Effects And Mechanisms Of Vascular Adventitial Fibroblasts MicroRNA-21on Vascular Remodeling

BackgroundVascular remodeling is the pathological endpoint of diverse cardiovascular diseases such as coronary heart diseases, post-angioplasty restenosis, atherosclerosis, and hypertension. The previous studies on vascular remodeling focused on endothelial cells (ECs) and medial vascular smooth muscle cells (VSMCs), however,the role of adventitial cells in vascular remodeling did not receive attention. Increasing evidence suggests that the adventitia is a critical regulator of vessel wall function in health and disease. Many Studies show that vascular adventitial fibroblasts (AFs) contribute to vascular remodeling through activation, differentiation and intraluminal migration. Under pathological stimuli such as injury and cytokines, AFs, the principal cell type in the adventitia, are activated and undergo phenotypic changes, including proliferation and differentiation to myofibroblasts (MFs). The characteristic of differentiation is that AFs acquire α-smooth muscle actin (α-SMA) expression. Increased proliferative capacity and increased secretion of the extracellular matrix (ECM) proteins are key hallmarks of MFs. Transient appearance of MFs is a feature of normal wound repair. The balance in adventitial cells (including AFs and MFs) proliferation and apoptosis is decisive for the physiology of the vasculature. When the MF life cycle is not regulated properly, absence of apoptosis leads to overproduction of ECM proteins deposition and pathological vascular remodeling. Moreover, these processes are accompanied by adventitial cells migration toward the lumen, thereby contribute to neointimal formation. Neointima formation is an important feature of pathological vascular remodeling. Thus, uncontrolled cell proliferation and suppression of apoptosis are critical cellular events in vascular remodeling. Vascular AFs and MFs have become2potential target cells for anti-vascular remodeling therapeutics.As a new layer of gene regulation mechanism, microRNAs (miRNAs) have been implicated in a multitude of important cellular processes, including regulation of cellular proliferation, apoptosis and differentiation. The abnormal proliferation and apoptosis play an important role during tumor occurrence and development process. Therefore, most previous research into the role of miRNAs has focused on tumor.MiR-21has attracted more attention than any other miRNA. Multiple studies have identified miR-21overexpressed in a wide variety of cancer cell lines and causally linked to cancer-related processes such as proliferation and apoptosis. miR-21was reported to be strongly overexpressed in a wide variety of cancer cell lines. On the basis of these findings, recently, miR-21has been referred to as an "oncomir"(i.e., a miRNA with oncogenic properties) with pro-proliferative and anti-apoptotic functions. Interfere with mir-21expression or with its interaction with their target genes has been suggested as a potential anticancer therapeutic approach.Related to tumor, the research of miRNA in cardiovascular started later. In recent years, the function of miR-21in cardiovascular also attracts much attention gradually. Next to tumor, miR-21appears to be strongly deregulated in cardiovascular disease. Under some cardiovascular disease conditions such as proliferative vascular disease, vascular walls balloon injury, cardiac hypertrophy and ischemic heart disease, the mir-21expression is deregulated. It was reported that miR-21was strongly overexpressed in vascular walls after balloon angioplasty of the rat carotid artery. MiR-21is expressed in VSMCs and regulates proliferation and apoptosis. MiR-21is also expressed in vascular ECs. It was reported that shear stress forces regulate the expression of miR-21in ECs, which influencing endothelial biology by decreasing apoptosis. However, the research of miRNA in vascular adventitia area is little at present. It is unclear whether miR-21is expressed in AFs and MFs and whether play a role in the proliferation and apoptosis of the2kinds cells.Numerous studies have demonstrated some characteristics of proliferative vascular disease are similar to those in tumor in cellular events and molecular pathways. Because proliferation and apoptosis are2important cellular events in vascular remodeling and tumor, miRNAs may play important roles in vascular remodeling.In recent years, many studies suggest that the intervention measures which treat vascular adventitial cells as therapeutic targets can improve vascular remodeling. A study showed that estrogen may attenuate the injury response in balloon injury model by inhibiting proliferation of adventitial cells and their contribution to neointima formation. A