MiR-125b is a Potential Protector of Arteriosclerosis Occlusive: Restricting the Proliferation and Migration of Vascular Smooth Muscle Cells via Decreasing the Level of AAMP and SRF.

Arteriosclerosis occlusive (ASO) is a manifestation of atherosclerosis (AS), and it is one of the main causes of lower limb ischemic necrosis in the elderly. Excessive proliferation and migration of vascular smooth muscle cells is one of the main causes of ASO, but the mechanism is unclear. MiR-125b is a tumor suppressor, which can inhibit the proliferation and migration in various kinds of tumors. At the same time, we nd miR-125b is the most obviously down-regulated microRNA in ASO. Therefore, we explore the role of miR-125b in ASO. This study shows that up-regulated miR-125b inhibits the proliferation of vascular smooth muscle cells through CCK8 assay and Brdu assay. Meantime, the up-regulation of miR-125b restricts the migration of vascular smooth muscle cells through trans-well assay and wound healing assay. Furthermore, this study nds that up-regulated miR-125b inhibits the expression level of angio-associated migratory cell protein (AAMP) and serum response factor (SRF), which may be an important way for miR-125b to regulate the proliferation and metastasis of smooth muscle cells.


Introduction
Atherosclerosis (AS) mainly refers to the deposition of lipids on blood vessel walls, which caused by abnormal metabolism [1]. Lower extremity arteriosclerosis occlusive (ASO) is the performance of AS in the lower limbs. To our knowledge, about 80% of the people, over 60 years old, undergo the ASO. The ASO can be rapidly relieved by interventional therapy. However, the 1-year-recurrence rate of stenosis up to 30%~50%, and about 12% of which who have severe lower limb ischemic necrosis even meet amputation [2]. So, it is a necessity to nd out the molecular mechanisms in ASO process, which might provide strategy for clinical treatments.
ASO is originally initiated by the accumulation of apolipoproteins in endothelium, which come from the abnormal metabolism of glucose and lipid [1,3]. Following, the accumulation of apolipoproteins leads a series of intracellular reactions, which eventually make vascular endothelial damage. Then, monocyte macrophages-induced in ammatory response appears [4]. In ammatory cells and in ammatory factors regulate the function of vascular smooth muscle cells (VSMCs), including cell proliferation, migration, differentiation and matrix secretion [4]. VSMCs, as the main cellular component of the middle layer of the arteries, its excessive proliferation and migration play pivotal roles in ASO and restenosis after interventional treatment [5]. Therefore, the prevention and relief of dysfunction of VSMC are important.
Recent studies have shown that microRNAs (miR) are involved in the occurrence and progression of cardiovascular diseases. For example, miR-124 promotes the degradation of TNF-α protein, which enhances in ammatory response and endothelial damage, and promotes the progression of ASO [6]. MiR-146a makes hyperproliferation in VSMCs and indirectly promotes the occurrence of ASO by upregulating the expression of BAX [7]. MiR-221 can signi cantly strengthen the migration and proliferation abilities of VSMCs [8]. However, there are microRNAs that inhibit ASO. MiR-143/145 regulate the expression of mammalian transcription factors and myocardinto, by serum response factors (SRF), to promote the differentiation of SVMC and to inhibit its proliferation, by which MiR-143/145 prevent VSMCs from further damage [9,10]. In addition, miR-21, miR-143and miR-221 are involved in the pathogenesis of ASO [11,12]. Nonetheless, those discovered microRNAs have made few contributions to the clinical treatment of ASO.
MiR-125b is down-regulated in a variety of tumor cells, including hepatocellular carcinoma, breast cancer, ovarian tumors, bladder cancer and leukemia [13][14][15][16][17]. When miR-125b expression is down-regulated, these cancer cells have enhanced proliferation and invasion capabilities. In ASO, miR-125b is one of the microRNAs with the most signi cant change in expression level [6]. Some research results suggest that miR-125b can inhibit the proliferation and migration of VSMCs. Meantime, some studies have shown that miR-125b can participate in the regulation of SRF, thereby promoting cellular migration [6]. However, in ASO, the molecular regulation mechanism of miR-125b on them is unclear. Therefore, we carry out experiments on how miR-125b regulates the arterial endothelial cell damage, and want to provide new strategies for clinical treatment of ASO.
In summary, our experiments obtain the following conclusions: 1. PDFG-BB can inhibit the expression of microNRA-125b; 2. Overexpression of miN-125b can inhibit the proliferation and migration of VSMC; 3.

Western Blot Assay
Cells with different treatments are harvested by cytology brush, followed by iced PBS washing, and lysed with RIPA lysis buffer (Sigma, USA), in which containing phosphorylase and protease inhibitor mixture (Thermo, USA). Then, total protein level is quanti ed by the BCA assay. Finally, speci c gels ware prepared for electrophoresis. A PVDF membrane is applied in immunoblotting after electrophoresis.
Secondary antibody incubation was performed in TBST solution with 5% skim milk, which contains different antibodies (antibodies against GAPDH, SRF and AAMP are purchased from Abcam (1:1000)).

