| BackgroundThe blood vessels are constantly subjected to mechanical forces in the form of shear stress and cyclic stretch due to pulsatile blood flow. Shear stress is mainly sensed by endothelial cells (ECs) lining the inner of the vessel wall, however, cyclic stretch can affect all cell types of the vessel wall. Vascular smooth muscle cells (VSMCs), in the media of the vessel wall, are the main target cells of mechanical stretch. A large number of evidence demonstrates that mechanical forces play critical roles in vascular development and equilibrium.Multiple in vitro and in vivo studies indicate that mechanical stretch almost affects all celluar functions of VSMCs, including proliferation, apoptosis, differentiation and migration. Generally, under normal conditions, moderate cyclic stretch (9%～12%elongation in human aorta) suppress the proliferation and apoptosis of VSMCs, maintaining the vessel wall structure and normal functions, however, in some pathological conditions, as occurred in hypertension and atherosclerosis, elevated mechanical stretch (>15%elongation)promote the proliferation, migration, apoptosis and extracellular matrix deposition of VSMCs, resulting in pathological vascular remodeling. In recent years, several biomechanical sensors such as receptors or ion channels on the surface of membrane, signaling pathways (MAPK, PI3K/Akt, et al.)and transcription factor (AP-1, NF-κB, Stat3, et al.) are found to be involved in biomechanical transduction, but the detail mechanisms underling the stretch mediated biological effects of VSMCs are still unclear because of the complex regulating network.microRNAs (miRNAs) are an class of endogenous small non-coding RNAs that negatively regulating gene expression at post-transcriptional level by directly degrading target mRNA or translation repression. Because miRNAs bind to their target mRNA3’UTR as either a perfect or imperfect manner, one miRNA could regulate multiple target genes and one mRNA also could be regulated by several miRNAs. To date, over1000miRNAs have been indemnified in human genome, and these miRNAs may target30%%～60%total genes. The deregulation of miRNAs plays very important roles in the pathogenesis of several cardiovascular disorders. In addition, recent studies demonstrate that miRNAs were also implicated in mechanical forces mediated cellular functions in cultured vascular cells. Compared to static controls, shear stress induced significant changes of several miRNAs expression in cultured ECs, and miR-19a, miR-23b were found to be involved in the progression of cell cycle; miR-92a played a critical role in regulating the production of nitric oxide (NO) via its target gene KLF2. Nevertheless, the effects of miRNAs on mechanical stretch mediated cellular functions of VSMCs are still not clear.MiR-21is a relatively well understood miRNA and considered as an onco-miRNA, its expression was found increased in many solid tumors with characteristics of promoting cell survival and migration. Besides, the expression of miR-21is abundant in vascular vessel walls. A vivo study revealed that the expression of miR-21was increased in balloon-injured carotid artery, and knockdown miR-21expression could attenuate the neointimal lesions via suppressing the VSMCs proliferation. Recently, several studies indicated miR-21was also a shear stress sensitive miRNA. Athero-protective laminar shear stress enhanced miR-21expression which was contribute to the NO production and ECs apoptosis, disturbed shear stress also induced sustained miR-21expression which was involved in endothelial inflammation.In light of the critical roles of mechanical stretch on VSMCs proliferation and apoptosis, we speculate that miR-21may be also sensitive to stretch stimuli and play an important role in stretch mediated VSMCs proliferation and apoptosis. In the past decade, most studies as to miRNA mainly focused on the biological effects of miRNA, however, little is known about the regulatory mechanisms of miRNA expression. In the present study, we also explore the molecular mechanisms of stretch induced miR-21expression.Objectives1. To observe the effects of mechanical stretch on miR-21expression in cultured human aortic smooth muscle cells.2. To clarify the role of miR-21in mechanical stretch mediated proliferation and apoptosis of human aortic smooth muscle cells.3. To explore the molecular mechanism of mechanical stretch induced miR-21expression.Materials and Methods1. Cell CultureHuman aortic smooth muscle cells (HASMCs) were obtained from ScienCell. When cells reached confluence, removed the medium and washed with PBS once, and then added pre-heated0.25%Trypsin to incubate1min at37℃, after that, removed the Trypsin and added fresh complete medium to collect cells. At last, transfer cells to a new cell culture bottle and maintain at37℃, in an incubator with5%CO2.2. Mechanical