study showed that smad7overexpression through gene transfer of smad7in the adventitial surface antagonized the TGF-β1signaling pathway and attenuated vascular remodeling and contribution of AFs to neointima formation after balloon angioplasty. Therefore, perivascular gene transfer which targets vascular adventitial cells represents a novel therapeutic strategy of vascular disease.Taken together, We put forward the hypothesis:miR-21was endogenously expressed in AFs and MFs; miR-21play an important role in regulating proliferation and apoptosis of AFs and MFs; moudulting miR-21expression could regulation proliferation and apoptosis of vascular AFs and MFs and further improve balloon injury-induced rat iliac artery remodeling.Objectives1. To established the cell model of MFs and determine the miR-21expression in AFs and MFs.2. To investigate the effects of miR-21modulation on proliferation and apoptosis of AFs and MFs.3. To investigate the effects of miR-21inhibition on neointima formation after iliac artery balloon injury.Materials 1. Cell cultureAFs were isolated from thoracic aortas of4-to6-week-old male Wistar-Kyoto rats weighing120to180g and cultured by the tissue adherence method. Cells from passage3to6were used.2. Grouping in vitro experiment2.1RT-PCR and Western blot were used to inspect the effection of TGF-β1on AF differentiation. In RT-PCR experiment, cell were grouped into6groups depending different time (0、6、12、24、36、48hours) or6groups at different concentrations (0、1、5、10、20、30ng/ml). In Western blot experiment, cell were grouped into4groups according to different time (0、12、24、36hours).2.2To determine whether the expression of miR-21in AFs and MFs was regulated, we used both gain-and loss-of-function approaches. Cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.2.3To determine the proliferation and apoptosis, cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.3. Myofibroblast modelSubconfluent AFs were incubated with different concentrations of TGF-β1in serum-free medium. The MF phenotype in TGF-β1-treated fibroblasts was identified by determining the expression of a-SMA by RT-PCR, Western blot analysis and fluorescent immunocytochemistry.4. Fluorescent immunocytochemistryFibroblasts with or without TGF-β1treatment were grown on glass coverslips, rinsed with PBS, fixed with3%paraformaldehyde, permeabilized with0.5%Triton-X100and blocked with2%BSA. Cells were incubated with the primary antibodies mouse anti-a-SMA and rabbit anti-vimentin. Immunofluorescent staining for a-SMA and vimentin involved incubation with FITC-conjugated anti-mouse IgG and TRITC-conjugated anti-rabbit IgG, respectively. Cellular nuclei were stained with DAPI. Images were examined under confocal microscope. 5. miRNA transfectionThe oligonucleotide was transfected into cells by use of Lipofectamine2000reagentas per the manufacturer’s instructions. Cells were transfected with pre-miR-21and miR-21inhibitor for miR-21overexpression and knockdown, respectively. The samples were assayed after48h. The transfection efficiency was determined by use of the negative control oligonucleotide with6-FAM at the5’end. After optimization, transfection complexes were added to cells at a final oligonucleotide concentration of50nM.6. Real-time quantitative RT-PCRTotal RNA was isolated for miR-21and a-SMA from cultured cells48h post-transfection with use of the mirVanaTM miRNA Isolation Kit and Trizol according to the manufacturer’s protocol. cDNA synthesis involved carried out in a thermal cycler. PCR reactions for miRNA and mRNA involved the ABI Prism7500Sequence Detection System and iQ5Real-Time PCR Detection System, respectively. Real-time RT-PCR analyses of miRNA expression involved use of TaqMan miRNA assays. RT-PCR analyses of the mRNA expression of a-SMA and (3-actin involved PrimeScript(?) RT-PCR kits. Analysis of relative miRNA and mRNA expression involved the△△CT method, with U6and β-actin used as endogenous controls, respectively.7. Western blot analysisCells were harvested with lysis buffer. Protein concentration was determined using the BCA method. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membrane, and blocked with5%nonfat milk/TBST and then incubated with primary antibodies. