MicroRNA-205Regulates The Calcification And Osteoblastic Differentiation Of Vascular Smooth Muscle Cells

BackgroudVascular calcification is the deposition of calcium phosphate crystals in cardiovascular tissues. It reduces vessel elasticity, resulting in impaired cardiovascular hemodynamics and increased morbidity and mortality associated with hypertension, aortic stenosis, cardiac hypertrophy, and other cardiovascular diseases. In the past, vascular calcification was considered a passive and degenerative process; however, recent research has shown that this process is regulated by a mechanism similar to that of bone formation. The identification of bone calcification regulatory factors in blood vessels and the differential expression of these factors in nondiseased and diseased vessels verified that vascular calcification is a regulated process analogous to skeletal bone formation but orchestrated by vascular smooth muscle cells (VSMCs).Vascular smooth muscle cells (SMCs) are main cells of the tunica media of artery and play an important role in the process of regulating vascular tone. Vascular SMCs are not terminally differentiated cells, which may transfer from the contractile phenotype in differentiated state to the synthetic phenotype in dedifferentiated state when the ambient signals change. This adjustment between different phenotypes above is an important pathogenesis of proliferative cardiovascular diseases. The vascular SMCs with contractile phenotype play an important role in maintaining the integrity of arterial wall structure and function. And the vascular SMCs with synthetic phenotype may accelerate the migration and thus result in the formation of vascular calcification. Vascular calcification is divided into four subtypes according to tissue dissection, including intimal calcification, medial artery calcification, cardiac valvular calcification and vascular calciphylaxis. Intimal calcification is closely associated with atherosclerosis, and medial calcification usually occurs in diabetes, uremia and other diseases, both of which tend to occur simultaneously. The former increases the risk of plaque disruption, while the latter results in arterial stiffness and affects hemodynamics.VSMCs release matrix vesicles with the capacity to concentrate calcium and phosphate, acting as nidus for mineralization, and undergo a phenotypic transition in which the expression of transcription factors associated with differentiated chondrocytes and osteoblasts induce the expression of mineralization regulating proteins such as alkaline phosphatase, osteopontin and osteocalcin.In this process, the vascular SMCs with contractile phenotype, pericytes and calcified vascular cells are clearly differentiated into "osteoblast-like" cells with secretory phenotype. These osteoblast-like SMCs have decreased expression of smooth muscle cell-specific markers, such as myocardin, smooth muscle myosin heavy chain and smooth muscle a-actin, and increased expression of bone-related proteins including alkaline phosphatase, osteocalcin and core-binding factor a-1(Cbfal/Runx2). But to date, the detail pathological mechanisms of vascular calcification still remain unclear.Runx2is a central transcription factor of inducing differentiation of osteoblasts and chondrocytes during the calcification process. By the way of targeted gene knockout, either intramembranous ossification or endochondral ossification occurred in Runx2-/-mice due to the failure of effective osteoblast differentiation, demonstrating that Runx2is an essential factor for differentiation into osteoblasts. Runx2overexpression may promote early osteoblast differentiation, and inhibit the maturation of osteoblasts resulting in increased number of early and immature osteoblasts. Runx2is generally not expressed in SMCs, but up-regulated under the action of stimulus causing calcification such as inflammation, oxidative stress, bone morphogenetic protein-2(BMP-2). Runx2is expressed in osteoblast-like SMCs, and the up-regulation of Runx2may drive vascular SMCs into osteoblast-like cells.BMP is one member of transforming growth factor-β (TGF-β) superfamily. Smads family includes proteins for intracellular BMP signal transduction. and divided into three subgroups according to its function, ie, receptor-regulated Smads (R-Smads), including Srmdl, Smad2, Smad3, Smad5, Smad8and Smad9; common-partner Smads (Co-Smads), only Smad4in mammals; inhibitory-Smads (I-Smads), including Smad6and Smad7. After BMP activating Smdl/5/8, R-Smads and Co-Smads form a complex, which thereby inducing Runx2gene expression. Smads induce the transduction of extracellular signals into the nucleus, which is subjected to multiple levels of membranal, intracellular and extracellular positive and negative feedback regulations.MicroRNAs (miRNAs) are a class of small (16-25nucleotides), single-stranded non-coding RNAs that negatively regulate gene expression through incomplete base-pairing to the3’untranslated region (3’-UTR) of target mRNAs, inducing mRNA degradation or blocking translation. miRNAs have been studied extensively because of their roles as regulators of cell