Stretch ApplicationPassage4-7HASMCs were seeded into Flexcell6-well plates coated with collagen I, when they reached80%～90%confluence, serum-free medium was replaced to induce quiescence for24hours, and then, fresh serum-free or complete medium was substituted, different levels of mechanical stretch was applied to HASMCs by use of a computer-controlled Flexcell5000-Tension system. The cultured HASMCs were divided into the following two groups:(1)10%cyclic stretch:HASMCs were exposed to cyclic stretch (10%elongation,1Hz)for0、3、6、12、24hours. (2)16%cyclic stretch:HASMCs were exposed to cyclic stretch (16%elongation,1Hz) for0、3、6、12、24hours.3. Real time quantitative PCR for miR-21After being stretched for indicated time, HASMCs were harvested and total RNA including miRNAs was extracted by use of TRIzol (invitrogen). The miR-21expression level was determined by real time quantitative PCR using miRNA universal cDNA synthesis kit and SYBR Green Mix kit (Exiqon) according to the manufacturers’instruction. Primers of rRNA U6and miR-21were obtained form Exiqon, and U6as a internal reference for miR-21expression.4. Transient TransfectionFor miR-21over-expression and inhibition, miR-21mimics and inhibitor were transfected into cultured HASMCs by use of Lipo fectamine2000at different concentrations. MiR-21mimics and inhibitor were purchased form GenePharm. For over-expression of PDCD4, plasmid encoding PDCD4gene which was gifted from professor Zhang LN(medical college of Shandong university) was transfected, and plasmid-GFP was used as a negative control.5. Cell Proliferation AnalysisAfter being stretched for12hours, HASMCs proliferation was determined by BrdU incorporation assay and cell count method, respectively. The detailed procedure was according to the manufactures’protocol.6. Western blot analysisHASMCs being stretched or maintained in static conditions were harvested, and protein was extracted and protein concentrations were determined by BCA method.15～20ug protein was separated by SDS-PAGE, and transferred to PVDF membrane, which was blocked by5%nonfat milk for1hour, and then following by incubating with primary antibody overnight, incubating with second antibody for1hour, washing with1×TBST three times. Visualization was performed by using an enhanced chemiluminscence-plus detection system. Relative protein band intensities were analyzed by use of Photoshop CS3software and β-actin was used as an internal reference. 7. FACS analysisWe used the AnnexinV-FITC apoptosis detection kit which was obtained from BIPEC to detect the apoptotic cells. HASMCs being stretched or in static conditions were harvested and every sample was re-suspended with400ul binding buffer, following with15-min AnnexinV-FITC staining and5-min PI labeling, and then were analyzed by FACS in1hour,10000cells were counted in each assay.8. Real time quantitative PCR for pri-miR-21, pre-miR-21, and PDCD4Total RNA was extracted from HASMCs by use of TRIzol and transcribed to cDNA using TAKARA cDNA synthesis kit. Real time quantitative PCR was performed using TAKARA SYBR Green Mix kit according to the manufactures’ instructions. Primers for pri-miR-21, pre-miR-21, PDCD4and β-actin were synthesized by GenePharm. β-actin was as an internal reference for pri/pre-miR-21, PDCD4mRNA levels.9. Immunofluorescence StainingHASMCs were exposed to16%stretch for3hours, and then were fixed with Immunol Staining solution for30min. After incubating with0.1%Triton X-100for8min and blocked with5%BCA for30min, HASMCs were incubated with primary antibody overnight at4℃. After washing with PBS3times, HASMCs were incubated with Fluor second antibody for1hour, and then DAPI for3min. Images were obtained by use of laser confocal microscopy.10. Electrophoretic Mobility Shift Assay (EMSA)HASMCs were subjected to16%stretch for indicated time points. Nuclear protein was extracted by use of a Pierce nuclear protein extraction kit and protein concentrations were determined by BCA method.8ug nuclear protein were mixed with biotin-labeled oligo for AP-1or NF-κB and other components in a total volume of20ul for30min, and then, separated by4%nondenaturing acrylamide PAGE. The detailed procedures followed the manufactures’instructions.11. Statistical AnalysisAll data are shown as mean±SEM, statistical significance was determined by one-way ANOVA or two-tail unpaired Student’s/test, P<0.05was considered statistically significant.Results1. The effects of mechanical stretch on miR-21expression in cultured HASMCsOur results demonstrated that elevated stretch (16%elongation,1Hz) increased miR-21expression significantly compared to static controls in the absence of serum, the peak was at12hour and was maintained at24hour (p<0.01); however, the moderate stretch (10%elongation,1Hz) had no effect on miR-21expression in the absence of serum (p>0.05). With serum,16%stretch still increased