Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANG Yangyang , PENG Xiaoxiang , SONG Wei , SUN Yanli , WANG Lujuan , LI Qian ,  ZHAO Ronglan

Department of Medical Laboratory, Weifang Medical University & Institutional Key Laboratory of Clinical Laboratory Diagnostics, 12th 5-Year Project of Shandong Province, WeifangMedical University & Key Discipline of Clinical Laboratory Medicine of Shandong Province, Affiliated Hospital of Weifang Medical University, Weifang Shangdong, 261053, P.R.China;

Corresponding author：

ZHAO Ronglan, Email: zhaoronglan76@sina.com

Keywords：

MicroRNA-140; chitosan; chondrocytes; signal peptide; rabbit

DOI：

10.7507/1002-1892.201705088

Video：

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Objective To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) (^NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Methods Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with ^NNSCS to form ^NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), ^NNSCS/GV268 empty plasmid transfection group (group B), and ^NNSCS/GV268-miR-140 transfection group (group C). ^NNSCS/GV268 and ^NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. Results RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B (P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated (P<0.05). There was no significant difference in above indexes between groups A and B (P>0.05). Conclusion Exogenous gene can be carried into the chondrocytes by ^NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.

Citation： ZHANG Yangyang, PENG Xiaoxiang, SONG Wei, SUN Yanli, WANG Lujuan, LI Qian, ZHAO Ronglan. Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(10): 1256-1261. doi: 10.7507/1002-1892.201705088 Copy

1.	Ortved KF, Begum L, Mohammed HO, et al. Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model. Mol Ther, 2015, 23(2): 363-373.
2.	Frisch J, Rey-Rico A, Venkatesan JK, et al. rAAV-mediated overexpression of sox9, TGF-β and IGF-I in minipig bone marrow aspirates to enhance the chondrogenic processes for cartilage repair. Gene Ther, 2016, 23(3): 247-255.
3.	Wang X, Guo Y, Wang C, et al. MicroRNA-142-3p inhibits chondrocyte apoptosis and inflammation in osteoarthritis by targeting HMGB1. Inflammation, 2016, 39(5): 1718-1728.
4.	Frisch J, Venkatesan JK, Rey-Rico A, et al. Effects of rAAV-mediated FGF-2 gene transfer and overexpression upon the chondrogenic differentiation processes in human bone marrow aspirates. J ExpOrthop, 2016, 3(1): 16.
5.	Zhao R, Peng X, Li Q, et al. Effects of phosphorylatable short peptide-conjugated chitosan-mediated IL-1Ra and igf-1 gene transfer on articular cartilage defects in rabbits. PLoS One, 2014, 9(11): e112284.
6.	Tao K, Rey-Rico A, Frisch J, et al. rAAV-mediated combined gene transfer and overexpression of TGF-β and SOX9 remodels human osteoarthritic articular cartilage. J Orthop Res, 2016, 34(12): 2181-2190.
7.	Wang GD, Zhao XW, Zhang YG, et al. Effects of miR-145 on the inhibition of chondrocyte proliferation and fibrosis by targeting TNFRSF11B in human osteoarthritis. Mol Med Rep, 2017, 15(1): 75-80.
8.	Gabler J, Ruetze M, Kynast KL, et al. Stage-specific miRs in chondrocyte maturation: differentiation-dependent and hypertrophy-related miR clusters and the miR-181 family. Tissue Eng Part A, 2015, 21(23-24): 2840-2851.
