood mononuclear cell microRNA signature of coronary artery disease. Biochem Bioph Res Co 2010; 394(3): 792-797.

[18] Wang G, Tam LS, Li EKM, Bonnie, Chow KM, Cathy CW, et al. Serum and urinary cell-free MiR-146a and MiR-155 in patients with systemic lupus erythematosus. J Rheumatol 2010; 37(12): 2516-2522.

[19] Kosaka N, Iguchi H, Ochiya T. Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis. Cancer Sci 2010; 101(10): 2087-2092.

[20] Wang HQ, Yu XD, Liu ZH, Xin C, Dino S, Lin TJ, et al. Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3. Am J Pathol 2011; 225(2): 232-242.

[21] Nazari-Jahantigh M, Wei Y, Noels H, Shamima A, Zhou Z, Rory RK, et al. MicroRNA-155 promotes atherosclerosis by repressing in macrophages. J Clin Invest 2012; 122(11): 4190-4202.

[22] Zhu J, Chen T, Yang L, Li Z, Wong M, Zheng X, et al. Regulation of microRNA-155 in atherosclerotic inflammatory responses by targeting MAP3K10. PloS one 2012; 7(11): e46551.

[23] O’Connell RM, Taganov KD, Boldin MP, Cheng, David B. MicroRNA-155 is induced during the macrophage inflammatory response. P Natl Acad Sci 2007; 104(5): 1604-1609.

[24] Hao L, Wang X, Cheng J, Sheng Y, Su M, Xia Z, et al. The upregulation of endothelin-1 and downregulation of miRNA-125a-5p,-155 and-199a/ b-3p in human atherosclerotic coronary artery. Cardiovasc Pathol 2014; 23(4): 217-223.

[25] Zhang E, Wu Y. Dual effects of miR-155 on macrophages at different stages of atherosclerosis: LDL is the key? Med Hypotheses 2014; 83(1): 74-78.

[26] Blanco RR, Austin H, Vest III RN, Ravinder V, Li W, Bernard L, et al. Angiotensin receptor type 1 single nucleotide polymorphism 1166A/C is associated with malignant arrhythmias and altered circulating miR-155 levels in patients with chronic heart failure. J Card Fail 2012; 18(9): 717-723.

[27] Cheng W, Liu T, Jiang F, Liu C, Zhao X, Gao Y, et al. microRNA-155 regulates angiotensin Ⅱtype 1 receptor expression in umbilical vein endothelial cells from severely pre-eclamptic pregnant women. Int J Mol Med 2011; 27(3): 393-399.

[28] Zhu G, Yang L, Guo R, Liu H, Shi Y, Wang H, et al. miR-155 inhibits oxidized low-density lipoprotein-induced apoptosis of RAW264. 7 cells. Mol Cell Biochem 2013; 382(1-2): 253-261.

ent heading

10.1016/S1995-7645(14)60318-7

*Corresponding author: Bo Zhang, Department of Ultrasound, East Hospital Affiliated to Medicine School, Tongji University, Shanghai 200120, China.

E-mail: zhangbodongfang@qq.com

△Co-first author.

Foundation project: It is supported by Shanghai Municipal Key Discipline Construction (ZK2012A27) and General Program Projects of Pudong Health Bureau of Shanghai (PW2013A-4).

Coronary heart disease

RAW264.7 macrophagocyte