miR-21expression compared to static controls (p<0.05), but the effect was moderate. With interesting,10%stretch significantly suppressed miR-21expression compared to static controls in the presence of serum as early as6hour, sustained for24hour (p<0.01).2. The role of miR-21in stretch mediated proliferation of cultured HASMCsBrdU incorporation assay and cell counting method were used to evaluate the proliferation of HASMCs. Our results indicated that16%stretch notably increased proliferation of cultured HASMCs compared to static controls in the absence of serum (p<0.01). Transfaction of miR-21inhibitor decreased miR-21expression42-fold compared to miRNA NC inhibitor (p<0.01), and miR-21inhibitor significantly suppressed16%stretch induced proliferation of HASMCs (p<0.01). Under the same conditions, results from cell counting were in accordance with that of BrdU incorporation assay. In the presence of serum,10%stretch suppressed serum-induced proliferation of HASMCs (p<0.01). Transfaction of miR-21mimics increased miR-21expression70-fold compared to miRNA NC mimics (p<0.01), and miR-21mimics significantly reduced the suppression effect of10%stretch on HASMCs proliferation (p<0.01).Western blot indicated that16%stretch reduced p27expression and increased p-Rb level compared to static controls (p<0.01). MiR-21inhibitor could partially reverse the effect of16%stretch on p27and p-Rb expression (p<0.01).3. The role of miR-21in stretch induced apoptosis of cultured HASMCs In the absence of serum,16%stretch increased apoptosis of cultured HASMCs, as compared with the static controls (p<0.01). When inhibition of miR-21,16%stretch induced apoptosis was further increased as compared with the NC inhibitor (p<0.01). We also investigated the effect of10%stretch on apoptosis of cultured HASMCs in the presence of serum, FACS analysis showed there was no difference between the stretched HASMCs and static controls (p>0.05).4. PDCD4is involved in stretch induced apoptosis of cultured HASMCsResults of real time PCR demonstrated that16%stretch significantly increased PDCD4mRNA expression (1.98-fold, p<0.05) in cultured HASMCs, but Western blot results indicated that PDCD4protein level was decreased by16%stretch as compared with static controls (0.55-fold, p<0.05). With inhibition of miR-21, the16%stretch induced PDCD4protein expression suppression was partially reversed (p<0.05).In order to evaluate the role of PDCD4in stretch induced HASMCs apoptosis, plasmid encoding PDCD4was transfected into HASMCs, and16%stretch induced apoptosis was greater than that of plasmid-GFP (p<0.01).5. The effect of16%stretch on AP-1and NF-κB activity in cultured HASMCsResults of real time PCR showed16%stretch increased pri-miR-21(1.73-fold, p<0.05) and pre--miR-21(2.85-fold, p<0.01) expression in cultured HASMCs, respectively. Western blot indicated16%stretch increased phosphor-NF-KB p65level and phosphor-c-jun as well as c-jun expression in nuclear. However, the c-fos expression was not affected by stretch. Immunofluorescence staining revealed that c-jun but not c-fos expression was increased in nucleus, c-fos expression was increased mainly in cytoplasm. Results of EMSA also indicated that the DNA binding activities of AP-1and NF-κB were enhanced by16%stretch as early as1hour and still maintained at3hour (p<0.01).6. AP-1(c-jun) was implicated in16%stretch induced miR-21expression in cultured HASMCsWe pretreated HASMCs with SN50and c-jun siRNA to inhibit16%stretch induced NF-κB and AP-1DNA binding activities, real time PCR indicated that c-jun significantly attenuated the16%stretch induced miR-21expression (p<0.01), but SN50had no effect on miR-21expression in stretched HASMCs (p>0.01).Conclusions1.16%stretch upregulated miR-21expression in cultured HASMCs, but in the presence of serum,10%stretch suppressed miR-21expression.2. miR-21was involved in mechanical stretch mediated proliferation and apoptosis in cultured HASMCs.3.16%stretch induced miR-21expression though promoting AP-1(c-jun) DNA binding activity. BackgroundSmooth muscle cells (SMCs) play a major role in maintaining normal vascular structure and functions, but they are not terminally differentiated cells as cardiac cells. Differentiated mature SMCs rarely proliferate and express a variety of SMC-specific contractile protein markers, including SM22a, Calponin, SMa-actin. In response to vessel injury, SMCs undergo significant phenotypic modulation, from a Differentiated mature state to a dedifferentiated immature state with characters of increased proliferation rate, migration and extracellular matrix synthesis, decreased expression of SMCs differentiation marker. The phenotypic modulation of SMCs plays a critical role in several vascular disorders, such as hypertension, atherosclerosis, and aneurysm. The molecular mechanisms of SMCs phenotypic modulation are partially understood, but