9.	Min Z, Zhang R, Yao J, et al. MicroRNAs associated with osteoarthritis differently expressed in bone matrix gelatin (BMG) rat model. Int J ClinExp Med, 2015, 8(1): 1009-1017.
10.	Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.
11.	Karlsen TA, de Souza GA, Ødegaard B, et al. MicroRNA-140 inhibits inflammation and stimulates chondrogenesis in a model of interleukin 1β-induced osteoarthritis. MolTher Nucleic Acids, 2016, 5(10): e373.
12.	Boonthum C, Namdee K, Boonrungsiman S, et al. Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor. Carbohydr Polym, 2017, 157: 311-320.
13.	Eslaminejad T, Nematollahi-Mahani SN, Ansari M. Cationic β-Cyclodextrin-Chitosan conjugates as potential carrier for pmCherry-C1 gene delivery. Mol Biotechnol, 2016, 58(4): 287-298.
14.	Zhao R, Sun B, Liu T, et al. Optimize nuclear localization and intra-nucleus disassociation of the exogene for facilitating transfection efficacy of the chitosan. Int J Pharm, 2011, 413(1-2): 254-259.
15.	赵荣兰, 彭效祥. 可磷酸化短肽偶联壳聚糖介导 IL-1RA 与 IGF-1 共转染对兔关节软骨细胞的作用. 中国生物化学与分子生物学报, 2014, 30(12): 1250-1256.0.
16.	Peng XX, Zhao RL, Song W, et al. Selection of suitable reference genes for normalization of quantitative real-time PCR in cartilage tissue injury and repair in rabbits.Int J MolSci, 2012, 13 (11): 14344-14355.
17.	Gibson G, Asahara H. microRNAs and cartilage. J Orthop Res, 2013, 31(9): 1333-1344.
18.	Tuddenham L, Wheeler G, Ntounia-Fousara S, et al.The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Letters, 2006, 580(17): 4214-4217.
19.	Gibson AL, Hui Mingalone CK, Foote AT, et al. Wnt7a inhibits IL-1β induced catabolic gene expression and prevents articular cartilage damage in experimental osteoarthritis. Sci Rep, 2017, 7:41823.
20.	Genemaras AA, Ennis H, Kaplan L, et al. Inflammatory cytokines induce specific time- and concentration-dependent MicroRNA release by chondrocytes, synoviocytes, and meniscus cells. J Orthop Res, 2016, 34(5): 779-790.
21.	闫虎, 苏友新, 林学义. IL-1β 诱导新西兰大白兔膝关节退变软骨细胞的体外培养及鉴定. 中国中西医结合杂志, 2014, 34(1): 81-86.0.
22.	Zhang M, Lu Q, Miller AH, et al. Dynamic epigenetic mechanisms regulate age-dependent SOX9 expression in mouse articular cartilage. Int J Biochem Cell Biol, 2016, 72: 125-134.
23.	Zhang Q, Ji Q, Wang X, et al. SOX9 is a regulator of ADAMTSs-induced cartilage degeneration at the early stage of human osteoarthritis. Osteoarthritis Cartilage, 2015, 23(12): 2259-2268.
24.	Simental-Mendía M, Lara-Arias J, Álvarez-Lozano E, et al. Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans. Braz J Med Biol Res, 2015, 48(12): 1063-1070.
25.	Lu J, Sun Y, Ge Q, et al. Histone deacetylase 4 alters cartilage homeostasis in human osteoarthritis. BMC MusculoskeletDisord, 2014, 15: 438.
26.	Makki MS, Haqqi TM. Histone deacetylase inhibitor vorinostat (SAHA) suppresses IL-1β-induced matrix metallopeptidase-13 expression by inhibiting IL-6 in osteoarthritis chondrocyte. Am J Pathol, 2016, 186(10): 2701-2708.
27.	Zamani S, Hashemibeni B, Esfandiari E,et al. Assessment of TGF-β3 on production of aggrecan by human articular chondrocytes in pellet culture system.Adv Biomed Res, 2014, 3: 54.
28.	Haller JM, Swearingen CA, Partridge D,et al. Intraarticular matrix metalloproteinases and aggrecan degradation are elevated after articular fracture. ClinOrthopRelat Res, 2015, 473(10): 3280-3288.