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG. Immunoreactive bands were visualized by ECL. The relative intensities of the bands were analyzed using P-actin as an internal control.8. Establishment of rat iliac artery balloon injury modelIliac artery balloon injury was performed according to Gabeler et al. Briefly, Wistar rats (320-350g) were generally anesthetized with sodium pentobarbital. A2F Fogarty catheter was inserted to the right iliac artery. To produce iliac artery injury, the balloon was inflated to3-4atm and withdrawn three times. The left iliac arteries were not injured and served as controls.9. Adventitial oligo transfer and animal grouping200βl of20%(w/v) F-127pluronic gel (Sigma) containing0.24mg antagomir against miR-21(miR-21antagomir) or PBS was applied to the adventitial around injured artery segments. Animals were divided into three groups:miR-21antagomir group (treated with miR-21antagomir dissolved in F-127pluronic gel, n=15), vehicle group (treated with PBS dissolved in F-127pluronic gel, n=20), and uninjured group (n=20). Animals of vehicle group were sacrificed at1days,3days, and21days after injury. Animals of miR-21antagomir group were sacrificed at3days and21days after injury. The vasclular sample were used to performe proliferation assay (3days after injury, n=5), apoptosis assay (1and3days after injury, n=5) and HE staining (21days after injury, n=5)10. Proliferation assayCell proliferation in vitro was determined by EdU. After48h of transfection, cell proliferation was quantified by the uptake of EdU into DNA by use of a Cell-LightTM EdU DNA Cell Proliferation Kit. All procedures for EdU incorporation experiments followed the kit’s protocol. Cell proliferation rate was calculated as the percentage of EdU-positive nuclei to total nuclei in high-power fields.To detect AF and MF proliferation in vivo, BrdU dissolved in PBS, was injected12and24hours before euthanasia. BrdU-incorporating nuclei were detected with a specific anti-BrdU antibody according to the manufacturer’s instructions. Cell proliferation rate was calculated as the percentage of BrdU-positive nuclei to total nuclei in high-power fields.11.Apoptosis assayThe level of apoptosis in cultured cells was evaluated by FACS of annexin-V-stained cells with use of the Annexin V Apoptosis Detection Kit and measurement of Caspase-3activity by use of the Caspase-3Colorimetric Assay Kit.Detection of apoptotic cells in vivo was performed by the TUNEL procedure with the In Situ Cell Death Detection Kit. Cell apoptotic rate was calculated as the percentage of TUNEL-positive nuclei to total nuclei in high-power field.12. ImmunocytochemistryTo detect AF differentiation in vivo, immunohistochemistry was performed in paraffin-embedded vessel sections. The expression of a-SMA was detected by immunohistochemistry SABC method.13. Morphometric analysisVascular remodeling was evaluated using morphometric analysis. Neointimal formation is expressed as the neointimal to medial area ratio (N/M). Morphometric analysis of N/M and lumen size was performed in sections stained with hematoxylin-eosin (H-E) using the Image Pro Plus computer software.14. Statistical analysisAll data are presented as mean±SEM from at least3independent experiments. Differences between2groups were analyzed by Student’s t test and between multiple groups by one-way ANOVA. Data analysis involved use of GraphPad Prism5.0. Differences were considered statistically significant at P<0.05.Results1. Differentiation of AFs to MFsRT-PCR and Western blot revealed that TGF-β1induced a time-and dose-dependent steady increase in a-SMA mRNA and protein expression expression in cultured AFs. Immunoblotting revealed a-SMA protein expression in AFs significantly increased with TGF-β1treatment (10ng/ml,24h). Double immunofluorescent staining revealed that AFs with TGF-β1(10ng/ml,24h) treatment co-expressed vimentin and a-SMA and formed numerous actin stress fibers, characteristic of MFs.2. MiR-21was endogenously expressed in AFs and markedly increased in MFsWe used real-time RT-PCR (TaqMan probes) to detect and quantify miRNAs expression of miR-21. MiR-21was found endogenously expressed in AFs and markedly increased in MFs.3. Modulating miR-21expression in AFs and MFs To determine whether the expression of miR-21in AFs and MFs was regulated, we used both gain-and loss-of-function approaches. The ectopic expression of miR-21was confirmed by real-time quantitative RT-PCR. Pre-miR-21significantly increased but miR-21inhibition decreased miR-21expression. Control oligos had no effect on miR-21expression. The results suggest that miR-21could be modulated both in AFs and MFs4. The effects of miR-21on proliferation of AFs and MFsTo determine the role of miR-21in the proliferation of AFs and MFs, EdU incorporation assay was used. MiR-21inhibitor decreased EdU incorporation while pre-miR-21significantly increased EdU incorporation in AFs and MFs, which indicated that miR-21promoted the proliferation of AFs and MFs. Moreover, MFs showed higher proliferation compared with AFs both at basal condition and with pre-miR-21treatment.5. The effects of miR-21on apoptosis of AFs and MFsWe further evaluated the effects of miR-21on the apoptosis of AFs and MFs by Annexin-V-staining and Caspase-3activity. Transfection with the