Effect of CPNE7 siRNA on cell proliferation and osteogenic differentiation in human periodontal ligament cells

Abstract

Abstract: PURPOSE: To investigate the effect and mechanism of CPNE7 siRNA on proliferation and osteogenic differentiation in human periodontal ligament cells (hPDLs). METHODS: hPDLs were isolated by enzyme digestion, transfected with pSUPER-CPNE7 in order to knock down CPNE7. The expression of CPNE7 mRNA and protein was detected by Western blot and RT-PCR; ELISA was carried out for the activity of NF-κB; hPDLs were pretreated with PDTC (10 μmol/L) for 30 min, CCK8 was used to evaluate the proliferation; ALP activity was assayed with ELISA; The expression of RUNX2, OSX and OCN was measured with RT-PCR and Western blot. The data were analyzed with SPSS11.0 software package. RESULTS: After transfection with pSUPER-CPNE7, CPNE7 expression was significantly decreased (P<0.05). ELISA assay indicated that CPNE7 siRNA enhanced NF-κB activity. CCK8 and RT-PCR assay showed that CPNE7 siRNA inhibited cell proliferation, ALP activity and decreased the expression of RUNX2, OSX and OCN in hPDLs; however, these effects were abolished by PDTC. CONCLUSIONS: CPNE7 siRNA inhibits the proliferation and osteogenic differentiation of hPDLs through NF-κB pathway.

Key words: Human periodontal ligament cells, Copine Ⅶ, Alkaline phosphatase, Osteogenic differentiation, NF-κB

LIU Cai-hong, HU Hai-yan. Effect of CPNE7 siRNA on cell proliferation and osteogenic differentiation in human periodontal ligament cells[J]. Shanghai Journal of Stomatology, 2016, 25(4): 420-425.

References

[1] Xia L, Zhang Z, Chen L, et al. Proliferation and osteogenic differentiation of human periodontal ligament cells on akermanite and β-TCP bioceramics [J]. Eur Cell Mater, 2011, 22: 68-82.
[2] Yu N, Prodanov L, te Riet Jt, et al. Regulation of periodontal ligament cell behavior by cyclic mechanical loading and substrate nanotexture [J]. J Periodontol, 2013, 84(10): 1504-1513.
[3] 曾婧, 王旭霞, 贾玉龙, 等. 帕米膦酸钠对大鼠正畸牙移动过程中破牙骨质细胞及破骨细胞分化因子表达的影响 [J]. 上海口腔医学, 2012, 21(5): 495-500.
[4] 郝轶, 李晶, 马佳音, 等. 牵张应变诱导人牙周膜细胞晚期凋亡的实验研究 [J]. 上海口腔医学, 2015, 24(4): 404-409.
[5] 王晓文, 范文红, 范明. Copines 蛋白家族的研究进展 [J]. 国外医学·生理病理科学与临床分册, 2005, 25(2): 153-155.
[6] Cowland JB, Carter D, Bjerregaard MD, et al. Tissue expression of copines and isolation of copines I and III from the cytosol of human neutrophils [J]. J Leukoc Biol, 2003, 74(3): 379-388.
[7] Lee J-H, Lee DS, Choung HW, et al. Odontogenic differentiation of human dental pulp stem cells induced by preameloblast-derived factors [J]. Biomaterials, 2011, 32(36): 9696-9706.
[8] Oh HJ, Choung HW, Lee HK, et al. CPNE7, a preameloblast-derived factor, regulates odontoblastic differentiation of mesenchymal stem cells [J]. Biomaterials, 2015, 37: 208-217.
[9] Chang J, Liu F, Lee M, et al. NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation [J]. Proc Natl Acad Sci USA, 2013, 110(23): 9469-9474.
[10] Jiang YX, Lu Y, Liu TJ, et al. Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification [J]. Mol Vis,2011,17: 291-299.
[11] Savino M, d'Apolito M, Centra M, et al. Characterization of copine VII, a new member of the copine family, and its exclusion as a candidate in sporadic breast cancers with loss of heterozygosity at 16q24. 3 [J]. Genomics, 1999, 61(2): 219-226.
[12] Ramsey CS, Yeung F, Stoddard PB, et al. Copine-I represses NF-κB transcription by endoproteolysis of p65 [J]. Oncogene, 2008, 27(25): 3516-3526.
[13] 王秋颖. 碱性磷酸酶特性及其应用的研究进展 [J]. 中国畜牧兽医, 2011, 38(1): 157-161.
[14] Liu TM, Lee EH. Transcriptional regulatory cascades in Runx2-dependent bone development [J]. Tissue En Part B Rev, 2013, 19(3): 254-263.
[15] Long F. Building strong bones: molecular regulation of the osteoblast lineage [J]. Nat Rev Mol Cell Biol, 2012, 13(1): 27-38.
[16] Zhao Y, Wang C, Li S, et al. Expression of Osterix in mechanical stress-induced osteogenic differentiation of periodontal ligament cells in vitro [J]. Eur J Oral Sci,2008, 116(3): 199-206.
[17] Matsubara T, Kida K, Yamaguchi A, et al. BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation[J]. J Biol Chem, 2008, 283(43): 29119-29125.
[18] Bai Y, Bai Y, Matsuzaka K, et al. Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells [J]. Cell Tissue Res, 2010, 342(2): 221-231.
[19] He H, Yu J, Cao J, et al. Biocompatibility and osteogenic capacity of periodontal ligament stem cells on nHAC/PLA and HA/TCP scaffolds [J]. J Biomater Sci Polym Ed, 2011, 22(1-3): 179-194.
[20] Krum SA, Chang J, Miranda-Carboni G, et al. Novel functions for NFκB: inhibition of bone formation [J]. Nat Rev Rheumatol, 2010, 6(10): 607-611.
[21] Eliseev RA, Schwarz EM, Zuscik MJ, et al. Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NFκB [J]. Exp Cell Res, 2006, 312(1): 40-50.
[22] Zhang J, Li ZG, Si YM, et al. The difference on the osteogenic differentiation between periodontal ligament stem cells and bone marrow mesenchymal stem cells under inflammatory microenviroments [J]. Differentiation, 2014, 88(4-5): 97-105.
[23] Lee SK, Choi HI, Yang YS, et al. Nitric oxide modulates osteoblastic differentiation with heme oxygenase-1 via the mitogen activated protein kinase and nuclear factor-kappaB pathways in human periodontal ligament cells [J]. Biol Pharm Bull, 2009, 32(8): 1328-1334.