Immuno uorescence
Brie y, A10 cells are seeded in 24-well plates for 24 h, and then xed by 4% paraformaldehyde, followed by permeabilization within 0.5% Triton X-100, and blocking within 5% bovine serum albumin (BSA, Sigma). Samples are incubated with primary antibody ( And the detection procedure was performed with crystal violet and microscope. Quanti cation of migrated cells were calculated by microscope. For wound healing assay, cells are seeded at least 80% fusion in 6-well plates, and scratched by 200 ul pipette tip, then wash with PBS to remove shed cells for extra 48 h culture. Scratch distance is also calculated by microscope.

Statistics
All experimental data are presented as the means ± SD. Statistical Package for the Social Sciences version 20.0 (SPSS Inc., USA) is used for statistical analyses. ANOVA, paired t-test, Chi-square () test and nonparametric test (Mann Whitney U) for statistical analysis of different situations. Statistical signi cance is considered when p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001). All histograms and curves are constructed with GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA).

Results
PDGF-BB plays a negative role in regulation of miR-125b in VSMCs.
Platelet-derived growth factor (PDGF) is a prominent inducer of vascular cell dysfunction, especially the PDGF-BB, which can trigger a variety of biological effects. By activating intracellular signal transduction pathways, PDGF-BB plays an important role in dysfunction of VSMCs, such as migration and proliferation. Therefore, PDGF-BB is used as an inducer in this study. In this paper, we explore the role of PDGF-BB in regulation of miR-125b. As the Fig. 1A showing, we set four groups: negative control group Interestingly, 20 ng/mL treatment of PDGF-BB abrogates the Mimic-125b-loaded liposomes-induced overexpression of miR-125b, which even down-regulates the level of miR-125b to fall below the normal level (60% down-regulation, compared with NC) (Fig. 1A, p < 0.001). In addition, we nd that Mimic-125bloaded liposomes exactly reverses PDGF-BB-induced down-regulation of miR-125b (Fig. 1b, p < 0.05).
MiR-125b restricts the cellular proliferation of VSMCs.
In order to exploring the role of miR-125b in regulation of cellular proliferation in VSMCs, we perform CCK assay and Brdu assay. Basing on the above results, we set two groups: NC group (treated with NC-loaded liposomes) and Mimic-125 group (treated with mimic-125b-loaded liposomes). As the results showing, Mimic-125b group holds lower OD value in 450 nm, which is about half of it in NC group ( Fig. 2A and B, p < 0.001). In other words, the up-regulation of miR-125b inhibits the cellular proliferation about 50% in VSMCs. For verifying the results, we perform Brdu experiment. As the Fig. 2C and D showing, transfection of mimic-125b makes nothing in cellular proliferation within 24 h, while it signi cantly inhibits the OD value in 450 nm at day 4 (about 40%, p < 0.001). It means the increasing of miR-125b can inhibit the cellular proliferation in VSMCs.
MiR-125b inhibits the cellular migration and invasion of VSMCs.
MiR-125b is an inhibitor in cellular proliferation and invasion in tumors. So, we attempt to nd out the relationship between miR-125 and cellular migration in VSMCs. Firstly, we treat the VSMCs with mimic-125b-loaded liposomes or NC-loaded liposomes for 24 h, which followed by transplantation in trans-well for another 48 h. Our three respective repeated data show that Mimic-125b group holds about 450 migrated cells in eld, while NC group holds about 650 migrated cells in eld (Fig. 3B). After normalization, we nd migrated cells of Mimic-125b group are about 70% of which in NC group (Fig. 3C). Furthermore, our experiments show that up-regulation of miR-125b decreases the migration ability about 30% in VSMCs (p < 0.05, Fig. 3B-D). In order to verifying the above results, we apply the wound healing assay for testing migration capability. VSMCs are seeded in 6-well plates, which are treated with NCloaded liposomes or mimic-125b-loaded liposomes for 24 h. Then refresh the medium for another 48 h culture after being scratched by 200 ul pipette tip. As the Fig. 4A showing, there are few differences in NC group and mimic-125b group at rst 24 h. However, at the second 24 h, we nd that the borders of the left and right parts almost coincide in NC group, while the gaps are still visible to the naked eye in Mimic-125b group. Through measuring the distance between the two borders of right and left parts, we make digital transformation. Just as the Fig. 4B showing, the distance between the two borders is about 750 µm in NC group at second 24 h, while it is about 700 µm in mimic-125b group. After normalization, we nd the migration ability in mimic-125b group is not more than 90% of which in NC group (p < 0.05, Fig. 4C). In other words, the up-regulation of miR-125b inhibits the migration in VSMCs, about 12% (p < 0.001, Fig. 4D).
AAMP and SRF are the targets of miR-125b for regulating the metastasis and proliferation in VSMCs.
Angio-associated migratory cell protein (AAMP) and serum response factor (SRF) are directly involved in cellular migration and proliferation, which plays pivotal role in ASO. So, we explore the inner relationship between the miR-125b and AAMP&SRF. Firstly, we use mimic-125-loaded liposomes or mimic-125b inhibitor-loaded liposomes (mimic-125bi-loaded liposomes) to make miR-125b up-or down-regulated cells. Just as Fig. 5 showing, Mimic-125b group holds more than 2-fold increasing of miR-125b when compares with NC group (p < 0.001). And the Mimic-125bi group almost doesn't have the expression of miR-125b, as comparing with NC group (p < 0.001, Fig. 5). Following, we test the protein level of AAMP and SRF after treatment of mimic-125b or mimic-125bi. Our results show that AAMP and SRF are signi cantly down-regulated in Mimic-125b group (AAMP: about 60%, SRF: 40%), while which are upregulated in Mimic-125bi group (AAMP: 1.25-fold, SRF: about 1.5-fold) ( Fig. 6A and B). However, in the IF assay, the mimic-125b-induced down-regulation of AAMP and SRF is not conspicuous, while the mimic-125bi-induced up-regulation of AAMP and SRF is signi cant. In summary, up-regulation of miR-125b inhibits the expression level of AAMP and SRF.