the detailed mechanisms remain elusive.Modulation of SMCs phenotype could occur at different regulating layers of gene expression. Recently, microRNAs (miRNAs) were reported to be involved in regulation of SMCs phenotype, miRNAs are a class of endogenous, small non-coding RNA, negatively regulate gene expression by directly degrading mRNA or translation inhibition. There is abundant miRNAs expression in vascular walls, almost participating in all cellular functions of vascular cells. The vascular vessels are constantly exposed to mechanical forces in the form of shear stress and stretch because of blood flow and blood pressure. Several recent researches revealed that the miRNAs expression profile in cultured ECs was significantly altered by shear stress, and miR-21,-19a,-23b,-92a were involved in shear stress mediated apoptosis, cell cycle, NO production, inflammation. In our previous study, we also found miR-21expression in cultured human aortic smooth muscle cells (HASMCs) was altered by cyclic stretch, implicating in proliferation and apoptosis. However, the roles of miRNAs in cyclic stretch induced other cellular functions in cultured SMCs are not clear. In the present study, we performed miRNA array to observe the altered miRNAs expression by stretch in cultured HASMCs, and several miRNAs expression were altered in stretched cells including miR-145, miR-21, miR-221, which were closely associated with SMCs phenotypic modulation.SMCs are the main target cells of cyclic stretch, and the latter also plays an important role in SMCs phenotypic modulation, but the results of the effects of stretch on SMCs phenotype in vitro were not consistent. The origin of SMCs, magnitude of stretch and the extracellular matrix used in experiments may contribute to the discrepancy, and our results indicated that elevated stretch (16%elongation,1Hz) decreased mature SMC-specific markers expression, promoting SMC phenotype transition form a differentiated state to a dedifferentiated state. Several in vitro and in vivo studies demonstrated that miRNAs, especially miR-143/145were critical regulators of SMC phenotype, however, the role of miRNA in stretch modulated SMCs phenotype transition was unclear until now. In this study, we will focus on the roles of screening out miRNAs in HASMCs phenotypic modulation. Besides, compared to our knowledge of miRNA functions, the regulatory mechanisms of miRNA expression was poor understood. In order better to understand the role of miRNAs in stretch mediated SMCs phenotypic modulation, the regulatory mechanism of miRNA expression by stretch was also investigated.Objectives1. To investigate the effect of stretch on miRNA expression profile in cultured HASMCs.2. To evaluate the role of miR-145in stretch mediated HASMCs phenotypic modulation.3. To investigate the regulatory mechanisms of miR-145expression by stretch. Methods1. Cell Culture and Stretch ApplicationHuman aortic smooth muscle cells (HASMCs) and smooth muscle cells medium (SMCM) were purchased from ScienCell (USA), passage4-7cells were used for experiment. HASMCs were seeded into Flexcell6-well plate coated with collagen I, when reached80%～90%confluence, serum-free SMCM was replaced for24hr culture, then exposed to cyclic stretch (16%elongation,1Hz) generated by a computer-controlled Tension system for indicated times. Pharmacological inhibitors for signaling molecules were added to SMCM1hour before stretch treatment.2. miRNA arrayHASMCs undergone16%stretch for12hrs or maintained in static conditions were collected, and total RNA samples were extracted by TRIzol method. Exiqon16.0miRNA assay chips were used for analysis of miRNA profile, the detailed works such as labeling with Hy3or Hy5, hybridization, scanning and data analysis were conducted by KANGCHEN (shanghai, China).3. qRT-PCRFor detecting miRNA expression, Exiqon SYBR Green miRNA assay kit was used according to the manufacturers’ protocol, levels of miRNAs were normalized by use of U6as a reference; for detecting mRNA expression levels, TAKARA cDNA synthesis kit and SYBR Green PCR kit were used according to the manufacturers’protocols, levels of target mRNA were normalized using GAPDH as a reference.4. Western blotHASMCs subjected to stretch or not were harvested and proteins in cytoplasm or nucleus were extracted. Determine the protein concentrations by BCA method. Western blot was performed using primary antibodies against SM22a, CNN, SMA-a-actin, et al. and HRP-conjugated secondary antibodies, followed by enhanced chemiluminescence detection and intensities analysis. 