miR-21inhibitor increased the percentage of Annexin V positive cells and Caspase-3enzymatic activities. However, pre-miR-21decreased and Caspase-3enzymatic activities. These results suggest that miR-21plays an anti-apoptosis role in AFs and MFs.6. The effects of antagonist of miR-21on proliferation and apoptosis of AFs and MFs in injured arteryImmunocytochemistry revealed that a-SMA positive cells were visible in the adventitia3days after balloon injury, which indicated some AFs differentiation into MFs.3days after injury, the adventitia cells in the injured arteries treated with vehicle showed higher proliferation rate compared with the uninjured arteries, however, treatment with miR-21antagomir led to a significant reduction in proliferation rate compared with vehicle group.TUNEL revealed that rare positive cells were detectable in the adventitia from uninjured vessels and many apoptotic cells could be seen in the adventitia from vehicle group at1day after injury. At3days after injury, numbers of apoptotic cells in adventitia from vehicle group were significantly reduced and showed no significant differences compared with uninjured group. However, the apoptosis level were significantly increased in miR-21antagomir group compared with the uninjured group and vehicle group (3days after injury).7. The effects of antagonist of miR-21on vascular remodeling induced by balloon injuryTo determine the effect of miR-21inhibition on vascular remodeling, morphometric analyses were carried out on the21days balloon-injured arteries. The injured arteries treated with vehicle showed extensive neointima formation and dramatic reduction in lumen size compared with the uninjured arteries. N/M (Neointimal/Medial area ratio) of the miR-21antagomir group was significantly lower than that of the vehicle group, while lumen size of the miR-21antagomir group was significantly larger than that of the vehicle group.Conclusions1. miR-21expressed in AFs and overexpressed in MFs dedifferentiated from AFs after treatment with TGF-β1.2. miR-21is an important regulator in proliferation and apoptosis of AFs and MFs.3. Antagonist of miR-21could improve vascular remodeling induced by balloon injury through regulating the proliferation and apoptosis of AFs and MFs. BackgroundRecent studies show that vascular adventitial fibroblasts (AFs), the most main cell components of adventitia, are the the important pathologic basis of neointima formation and pathological vascular remodeling after vascular injury. When blood vessels suffere the pathological injury or stimulation from both inside and outside, AFs will be "activated". The "activated" AFs show higher proliferative activity, and at the same time undergo phenotypic conversion into myofibroblasts (MFs). MFs display stronger ability in proliferation, synthesis of extracellular matrix (ECM) and migration. A part of the MFs will migrate to vascular intima and secrete ECM, which actively involved in neointimal formation. Therefore, AF and MF are two important therapeutic targets for cardiovascular disease.The balance in proliferation and apoptosis of AFs and MFs is decisive for the physiology of the vasculature. In vascular proliferative diseases, the balance is disturbed, which causes AFs and MFs excessive proliferation and apoptosis defects, excessive collagen secretion, deposition of ECM, finally results in adverse vascular remodeling. Therefore, to clarify the mechanism of regulating the proliferation and apoptosis of the2type cells has important significance for the prevention and improvement of adverse vascular remodeling.MicroRNA (miRNA) is endogenous small RNA molecule that plays important role in gene regulation. The recent studies show that miRNA is widely involved in multiple cell biological processes, including cell proliferation, apoptosis and differentiation. Among the miRNAs which have been found and identified, microRNA-21(miR-21) has attracted much attention. Much research showed that miR-21expression was up-regulated in a wide variety of tumor, which were closely related with tumor occurrence and development. Our study showed that miR-21was also expressed in vascular adventitia cells and played an important role in regulating proliferation and apoptosis of AFs and MFs. We found that antagonist of miR-21attenuated balloon injury-induced neointimal formation by regulating proliferation and apoptosis of AFs and MFs. However, the mechanism of miR-21regulation proliferation and apoptosis of AFs and MFs remains unclear.MiRNA exert their functions mainly through regulating target genes. PDCD4is a new kind of tumor suppressor gene found in recent years. Recent research shows that PDCD4plays an important role in the regulation of cell proliferation and apoptosis, which are closely