Discussion
Lower extremity arteriosclerotic occlusive (ASO) is induced by atherosclerotic thrombosis, which causes narrowing or occlusion of the lower extremity arteries. And the persistent ischemia eventually leads to tissue necrosis. Most people with ASO don't have intermittent claudication symptoms, and only 10 ~ 30% of which show these symptoms. In the preclinical stage of ASO, it's different to make early diagnosis through clinical routine screening. This makes misunderstanding that ASO is an uncommon disease in people under 55 years old. However, with the increasing of the age, the prevalence of ASO is dramatically increased, which attacks about 8-10% of people over 65 years old and about 20% of people over 80 years old [18]. Therefore, early diagnosis provides early treatment opportunities for relieving lower limb arterial ischemia caused by ASO.
Vascular smooth muscle cells (VSMCs) are the major cellular components that make up the middle layer of the arteries and are involved in the process of vascular diseases. At present, it is believed that the excessive proliferation and migration of VSMCs is one of the main causes of ASO, and it is also the main cause of restenosis after interventional treatment [5]. Recently, more and more evidences imply that microRNA can regulate the function of VSMCs, endothelial cells and macrophages, thereby affecting the pathogenesis and progression of ASO [19]. Previous studies have con rmed that miR-21, miR-143 and miR-221 are involved in the pathogenesis of ASO [11,12]. For example, miR-21 can regulate the proliferation and migration of VSMCs by targeting TPM1, thereby affecting the pathogenesis and progression of ASO. The deletion of miR-1298 is related to the CpG hypermethylation of ASO, and the down-regulation of miR-1298 regulates the proliferation and migration of VSMCs by directly targeting connexin-43, which plays a role in angiogenesis [20]. MiR-125b is one of the microRNAs which are the signi cantly down-regulated in ASO. Some research results suggest that miR-125b can inhibit the proliferation and migration of VSMCs, but the mechanism is unclear. In this study, we nd that upregulation of PDGF-BB in VSMCs can signi cantly inhibit the expression level of miR-125b (Fig. 1A, p < 0.001). At the same time, the decreasing of miR-125b leads to signi cant up-regulation of AAMP and SRF proteins in VSMCs (Fig. 6A-D). In the following experiments, we nd that VSMCs in NC group, with lower expression level of miR-125b, holds stronger migration and invasion ability, as comparing with the cells in experimental group (p < 0.05, Figs. 3 and 4). On the other hand, transfection of mimic-125 increases the level of miR-125b in VSMCs (p < 0.05, Fig. 1b). And the up-regulated protein level of miR-125b signi cantly restricts the growth and proliferation capability of VSMCs (about 50%, p < 0.001, Fig. 2). Meantime, the up-regulation of miR-125b signi cantly inhibits the migration and invasion of VSMCs (through trans-well assay and wound healing assay) (p < 0.05, Figs. 3 and 4). Furthermore, the upregulation of miR-125b signi cantly down-regulates the level of AAMP and SRF in VSMCs (Fig. 6).

Conclusion
Therefore, for the above data, we can conclude that miR-125b may inhibit the proliferation and migration of vascular smooth muscle cells by inhibiting the expression of AAMP and SRF. In other words, the decreasing of miR-125b may be an inducer of ASO. Therefore, observing the expression of miR-125b probably can provide evidence for predicting the occurrence of ASO in the preclinical stage. And the increasing of miR-125b may provide a therapeutic strategy for preventing the progression of ASO.     miR-125b is regulated by mimic-125b and mimic-125b inhibitor. 10A cells are seeded in 6-well plates for 24h, then treated with mimic-125b-loaded liposomes, mimic-NC-loaded liposomes or mimic-125b inhibitor (Mimic-125bi) for 24h, which followed by qT-PCR assay. Mimic-125b group shows about 2-fold up-regulation of miR-125b (p<0.001), while the Mimic-125bi group shows about 100% down-regulation of miR-125b (p<0.001). Same treatments are applied in 10A cells, which are followed by IF assay.