<i>BMP7</i> Gene Silencing Inhibits Osteogenic Differentiation of Porcine Arotic Valve Interstitial Cells Induced by Osteogenic Induction Medium

doi:10.13523/j.cb.20190507

China Biotechnology

2019, Vol. 39

Issue (5): 63-71 DOI: 10.13523/j.cb.20190507

BMP7 Gene Silencing Inhibits Osteogenic Differentiation of Porcine Arotic Valve Interstitial Cells Induced by Osteogenic Induction Medium

Yu CHENG,Qiong SHI,Li-qin AN,Meng-tian FAN,Gai-gai HUANG,Ya-guang WENG(

)

Key Laboratory of Clinical Laboratory Diagnostics of Ministry Education, Faculty of Laboratory Medicine,Chongqing Medicine University, Chongqing 400016, China

Download:

HTML

PDF(1598KB) HTML
Export: BibTeX | EndNote (RIS)

Abstract

Objective: To investigate the effect of small interfering RNA (siRNA) -mediated bone morphogenetic protein7 (BMP7) gene silencing on osteogenic differentiation of porcine aortic valve interstitial cells induced by osteogenic induction medium,and to provide a theoretical basis for the intervention and treatment of calcific aortic valve disease.Methods:The non-CAVD valvular tissues (non-CAVD group) were taken from patients with surgical treatment of aortic dissection,the CAVD valvular tissues (CAVD group) were taken from patients undergoing aortic valve replacement because of calcified aortic valve stenosis.The expression levels of BMP7 and Runx2 in the non-CAVD group and CAVD group were tested by immunohistochemistry and Western blot. Healthy domestic pigs were sacrificed and the aortic valve leaflets were aseptically removed immediately.The aortic valve interstitial cells(VICs) were isolated by continuous collagenase digestion,its morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining. VICs were transfected with BMP7-siRNA by liposome method.The expression of BMP7 at mRNA and protein levels in the VICs transfected with BMP7-siRNA was detected by qPCR and Western blot,respectively.The conditioned medium induced osteogenic differentiation of VICs for the establishment of calcification model of aortic valve interstitial cells in vitro,then Alkaline phosphatase (ALP)staining and Alizarin red S staining was used to evaluate the cell early and late osteogenic differentiation abilities.The mRNA and protein levels of Runx2,OCN and OPN were determined by qPCR and Western blot,respectively.The protein levels of p-drosophila mothers against de-capentaplegic 1/5/8 (p-Smad1/5/8) was also determined by Western blot.Results:The expression of BMP7 and Runx2 in CAVD group was significantly higher than that in non-CAVD group.The primary porcine aortic valve interstitial cells were successfully isolated and the staining of α-smooth muscle actin (α-SMA) and Vimentin were positive,the staining of von Willebrand factor (vWF) was negative.The expression of BMP7 at mRNA and protein levels in the VICs transfected with BMP7-siRNA was significantly decreased,and the cell early and late osteogenic differentiation abilities were significantly decreased.The mRNA and protein levels of Runx2,OCN and OPN were significantly reduced.Meanwhile,the protein levels of p-Smad1/5/8 were down-regulated.Conclusion:BMP7 gene silencing obviously inhibits the osteogenic differentiation of aortic valve interstitial cells induced by osteogenic induction medium.The BMP7/Smads signaling pathways may play an important role in these processes.

Key words： Calcific aortic valve disease Valve interstitial cell BMP7 Osteogenic differentiation RNA interference

Received: 24 October 2018 Published: 04 June 2019

ZTFLH:

Q819

Corresponding Authors: Ya-guang WENG E-mail: yaguangweng@126.com

	Service
	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	Yu CHENG
	Qiong SHI
	Li-qin AN
	Meng-tian FAN
	Gai-gai HUANG
	Ya-guang WENG

Cite this article:

Yu CHENG,Qiong SHI,Li-qin AN,Meng-tian FAN,Gai-gai HUANG,Ya-guang WENG. BMP7 Gene Silencing Inhibits Osteogenic Differentiation of Porcine Arotic Valve Interstitial Cells Induced by Osteogenic Induction Medium. China Biotechnology, 2019, 39(5): 63-71.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190507 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I5/63

Table 1 The sequence of Primers for PCR(pig)

Fig.1 The expression of BMP7 and Runx2 protein in CAVD group was significantly higher than that in non-CAVD group (a)The expression of BMP7 and Runx2 in CAVD group and non-CAVD group detected by immunohistochemistry(×200) scale bar represented 50μm (b)The expression of BMP7 and Runx2 detected by Western blot Mean±SD,n=3.*** P<0.001 vs non-CAVD group

Fig.2 Morphological and phenotypic characterization of porcine aortic valve interstitial cells (a)Light microscopy image(×100) of VICs (b)Immunofluorescence staining of α-SMA(×200) (c)Immunofluorescence staining of Vimentin(×200) (d)Immunofluorescence staining of vWF (×200)

Fig.3 Transfection with BMP7-siRNA decreased BMP7 expression in the valve interstitial cells (a)The mRNA expression of BMP7 was detected by qPCR (b)The protein expression was determined by Western blot Mean±SD,n=3.** P<0.01 vs control group or negative control group

Fig.4 The effect of BMP7-siRNA on the early and late osteogenic differentiation abilities of the valve interstitial cells (a)The cell early osteogenic differentiation ability was measured by ALP staining(×100) (b)The cell late osteogenic differentiation ability was measured by Alizarin red S staining(×100)

Fig.5 The downregulation of BMP7 inhibits the expression of osteoblast-related genes and proteins in valve interstitial cells (a)The mRNA expression of Runx2,OCN and OPN was detected by qPCR (b)The protein expression was determined by Western blot Mean±SD,n=3.* P<0.05, ** P<0.01, *** P<0.001 vs control group or negative control group

Fig.6 The effect of BMP7-siRNA on BMP-Smad1/5/8 signaling pathway The expression and activation of Smad1/5/8 protein was detected by Western blot P<0.01 vs control group or negative control group


[1]	Yutzey K E, Demer L L, Body S C , et al. Calcific aortic valve disease a consensus summary from the alliance of investigators on calcific aortic valve disease.Arteriosclerosis, Thrombosis, and Vascular Biology, 2014,34(11):2387-2393.

[2]	Peltonen T, Ohukainen P, Ruskoaho H , et al. Targeting vasoactive peptides for managing calcific aortic valve disease. Ann Med, 2017,49(1):63-74. doi: 10.1080/07853890.2016.1231933

[3]	李康, 杨重庆, 鲁安怀 , 等. 心脏瓣膜钙化的增龄性改变. 中华老年医学杂志, 2013,32(9):934-936. doi: 10.3760/cma.j.issn.0254-9026.2013.09.003

[3]	Li K, Yang C Q, Lu A H , et al. Age-related changes in calcification of heart valves. Chin J Geriatr, 2013,32(9):934-936. doi: 10.3760/cma.j.issn.0254-9026.2013.09.003

[4]	Horne A, Reineck E A, Hasan R K , et al. Transcatheter aortic valve replacement:Historical perspectives,current evidence,and future directions. Am Heart J, 2014,168(4):414-423. doi: 10.1016/j.ahj.2014.07.017

[5]	Adams D H, Popma J J, Reardon M J , et al. Transcatheter aortic valve replacement with a self-expanding prosthesis. N Engl J Med, 2014,370(19):1790-1798. doi: 10.1056/NEJMoa1400590

[6]	陆洋, 傅巧美 . 心脏瓣膜置换术后患者抗凝治疗依从性的调查分析. 解放军护理杂志, 2016,33(8):62-64.

[6]	Lu Y, Fu Q M . Investigation and analysis of the compliance of anticoagulant therapy for patients underwent cardiac valve replacement. Nursing Journal of Chinese People’s Liberation Army, 2016,33(8):62-64.