related whith tumor development. Until now, there are no research reports about PDCD4in vascular adventitia. PDCD4is confirmed as miR-21target genes and played an important role in regulating of cell proliferation, apoptosis in a variety of tumor cells. The expression and gene regulation of miRNA and its target genes exhibit tissue specificity, therefore, whether PDCD4is a target gene of miR-21in the AFs and MFs still need to be identificated. The JNK/c-Jun signaling pathway is closely related to the cell growth. There is little study on the relationship between PDCD4and JNK pathway. Studies have reported PDCD4can interfere with JNK mediated phosphorylation of c-Jun and inhibit tumor transformation through inhibition of c-Jun activation and subsequent AP-1dependent transcription. However, at present, whether miR-21can regulat JNK pathway and PDCD4is involved in the regulation process are still not reported.In view of the above problems, we propose the following hypothesis:in vascular AFs and MFs, PDCD4is a target gene of miR-21; PDCD4can effectively regulate AFs and MFs proliferation and apoptosis, and directly involved in miR-21-mediated cell events; JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.Objectives1. To verify whether PDCD4is a direct target of miR-21in AFs and MFs.2. To investigate the effects of PDCD4on proliferation and apoptosis of AFs and MFs.3. To investigate whether PDCD4participates in miR-21-mediated proliferation and apoptosis in AFs and MFs.4. To investigate the effects of miR-21/PDCD4on JNK activity and determine the role of JNK/c-Jun pathway in miR-21-mediated cellular effect in AFs and MFs.Methods1. AFs culture and establishment of myofibroblast model AFs were isolated from thoracic aortas of4-to6-week-old male Wistar-Kyoto rats weighing120to180g and cultured by the tissue adherence method. Cells from passage3to6were used.MF differentiation was induced with TGF-(31and identified by determining the expression of a-SMA by RT-PCR, Western blot analysis and fluorescent immunocytochemistry.2. Experimental grouping2.1The dual-luciferase reporter assay was performed to confirm that miR-21can directly bind to PDCD4. Cells were grouped into2groups:luciferase vector group (pMIR-REPORT luciferase plasmids) and vehicle vector group. Each group was further divided into3subgroups:vehicle control group, pre miR control group and pre-miR-21group.2.2To investigate whether PDCD4is regulated by miR-21, Western blot analysis was performed to detect PDCD4protein expression after miR-21overexpression and miR-21inhibition. Cells were grouped into5groups:vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.2.3To determine the proliferation and apoptosis, cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, PDCD4siRNA group, miR-21inhibitor+PDCD4siRNA group.2.4To investigate the effection of miR-21overexpression on JNK activity, cells were grouped into3groups:vehicle control group, pre miR control group and pre-miR-21group.2.5To investigate the effection of miR-21inhibition on JNK activity and whether PDCD4participates in the regulation of miR-21on JNK activity, cells were grouped into4groups:vehicle control group, miR inhibitor control group, miR-21inhibitor group, miR-21inhibitor+PDCD4siRNA group.3. miRNA and siRNA transfectionThe oligonucleotide was transfected into cells by use of Lipofectamine2000reagentas per the manufacturer’s instructions. Cells were transfected with pre-miR-21and miR-21inhibitor for miR-21overexpression and knockdown, respectively. Transfection complexes were added to cells at a final oligonucleotide concentration of50nM. For PDCD4knockdown, PDCD4siRNA was transfected into cells by use of lipofectamine2000reagent. The final PDCD4siRNA concentration was80nM.4. Proliferation assayCell proliferation was determined by EdU proliferation assay. After48h of transfection, cell proliferation was quantified by the uptake of EdU into DNA by use of EdU DNA Cell Proliferation Kit. All procedures for EdU incorporation experiments followed the kit’s protocol. Cell proliferation rate was calculated as the percentage of EdU-positive nuclei to total nuclei in high-power fields.5. Apoptosis assayThe level of apoptosis in cultured cells was evaluated by measurement of Caspase-3activity by use of the Caspase-3Colorimetric Assay Kit. All procedures followed the kit’s protocol.6. Western blot analysisCells were harvested with lysis buffer. Protein concentration was determined using the BCA method. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membrane, and blocked with5%nonfat milk/TBST and then incubated with primary antibodies. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG. Immunoreactive bands were visualized by ECL. The relative intensities of the bands were analyzed using β-actin as an internal control.7. Dual-luciferase reporter assayTo confirm that miR-21can directly bind to PDCD4, the dual-luciferase reporter assay was performed, we cloned a construct with a fragment of the3’-UTR of PDCD4mRNA with the putative miR-21binding sequence into pMIR-REPORT luciferase plasmids (luciferase reporter vector) for co-transfection with pre-miR-21into293T cells. The vector without the putative miR-21binding sequence (vehicle vector) was the negative control. The samples were co-transfected with the Renilla luciferase-expressing plasmid pRL-TK (internal control plasmid). At24h after transfection, firefly and renilla luciferase activities were measured by use of the dual-luciferase reporter assay system on a luminometer according to the manufacturer’s instructions.8. Statistical analysisAll data are presented as mean±SEM from at least3independent experiments. Differences between2groups were analyzed by Student’s t test and between multiple groups by one-way ANOVA. Data analysis involved use of GraphPad Prism5.0. Differences were considered statistically significant at P<0.05.Results1. PDCD4is a direct target of miR-21in AFs and MFs1.1The expression level of PDCD4in AFs and MFsWestern blot analysis revealed PDCD4protein expression level is significantly lower in MFs than in AFs. However, miR-21expression level markedly increased in MFs compared with AFs. The inverse correlation between miR-21and Pdcd4protein suggests that PDCD4may be a target of miR-21in AFs and MFs. 1.2Dual-luciferase reporter assayTo confirm that miR-21can directly bind to PDCD4, the dual-luciferase reporter assay was performed. Our results indicated that pre-miR-21significantly inhibited luciferase activity as compared with control treatments. Therefore, miR-21could directly bind to PDCD4.1.3The miR-21regulation on PDCD4protein expressionTo investigate whether PDCD4is regulated by miR-21, western blot analysis was performed after miR-21overexpression and miR-21inhibition. Our results indicated that miR-21inhibitor increased but pre-miR-21decreased PDCD4protein level in AFs and MFs.Therefore, the above study results proved that PDCD4is a direct target gene of miR-21in AFs and MFs.2. The effects of PDCD4inhibition on miR-21-mediated proliferation in AFs and MFsKnockdown of PDCD4by siRNA significantly increased proliferation in AFs and MFs as determined by EDU proliferation assay. miR-21inhibitor decreased the proliferation of AFs and MFs, however, PDCD4siRNA partly rescued the reduced cellular proliferation with miR-21inhibition. Our data showed that miR-21inhibitor+PDCD4siRNA group demonstrated higher proliferation activity compared with miR-21inhibitor group. The results suggest PDCD4not only plays an essential role in regulaing proliferation of AFs and MFs, but also directly participates in miR-21-mediated proliferation in AFs and MFs3. The effects of PDCD4inhibition on miR-21-mediated apoptosis in AFs and MFsKnockdown of PDCD4by siRNA decreased apoptosis as determined by caspase-3activity. MiR-21inhibitor increased the caspase-3activity of AFs and MFs, however, PDCD4siRNA partly alleviated the increase in apoptosis with mir-21inhibition. Our data showed that miR-21inhibitor+PDCD4siRNA group demonstrated a significantly lower proliferation activity compared with miR-21inhibitor group. The results suggest PDCD4is a regulator of apoptosis in AFs and MFs and directly participates in miR-21-mediated apoptosis in AFs and MFs.4. The effects of PDCD4inhibition on JNK activityWstern blot analysis demonstrated that knockdown of PDCD4by siRNA significantly increased the JNK/c-Jun activity, the of p-JNK and p-c-Jun protein showed higher expression level compared with control siRNA.5. The effects of miR-21on JNK activityMiR-21inhibitor increased PDCD4expression in AFs and MFs, while decreased the level of p-JNK and p-c-Jun. In contrast, pre-miR-21decreased PDCD4expression, while increased the level of p-JNK and p-c-Jun. The results suggested that miR-21could enhance JNK activity while miR-21inhition could decrease JNK activity.6. The effects of PDCD4inhibition on the regulation of miR-21in JNK activityMiR-21inhibition decreased JNK activity in AFs and MFs, however, miR-21inhibitor+PDCD4siRNA group demonstrated a significantly higher JNK activity compared with miR-21inhibitor group. The results suggested that PDCD4not only affected JNK activity, but also affected the regulation of miR-21on JNK activity. It also suggests that JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.Conclusions1. PDCD4is a direct target of miR-21in AFs and MFs.2. PDCD4could regulate proliferation and apoptosis of AFs and MFs, and directly participates in miR-21-mediated cellular effect.3. JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.