differentiation, growth, proliferation and apoptosis, and therefore their involvement in several diseases, in particular their role in cancer. Importantly, miRNAs are highly expressed in the cardiovascular system and their involvement not only in cardiovascular development, but also in cardiovascular diseases including atherosclerosis and pulmonary arterial hypertension has only recently began to be understood. Several miRNAs, such as miR-125b, miR-204, miR-29a/b, miR-30b/c and miR-133a, have been identified that are involved in VSMC calcification. However, the mechanisms underlying the effect of miRNAs and their targets in the modulation of VSMC differentiation leading to vascular calcification remain to be elucidated.Studies have revealed that miR-205was highly expressed in head and neck cancer, ovarian cancer and breast cancer and closely related to invasive squamous cell carcinoma. miR-205could target JPH4genes by AKT phosphorylation, inhibit cancer cell apoptosis, thus playing an important role in tumorigenesis and progression. It was also reported that miR-205as a tumor suppressor gene inhibited breast cancer by targeting HER3, inhibited cancer cell migration and invasion by inhibiting the epithelium and mesenchymal transformation, thus inhibiting tumor progression. The recent studies revealed that miR-205could regulate osteoblast differentiation and osteogenesis via targeted regulation of Runx2. So, whether miR-205is possible to participate in osteoblast differentiation of vascular SMCs through above ways, thereby regulating the process of vascular calcification?In our research, human aortic SMCs were used to observe the expression and change of miR-205in the process of differentiation of vascular SMCs into osteoblasts and the function pathway of miR-205in regulating vascular SMC calcification by the means of real-time quantitative reverse transcription PCR, plasmid construction and transfection, and luciferase assay, further revealing the pathological mechanism of vascular calcification.miR-205was previously shown to be among a series of miRNAs that regulate osteoblast differentiation and bone formation by directly targeting Runx2. Whether miR-205is also involved in VSMC osteogenic differentiation has not yet been explored. In this study, we found that miR-205is a negative regulator of osteogenic differentiation in VSMCs, and identified Runx2and Smadl as its direct targets.Material and Methods1Cell culture and calcification modelHuman aortic smooth muscle cells (HASMCs) were purchased from Promocell and cultured in DMEM supplemented with10%fetal bovine serum (FBS) and100units/ml penicillin/streptomycin at37℃and5%CO2. To induce calcification, the cells were incubated in DMEM containing15%FBS supplemented with10mM β-glycerophosphate (β-GP).2Measurements of ALP activity and osteocalcin secretionTo determine the activity of alkaline phosphatase (ALP), cells were washed with PBS and scraped into a solution containing20mM Tris-HCl,150mM NaCl,1%Triton X-100,0.02%NaN3, and1μg/mL aprotinin. Lysates were homogenized and assayed for ALP activity by measuring the release of p-nitrophenol at37℃using a spectrophotometer. Results were normalized to total cellular protein content. Osteocalcin secretion into the culture media was measured by using a specific radioimmunoassay kit according to the manufacturer’s instructions. Protein expression was normalized to total cellular protein measured by the Bradford protein assay.3Alizarin Red S stainingThe formation of a mineralized matrix was determined by Alizarin Red S staining. HASMCs cultured in DMEM supplemented with10mM β-GP were fixed in70%ethanol for1h at room temperature and stained with40mM Alizarin Red S for10min. Then, cells were washed with PBS to eliminate nonspecific staining and the stained matrix was photographed using a digital microscope. For quantification of staining, the Alizarin Red S stain was released from the cell matrix by incubation in cetyl-pyridinium chloride for15min and the amount of released dye was measured by spectrophotometry at540nm. The results were normalized to total cellular protein content.4Quantitative reverse transcriptase PCR (qRT-PCR)Total RNA was extracted from HASMCs. In the in vitro experiment, the mRNA expression of miR-205, Runx2and Smadl in HASMCs was analyzed. Relative expression of miRNA or mRNA was normalized to the expression of U6or β-actin and evaluated by the2-△△Ct method.5Western blottingTotal proteins were extracted from HASMCs. The protein expression of Runx2and Smadl was analyzed in our experiment. Band intensity was quantified by using Image J software.6Plasmid constructionThe full-length open reading frame of Runx2or Smadl was cloned into pcDNA3.1(+) to generate their expression vectors. The wild-type Runx2or Smadl3’-UTR (WT) was cloned into the pGL3-basic vector. Site-directed mutagenesis of the miR-205seed sequence in the3’-UTR (Mut) was performed using the QuikChangeTM Site-Directed Mutagenesis Kit.7Oligonucleotide transfection Cells were seeded into6-well plates, transfected with miR-205mimics, anti-miR-205, Runx2siRNA, Smad1siRNA or the respective controls using LipofactamineTM RNAiMAX, or