[7]	He C, Tang H, Mei Z , et al. Human interstitial cellular model in therapeutics of heart valve calcification. Amino Acids, 2017,49(12):1981-1997. doi: 10.1007/s00726-017-2432-3

[8]	Leopold J A . Cellular mechanisms of aortic valve calcification. Circ Cardiovasc Interv, 2012,5(4):605-614. doi: 10.1161/CIRCINTERVENTIONS.112.971028

[9]	Liu A C, Joag V R, Gotlieb A I . The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am J Pathol, 2007,171(5):1407-1418. doi: 10.2353/ajpath.2007.070251

[10]	Mohler E R, Gannon F, Reynolds C , et al. Bone formation and inflmmation in cardiac valves. Circulation, 2001,103(11):1522-1528. doi: 10.1161/01.CIR.103.11.1522

[11]	Osman L, Yacoub M H, Latif N , et al. Role of human valve interstitial cells in valve calcification and their response to atorvastatin. Circulation, 2006,114(1 Suppl):I547-1552.

[12]	Katz R, Budoff M J, Takasu J , et al. Relationship of metabolic syndrome with incident aortic valve calcium and aortic valve calcium progression:the Multi-Ethnic Study of Atherosclerosis (MESA). Diabetes, 2009,58(4):813-819 doi: 10.2337/db08-1515

[13]	Wang S N, Lapage J, Hirschberg R . Loss of tubular bone morphogenetic protein-7 in diabetic nepropathy. J Am Soc Nephrol, 2001,12(11):2392-2399.

[14]	Morrissey C, Brown L G, Pitts T E , et al. Bone morphogenetic protein 7 is expressed in prostate cancer metastases and its effects on prostate tumor cells depend on cell phenotype and the tumor microenvironment. Neoplasia, 2010,12(2):192-205. doi: 10.1593/neo.91836

[15]	Ye L, Lewis-Russell J M, Sanders A J ,et al. HGF /SF up-regulates the expression of bone morphogenetic protein 7 in prostate cancer cells. Urol Oncol, 2008,26(2):190-197. doi: 10.1016/j.urolonc.2007.03.027

[16]	Miyazonok K, Kusanagi K, Inoue H . Divergence and convergence of TGFb/ BMP signaling. J Cell Physiol, 2001,187(3), 265-276. doi: 10.1002/(ISSN)1097-4652

[17]	Bowler M A, Merryman W D . In vitro models of aortic valve calcification:solidifying a system. Cardiovasc Pathol, 2015,24(1):1-10. doi: 10.1016/j.carpath.2014.08.003

[18]	Pawade T A, Newby D E, Dweck M R . Calcification in aortic stenosis:The skeleton key. J Am Coll Cardiol, 2015,66(5):561-577. doi: 10.1016/j.jacc.2015.05.066

[19]	Sider K L, Zhu C, Kwong A V , et al. Evaluation of a porcine model of early aortic valve sclerosis. Cardiovasc Pathol, 2014,23(5):289-297. doi: 10.1016/j.carpath.2014.05.004

[1]	FENG Zhao,LI Jiang-hao,WANG Jia-hua. Functional Analysis of RpRPL22, a Ribosomal Protein Homologous Gene, in the Symbiotic Nodulation Process of Robinia Pseudoacacia[J]. China Biotechnology, 2021, 41(7): 10-21.

[2]	ZHU Ying,FAN Meng-tian,LI Ju-qiong,CHEN Bin,ZHANG Meng-hao,WU Jing-hong,SHI Qiong. Effect of Chemokine Receptor CX3CR1 on Osteogenic Differentiation of Human Aortic Valve Interstitial Cells[J]. China Biotechnology, 2019, 39(8): 7-16.

[3]	Zhao FENG,Min-xia CHOU. *Functional Analysis of Rpfan37* in the Symbiotic Nodulation Process of Robinia Pseudoacacia**[J]. China Biotechnology, 2018, 38(5): 47-55.