1. Ortved KF, Begum L, Mohammed HO, et al. Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model. Mol Ther, 2015, 23(2): 363-373.
2. Frisch J, Rey-Rico A, Venkatesan JK, et al. rAAV-mediated overexpression of sox9, TGF-β and IGF-I in minipig bone marrow aspirates to enhance the chondrogenic processes for cartilage repair. Gene Ther, 2016, 23(3): 247-255.
3. Wang X, Guo Y, Wang C, et al. MicroRNA-142-3p inhibits chondrocyte apoptosis and inflammation in osteoarthritis by targeting HMGB1. Inflammation, 2016, 39(5): 1718-1728.
4. Frisch J, Venkatesan JK, Rey-Rico A, et al. Effects of rAAV-mediated FGF-2 gene transfer and overexpression upon the chondrogenic differentiation processes in human bone marrow aspirates. J ExpOrthop, 2016, 3(1): 16.
5. Zhao R, Peng X, Li Q, et al. Effects of phosphorylatable short peptide-conjugated chitosan-mediated IL-1Ra and igf-1 gene transfer on articular cartilage defects in rabbits. PLoS One, 2014, 9(11): e112284.
6. Tao K, Rey-Rico A, Frisch J, et al. rAAV-mediated combined gene transfer and overexpression of TGF-β and SOX9 remodels human osteoarthritic articular cartilage. J Orthop Res, 2016, 34(12): 2181-2190.
7. Wang GD, Zhao XW, Zhang YG, et al. Effects of miR-145 on the inhibition of chondrocyte proliferation and fibrosis by targeting TNFRSF11B in human osteoarthritis. Mol Med Rep, 2017, 15(1): 75-80.
8. Gabler J, Ruetze M, Kynast KL, et al. Stage-specific miRs in chondrocyte maturation: differentiation-dependent and hypertrophy-related miR clusters and the miR-181 family. Tissue Eng Part A, 2015, 21(23-24): 2840-2851.
9. Min Z, Zhang R, Yao J, et al. MicroRNAs associated with osteoarthritis differently expressed in bone matrix gelatin (BMG) rat model. Int J ClinExp Med, 2015, 8(1): 1009-1017.
10. Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.
11. Karlsen TA, de Souza GA, Ødegaard B, et al. MicroRNA-140 inhibits inflammation and stimulates chondrogenesis in a model of interleukin 1β-induced osteoarthritis. MolTher Nucleic Acids, 2016, 5(10): e373.
12. Boonthum C, Namdee K, Boonrungsiman S, et al. Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor. Carbohydr Polym, 2017, 157: 311-320.
13. Eslaminejad T, Nematollahi-Mahani SN, Ansari M. Cationic β-Cyclodextrin-Chitosan conjugates as potential carrier for pmCherry-C1 gene delivery. Mol Biotechnol, 2016, 58(4): 287-298.
14. Zhao R, Sun B, Liu T, et al. Optimize nuclear localization and intra-nucleus disassociation of the exogene for facilitating transfection efficacy of the chitosan. Int J Pharm, 2011, 413(1-2): 254-259.
15. 赵荣兰, 彭效祥. 可磷酸化短肽偶联壳聚糖介导 IL-1RA 与 IGF-1 共转染对兔关节软骨细胞的作用. 中国生物化学与分子生物学报, 2014, 30(12): 1250-1256.0.
16. Peng XX, Zhao RL, Song W, et al. Selection of suitable reference genes for normalization of quantitative real-time PCR in cartilage tissue injury and repair in rabbits.Int J MolSci, 2012, 13 (11): 14344-14355.
17. Gibson G, Asahara H. microRNAs and cartilage. J Orthop Res, 2013, 31(9): 1333-1344.
18. Tuddenham L, Wheeler G, Ntounia-Fousara S, et al.The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Letters, 2006, 580(17): 4214-4217.
19. Gibson AL, Hui Mingalone CK, Foote AT, et al. Wnt7a inhibits IL-1β induced catabolic gene expression and prevents articular cartilage damage in experimental osteoarthritis. Sci Rep, 2017, 7:41823.
20. Genemaras AA, Ennis H, Kaplan L, et al. Inflammatory cytokines induce specific time- and concentration-dependent MicroRNA release by chondrocytes, synoviocytes, and meniscus cells. J Orthop Res, 2016, 34(5): 779-790.
21. 闫虎, 苏友新, 林学义. IL-1β 诱导新西兰大白兔膝关节退变软骨细胞的体外培养及鉴定. 中国中西医结合杂志, 2014, 34(1): 81-86.0.
22. Zhang M, Lu Q, Miller AH, et al. Dynamic epigenetic mechanisms regulate age-dependent SOX9 expression in mouse articular cartilage. Int J Biochem Cell Biol, 2016, 72: 125-134.
23. Zhang Q, Ji Q, Wang X, et al. SOX9 is a regulator of ADAMTSs-induced cartilage degeneration at the early stage of human osteoarthritis. Osteoarthritis Cartilage, 2015, 23(12): 2259-2268.
24. Simental-Mendía M, Lara-Arias J, Álvarez-Lozano E, et al. Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans. Braz J Med Biol Res, 2015, 48(12): 1063-1070.
25. Lu J, Sun Y, Ge Q, et al. Histone deacetylase 4 alters cartilage homeostasis in human osteoarthritis. BMC MusculoskeletDisord, 2014, 15: 438.
26. Makki MS, Haqqi TM. Histone deacetylase inhibitor vorinostat (SAHA) suppresses IL-1β-induced matrix metallopeptidase-13 expression by inhibiting IL-6 in osteoarthritis chondrocyte. Am J Pathol, 2016, 186(10): 2701-2708.
27. Zamani S, Hashemibeni B, Esfandiari E,et al. Assessment of TGF-β3 on production of aggrecan by human articular chondrocytes in pellet culture system.Adv Biomed Res, 2014, 3: 54.
28. Haller JM, Swearingen CA, Partridge D,et al. Intraarticular matrix metalloproteinases and aggrecan degradation are elevated after articular fracture. ClinOrthopRelat Res, 2015, 473(10): 3280-3288.

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Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes

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