过表达miR-155抑制BMP9诱导间充质干细胞C3H10T1/2成骨分化
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摘要
目的:研究过表达miR-155对BMP9诱导间充质干细胞C3H10T1/2成骨分化的影响。方法:(1)用重组腺病毒Ad-BMP9(BMP9)诱导C3H10T1/2细胞成骨分化,定量PCR(qPCR)检测miR-155的表达,RT-PCR检测Runx2和ALP的表达。(2)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测miR-155的表达,ALP活性和染色检测早期成骨能力。(3)miR-155和BMP9共同处理C3H10T1/2细胞,诱导分化14d茜素红S染色检测晚期成骨能力。(4)miR-155和BMP9共同处理C3H10T1/2细胞,qPCR检测成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN的表达。(5)miR-155和BMP9共同处理C3H10T1/2细胞,Western blot检测p-Smad1/5/8、OCN和OPN蛋白水平的表达。(6)qPCR和Western blot分别检测HIF1α和VEGF的mRNA表达水平和蛋白质表达水平。(7)应用荧光素酶报告基因对miR-155的靶基因进行筛选和验证。结果:在BMP9诱导C3H10T1/2细胞成骨分化过程中,过表达miR-155降低ALP活性及染色;减少钙盐沉积;成骨分化相关基因Runx2、OSX、COL1A1、ALP、OCN和OPN表达降低;抑制p-Smad1/5/8、OCN和OPN蛋白水平的表达;HIF1α和VEGF的mRNA和蛋白表达水平减少。在对靶基因的检测中,过表达miR-155可以抑制HIF1α蛋白水平的表达,但对其mRNA水平无明显影响。结论:miR-155过表达减弱BMP9诱导间充质干细胞C3H10T1/2成骨分化,可能是通过抑制Smad/BMP信号通路发挥作用,也有可能是通过抑制靶基因HIF1α的表达来发挥作用。
Purpose: To investigate the effect of overexpressed miR-155 on the osteogenic differentiation of mesenchymal stem cells C3H10T1/2 cells induced by BMP9,and the related mechanisms contained in this process. Methods:(1) Used adenovirus BMP9( BMP9) to induced the osteogenic differentiation of C3H10T1/2cells,tested the expression of miR-155 by quantitative PCR( q PCR),detected the expression of Runx2 and ALP use RT-PCR.(2) Cotransfected BMP9 and miR-155 into C3H10T1/2 cells,measured the expression of miR-155 by q PCR, ALP activity and ALP staining evaluated the early osteogenesis ability of C3H10T1/2 cells.(3) Cotransfected BMP9 and miR-155 into C3H10T1/2 cells,used Alizarin red S staining to estimate the osteogenesis ability in the later stage of differentiation in 14 d.(4) Treated C3H10T1/2 cells with BMP9 and miR-155,tested osteogenesis-related genes by q PCR,include Runx2, OSX, COL1A1, ALP, OCN and OPN.(5) Treated C3H10T1/2 cells with BMP9 and miR-155,detected the expression of p-Smad1/5/8,OCN and OPN on protein levels used Western blot.(6) q PCR and Western blot examined the expression of HIF1α and VEGF on mRNA and protein levels respectively.(7) Confirmed the target gene of miR-155 by luciferase reporter assay.Results: During the process of osteogenic differentiation of C3H10T1/2 cells induced by BMP9,overexpressed miR-155 decreased ALP activity,ALP staining and Alizarin red S staining. Repressed the expression of bonerelated genes,such as Runx2,OSX,COL1A1,ALP,OCN and OPN,also. On the protein levels,overexpressed miR-155 inhibited the expression of p-Smad1/5/8,OCN and OPN,the expression of HIF1α and VEGF were decreased on mRNA and protein levels,too. Through luciferase reporter assay,confirmed that HIF1α is one of the target genes of miR-155,miR-155 decreased the expression of HIF1α on protein level,not on mRNA level,yet. Conclusion: Overexpressed miR-155 could inhibit the osteogenic differentiation induced by BMP9 in mesenchymal stem cells C3H10T1/2 cells,it may through Smad/BMP signaling pathway and its target gene HIF1α to play the inhibitory effect in this process.