[4]	DONG Wei-peng,ZHANG Shao-hua,XU Xiang,YAN Jiong. *The Effect and Mechanism of Down-Regulated Fsp27* Gene Combined with Myricetin on Lipolysis of 3T3-L1 Adipocytes**[J]. China Biotechnology, 2018, 38(12): 7-13.

[5]	LIU Hong-xia, SHI Qiong, ZHOU Yi-qing, AN Li-qin, YAN Shu-juan, ZHANG Ru-yi, WENG Ya-guang. Overexpression of miR-155 Inhibits the Osteogenic Differentiation of Mesenchymal Stem Cells C3H10T1/2 Induced by BMP9[J]. China Biotechnology, 2017, 37(5): 9-18.

[6]	LIU Xiao-hua, JI Cai-xia, XU Li, DONG Chao-qun, LUO Jin-yong. Hmox1 Can Promote BMP9-induced Osteogenesis in C3H10 T1/2[J]. China Biotechnology, 2017, 37(4): 33-39.

[7]	CAO Jun-jie, LI Ai-fang, WEI Ya-lin, LIAN Jing, TANG Min. Role of Notch Signaling Pathway in Bone Morphogenetic Protein 4-induced Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells and Its Mechanism[J]. China Biotechnology, 2017, 37(4): 48-55.

[8]	JI Cai-xia, LIU Xiao-hua, XU Li, DONG Chao-qun, LUO Jin-yong. Runx1 Enhances BMP9-induced Osteogenic Differentiation in Mesenchymal Stem Cell Line MEFs[J]. China Biotechnology, 2017, 37(3): 10-17.

[9]	XU Li, JI Cai-xia, LIU Xiao-hua, YU Ting-ting, LUO Jin-yong. Effects of DLX1 on BMP9-induced Osteogenic Differentiation of C3H10T1/2 Mesenchymal Stem Cells[J]. China Biotechnology, 2017, 37(10): 8-15.

[10]	LI Ya-sha, LIU Xing, BI Yang, YANG Ke, ZHAO Li, GONG Meng-jia, GUO Qi. BMP9 Induced the Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells in vitro and vivo[J]. China Biotechnology, 2016, 36(5): 20-26.

[11]	LIU Yi-xuan, BIAN Zhen, MA Hong-mei. Progress and Prospect of Cancer Gene Therapy[J]. China Biotechnology, 2016, 36(5): 106-111.

[12]	HU Na, LIU Qing, TANG Zhao-yong, TANG He-jing, AO Lan, ZHAO Zi-hao, FANG Liao-qiong. *siRNA Inhibits the Growth and Migration of Mouse Melanoma by MMP-9* and FAK Gene**[J]. China Biotechnology, 2016, 36(5): 34-39.

[13]	ZHAO Zhi-wu, WANG Jun-shi, MA Min, ZHANG Shao-hua, YAN Jiong. *Effect of Down-regulated Perilipin 1* Gene Expression on Lipolysis of 3T3-L1 Adipocytes**[J]. China Biotechnology, 2016, 36(3): 17-22.

[14]	SONG Qi-ling, SHI Qiong, CHEN Chu, TANG Zu-chuan, LIU Hong-xia, ZHOU Yi-qing, ZHANG Ru-yi, YAN Shu-juan, WENG Ya-guang. MiR-21 Synergizes with BMP9 in Osteogenic Differentiation During Mesenchymal Stem Cells Line C3H10T1/2[J]. China Biotechnology, 2016, 36(2): 22-29.

[15]	TANG Da-gang, WANG Miao, ZHANG Yan-liang, WANG Xiao-lin, LI Gui-qiang, LUO Xiao-ji. Hey1 Gene Is Involved in Regulating BMP9-induced Osteogenic Differentiation of C3H10T1/2 Cells[J]. China Biotechnology, 2015, 35(6): 14-20.

Viewed

Full text

Abstract

Cited

Shared

Discussed