5. Transient TransfectionChemical modified miRNA inhibitor or mimics were obtained from GenePharm (shanghai, China), miRNA inhibitor or mimics specific for miR-145,-21,221were transfected into cultured HASMCs by use of Lipofectamine2000at a final concentration of90nM.48hours later, HASMCs were exposed to16%stretch or maintained in static conditions. siRNA aim at ACE was transfected into HASMCs using Lipo2000to suppress ACE expression, after48hrs, HASMCs were exposed to16%stretch or maintained in static conditions.6. Statistical analysisAll data was shown as means±SEM. statistical significance was determined by one-way ANOVA or two-tail unpaired Student’s t test, P<0.05was considered statistically significant.Results1. Differential miRNAs profile in stretched HASMCsUsing microarray, we compared the miRNAs expression profile in HASMCs undergoing16%stretch for12hrs with those kept in static controls. Our results indicated that several miRNA levels were altered by stretch, including miR-21, miR-330, let-7c, which were significantly up-regulated (p<0.05); and miR-145, miR-221, miR-29b, which was up-regulated (p<0.05). Interesting, miR-145, miR-21, miR-221were reported to play important roles in SMC phenotypic modulation.2.16%stretch reduced miR-145, miR-221expression and increased miR-21levels.We used qRT-PCR to verify the results of microarray. Compared to static controls,16%stretch significantly reduced miR-145expression (p<0.05), but had no effect on miR-143level (p>0.05), and the expression of miR-221was also decreased (p<0.01); miR-21expression was increased significantly (p<0.01), which was consistent with our previous study.3.16%stretch promotes the transition of HASMCs from a differentiated phenotype to a dedifferentiated state, After12hr16%stretch treatment, the protein and mRNA expression of calponin, SM22a, SMA-a-actin were also decreased compared to static controls (p<0.05). The SRP and myocardin protein expression in nucleus were also reduced by16%stretch (p<0.05).4. miR-145was involved in16%stretch mediated phenotypic modulation of HASMCsMiR-145, miR-21, miR-221were all reported to associated with SMC phenotype transition in varies of conditions, but which plays roles under stretch stimuli was unclear. Firstly, we used miR-21inhibitor to repress stretch induced miR-21up-regulation, however, the protein and mRNA levels of SMCs differentiated marker were not affected (p>0.05). MiR-145mimics significantly reversed stretch-induced miR-145suppression, and the protein levels of SMC-specific marker increased notably in HASMCs with or without stretch treatment (p<0.05). MiR-221mimics had no effect on stretch induced phenotype transition of cultured HASMCs (p>0.05). Transfected miR-221inhibitor or mimics into HASMCs kept in static conditions, the protein and mRNA expression of SMC differentiated marker were also not affected (p>0.05). These results indicate that miR-145plays a critical role in stretch induced phenotypic modulation of HASMCs.5. KLF4was involved in phenotypic modulation of HASMCs by stretch16%stretch increased KLF4protein expression (p<0.05) but had no effect on KLF4mRNA level (p>0.05). Recovery of stretch induced miR-145inhibition by miR-145mimics significantly reduced KLF4protein expression in stretched HASMC (p<0.05), and stretch induced inhibition of myocardin expression in nucleus was almost abolished (p<0.05), but the SRF protein level was not affected by miR-145mimics (p>0.05).6. Activation of ERK1/2signaling pathway was responsible to decreased miR-145level by stretch.Pri-miR-143/145level was reduced by16%stretch (p<0.05), indicating that stretch induced miR-145suppression at least partially occurred at transcriptional level. In response to16%stretch, p38MAPK, ERK1/2and Akt signaling pathway were activated demonstrated by increased p-ERK1/2and p-Akt levels. We used Pharmacological inhibitors SB203580, PD98058and LY294002to block activation of p38MAPK, ERK1/2and Akt pathway, respectively. PCR results revealed that PD98058significantly increased miR-145expression in stretched HASMCs (p<0.05), accompanied by increased levels of SM22a, calponin, SMA-a-actin(p<0.01).7. Up-regulation of ACE was implicated in regulation of miR-145level by16%stretchCompared to static controls,16%stretch promoted ACE mRNA and protein expression significantly (p<0.01). Knockdown ACE protein expression by use of siRNA repressed16%stretch induced miR-145inhibition (p<0.05) and partially recovered the SMCs differentiated markers expression in stretched cells (p<0.05). Inversely, added miR-145mimics into HASMCs48hrs before16%stretch treatment, the stretch induced ACE protein expression was partially abolished (p<0.05), but mRNA level was not affected (p>0.05).Conclusion1.16%stretch induced several miRNAs expression changes in cultured HASMCs, including miR-145,-21,-221,-24which were related to SMC phenotyptic modulation.2.16%stretch suppressed miR-145expression in cultured HASMCs, promoting the transition from a contractile phenotype to a synthetic state.3. Activation of ERK1/2pathway and up-regulation of ACE contribute to16%stretch-induced miR-145suppression. |
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