transfected with plasmids using Lipofactamine2000reagent. The cells were collected for assays48h after transfection.8Luciferase assaysFor luciferase assays, HASMCs were cultured’in24-well plates and co-transfected with luciferase reporter plasmid and miR-205mimics and pRL-TK vector using Lipofectamine2000. Cells were harvested and lysed48h after transfection, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System. Renilla-luciferase was used for normalization. The experiments were performed independently in triplicate.9Statistical analysisData are expressed as mean±SD from at least three independent experiments. Statistical analysis was performed using Student’s t test. P<0.05was considered statistically significant.Results1To induce the calcification of HASMCs, cells were incubated in DMEM supplemented with10mM β-GP. Increased matrix mineralization, Runx2expression, ALP activity and osteocalcin levels were induced when cells were cultured under calcification conditions.2To examine the possible association between miR-205and HASMC calcification, the expression levels of miR-205were determined by qRT-PCR, which showed a statistically significant downregulation of the expression of miR-205in response to β-GP treatment, indicating that miR-205is downregulated during the calcification of HASMCs.3To further investigate the role of miR-205in the osteoblastic differentiation of HASMCs, cells were transfected with miR-205mimics or anti-miR-205or the respective controls, and the levels of miR-205were determined by qRT-PCR. miR-205up-and down-regulation could be maintained for approximately8days in these cells. 4miR-205silencing significantly enhanced osteoblastic differentiation, which was indicated by enhanced in vitro matrix mineralization, up-regulated Runx2protein expression, increased ALP activity and osteocalcin secretion in anti-miR-205-transfected HASMCs compared with cells transfected with miR-ctrl. In contrast, matrix mineralization, Runx2protein expression, ALP activity and osteocalcin secretion were reduced in miR-205minics-treated HASMCs. Together, these results indicate that miR-205plays a negative role in the regulation of osteoblastic differentiation of HASMCs.5Given that Runx2and Smadl, which are key downstream mediators of bone morphogenetic protein-2signaling, have been previously reported to be target genes of miR-205, we next aimed to determine whether they are involved in the regulation of osteoblastic differentiation of HASMCs. We found the putative target sites for miR-205in the3’-UTRs of Runx2and Smadl. To analyze the relationship between miR-205and Runx2and Smadl, HASMCs were co-transfected with miR-205mimics and luciferase reporter constructs containing the wild-type (wt) or mutant (mut) miR-205target sites in the Runx2or Smadl3’-UTR, and luciferase activities were measured48h after transfection. The results showed that overexpression of miR-205significantly decreased the luciferase activity of the wt-3’-UTRs of Runx2and Smadl compared to miR-control transfected cells whereas it had no effect on the mut-3’-UTRs, indicating that miR-205suppressed the expression of Runx2and Smadl by directly binding to target sites in their3’-UTRs. Furthermore, qRT-PCR analysis showed that miR-205overexpression had no significant effect on Runx2and Smadl mRNA levels. However, miR-205overexpression significantly downregulated the expression of the Runx2and Smad1proteins compared to untransfected controls or miR-control transfected cells. These results support the bioinformatics predictions indicating Runx2and Smadl3’-UTRs as direct targets of miR-205.6We further explored whether Runx2and Smadl deregulations were required for the suppressive effect of miR-205in regulating the osteogenic differentiation of HASMCs. We restored the expression of Runx2and Smadl in miR-205-overexpressing HASMCs by transfection of Runx2and Smadl ORF constructs without3’-UTRs, and assessed their effect on ALP activity and osteocalcin levels. overexpression of Runx2or Smad1significantly increased ALP activity and osteocalcin secretion, suggesting that Runx2or Smadl overexpression impaired the effect of miR-205during osteoblastic differentiation of HASMCs. Furthermore, cotransfection of Runx2siRNA or Smadl siRNA with anti-miR-205almost completely blocked the positive role of anti-miR-205on these parameters. Taken together, these results suggested that Runx2and Smad1are functional targets of miR-205.Conclusion1Four indicators including alkaline phosphatase, osteocalcin, Runx2and calcium deposition demonstrated that β-glycerolphosphate could successfully induce human aortic SMC differentiation into osteoblasts.2The endogenous miR-205showed decreased expression in the process of vascular SMC differentiation into osteoblasts, and further studies revealed that miR-205acted as a negative regulator in the process of differentiation.3Runx2and Smadl are associated with the process of miR-205in regulating human aortic SMC calcification.4miR-205directly inhibited the expression of Runx2and Smadl by combining with the target sites of Runx2and Smadl3’-UTRs, thus inhibiting the transformation of human aortic SMCs into osteoblasts.