Purpose: To investigate the effect of overexpressed miR-155 on the osteogenic differentiation of mesenchymal stem cells C3H10T1/2 cells induced by BMP9,and the related mechanisms contained in this process. Methods:(1) Used adenovirus BMP9( BMP9) to induced the osteogenic differentiation of C3H10T1/2cells,tested the expression of miR-155 by quantitative PCR( q PCR),detected the expression of Runx2 and ALP use RT-PCR.(2) Cotransfected BMP9 and miR-155 into C3H10T1/2 cells,measured the expression of miR-155 by q PCR, ALP activity and ALP staining evaluated the early osteogenesis ability of C3H10T1/2 cells.(3) Cotransfected BMP9 and miR-155 into C3H10T1/2 cells,used Alizarin red S staining to estimate the osteogenesis ability in the later stage of differentiation in 14 d.(4) Treated C3H10T1/2 cells with BMP9 and miR-155,tested osteogenesis-related genes by q PCR,include Runx2, OSX, COL1A1, ALP, OCN and OPN.(5) Treated C3H10T1/2 cells with BMP9 and miR-155,detected the expression of p-Smad1/5/8,OCN and OPN on protein levels used Western blot.(6) q PCR and Western blot examined the expression of HIF1α and VEGF on mRNA and protein levels respectively.(7) Confirmed the target gene of miR-155 by luciferase reporter assay.Results: During the process of osteogenic differentiation of C3H10T1/2 cells induced by BMP9,overexpressed miR-155 decreased ALP activity,ALP staining and Alizarin red S staining. Repressed the expression of bonerelated genes,such as Runx2,OSX,COL1A1,ALP,OCN and OPN,also. On the protein levels,overexpressed miR-155 inhibited the expression of p-Smad1/5/8,OCN and OPN,the expression of HIF1α and VEGF were decreased on mRNA and protein levels,too. Through luciferase reporter assay,confirmed that HIF1α is one of the target genes of miR-155,miR-155 decreased the expression of HIF1α on protein level,not on mRNA level,yet. Conclusion: Overexpressed miR-155 could inhibit the osteogenic differentiation induced by BMP9 in mesenchymal stem cells C3H10T1/2 cells,it may through Smad/BMP signaling pathway and its target gene HIF1α to play the inhibitory effect in this process.
引文
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[22]Kristina K,Anne M D.MicroRNA biogenesis and regulation of bone.Arthritis Research&Therapy,2011,13(3):220.
[23]Wang R N,Green J,Wang Z,et al.Bone morphogenetic protein(BMP)signaling in development and human diseases.Genes&diseases,2014,1(1):87-105.
[24]Inose H,Ochi H,Kimura A,et al.microRNA regulatory mechanism of osteoblast differentiation.Proceedings of the National Academy of Sciences of the United States of America,2009,106(49):20794-20799.
[25]Li Z,Hassan M Q,Volinia S,et al.microRNA signature for a BMP2-induced osteoblast lineage commitment program.Proceedings of the National Academy of Sciences of the United States of America,2008,105(37):13906-13911.
[2]Zuo B,Zhu J,Li J,et al.microRNA-103a functions as a mechanosensitive microRNA to inhibit boneformation through targeting Runx2.Journal of Bone and Mineral Research:The Official Journal of the American Society for Bone and Mineral Research,2015,30(2):330-345.
[3]Zeng Y,Qu X,Li H,et al.MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2.FEBS Letters,2012,586(16):2375-2381.
[4]Taipaleenmaki H,Bjerre Hokland L,Chen L,et al.Mechanisms in endocrinology:micro-RNAs:targets for enhancing osteoblast differentiation and bone formation.European Journal of Endocrinology/European Federation of Endocrine Societies,2012,166(3):359-371.
[5]Li Y,Kowdley K V.MicroRNAs in common human diseases.Genomics,Proteomics&Bioinformatics,2012,10(5):246-253.
[6]Ivey K N,Srivastava D.MicroRNAs as regulators of differentiation and cell fate decisions.Cell Stem Cell,2010,7(1):36-41.
[7]Itoh T,Takeda S,Akao Y.MicroRNA-208 modulates BMP-2-stimulated mouse preosteoblast differentiation by directly targeting V-ets erythroblastosis virus E26 oncogene homolog 1.The Journal of Biological Chemistry,2010,285(36):27745-27752.
[8]Hwang H W,Mendell J T.MicroRNAs in cell proliferation,cell death,and tumorigenesis.British Journal of Cancer,2006,94(6):776-780.
[9]Faraoni I,Antonetti F R,Cardone J,et al.miR-155 gene:a typical multifunctional microRNA.Biochimica et Biophysica Acta,2009,1792(6):497-505.
[10]Du F,Yu F,Wang Y,et al.MicroRNA-155 deficiency results in decreased macrophage inflammation and attenuated atherogenesis in apolipoprotein E-deficient mice.Arteriosclerosis,Thrombosis,and Vascular Biology,2014,34(4):759-767.
[11]Vigorito E,Perks K L,Abreu-Goodger C,et al.microRNA-155regulates the generation of immunoglobulin class-switched plasma cells.Immunity,2007,27(6):847-859.
[12]O’Connell R M,Kahn D,Gibson W S,et al.MicroRNA-155promotes autoimmune inflammation by enhancing inflammatory T cell development.Immunity,2010,33(4):607-619.
[13]Nazari-Jahantigh M,Wei Y,Noels H,et al.MicroRNA-155promotes atherosclerosis by repressing Bcl6 in macrophages.The Journal of Clinical Investigation,2012,122(11):4190-4202.
[14]Wang M,Tan L P,Dijkstra M K,et al.miRNA analysis in Bcell chronic lymphocytic leukaemia:proliferation centres characterized by low miR-150 and high BIC/miR-155 expression.The Journal of Pathology,2008,215(1):13-20.
[15]Lawrie C H.MicroRNAs and lymphomagenesis:a functional review.British Journal of Haematology,2013,160(5):571-581.
[16]Costinean S,Zanesi N,Pekarsky Y,et al.Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice.Proceedings of the National Academy of Sciences of the United States of America,2006,103(18):7024-7029.
[17]Yin Q,Wang X,Fewell C,et al.MicroRNA miR-155 inhibits bone morphogenetic protein(BMP)signaling and BMP-mediated Epstein-Barr virus reactivation.Journal of Virology,2010,84(13):6318-6327.
[18]Yanaihara N,Caplen N,Bowman E,et al.microRNA molecular profiles in lung cancer diagnosis and prognosis.Cancer Cell,2006,9(3):189-198.
[19]Volinia S,Galasso M,Costinean S,et al.Reprogramming of miRNA networks in cancer and leukemia.Genome Research,2010,20(5):589-599.
[20]Jiang S,Zhang H W,Lu M H,et al.MicroRNA-155 functions as an OncomiR in breast cancer by targeting the suppressor of cytokine signaling 1 gene.Cancer Research,2010,70(8):3119-3127.
[21]Omrane I,Benammar-Elgaaied A.The immune microenvironment of the colorectal tumor:Involvement of immunity genes and microRNAs belonging to the TH17 pathway.Biochimica et Biophysica Acta,2015,1856(1):28-38.
[22]Kristina K,Anne M D.MicroRNA biogenesis and regulation of bone.Arthritis Research&Therapy,2011,13(3):220.
[23]Wang R N,Green J,Wang Z,et al.Bone morphogenetic protein(BMP)signaling in development and human diseases.Genes&diseases,2014,1(1):87-105.
[24]Inose H,Ochi H,Kimura A,et al.microRNA regulatory mechanism of osteoblast differentiation.Proceedings of the National Academy of Sciences of the United States of America,2009,106(49):20794-20799.
[25]Li Z,Hassan M Q,Volinia S,et al.microRNA signature for a BMP2-induced osteoblast lineage commitment program.Proceedings of the National Academy of Sciences of the United States of America,2008,105(37):13906-13911.