Li Feng-Xia, Wang Jun, Ma Yu-Yun
Department of Stomatology,Ankang Hospital of Traditional Chinese Medicine. Ankang 725000, Shaanxi Province, China. E-mail:
Shanghai Kou Qiang Yi Xue. 2017 Oct;26(5):504-509.
To investigate the effect of CCN3 on proliferation and apoptosis in periodontal ligament fibroblasts (PDLFs) and related mechanism.
Recombinant vector pcDNA.3.1-CCN3 was constructed and transfected into human PDLFs to overexpress CCN3. CCN3 siRNA was transfected to inhibit CCN3. Fra-1 siRNA was transfected into the PDLFs with CCN3 inhibition to realize the inhibition of CCN3 and Fra-1 in the meantime. mRNA expressions of CCN3 and Fra-1 were measured by quantitative real-time PCR (qRT-PCR). The protein expressions of CCN3, Fra-1and Bcl-2 were detected by Western blot. Cell growth and viability and proliferation of PDLFs were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assays; Caspase-3 activity was tested by using the available kit. The data were analyzed with SPSS20.0 software package.
The results showed that the mRNA (P<0.05) and protein (P<0.05) expressions of CCN3 were significantly up-regulated in the experimental group of pcDNA.3.1-CCN3 transfection. In addition, the mRNA (P<0.05) and protein (P<0.05) expressions of CCN3 were significantly decreased in the experimental group of CCN3 siRNA transfection. Cell growth (P<0.05) and viability, proliferation (P<0.05) and Bcl-2 (P<0.05) protein expression were increased, while caspase-3 activity (P<0.05) decreased in the PDLFs with CCN3 inhibition. However, CCN3 overexpression exhibited reversed effect. CCN3 overexpression or inhibition could remarkably constrain (P<0.05) or promote (P<0.01) the expression of Fra-1, respectively. Moreover, co-inhibition of CCN3 and Fra-1 could promote apoptosis (P<0.01) and inhibit proliferation (P<0.05) in PDLFs.
The results suggest that inhibition of CCN3 could accelerate proliferation and constrain apoptosis via up-regulating expression of Fra-1 in PDLFs.
研究CCN3对牙周膜成纤维细胞（PDLFs）增殖和凋亡的影响及其相关机制。
构建重组载体pcDNA.3.1-CCN3并转染至人PDLFs中以过表达CCN3。转染CCN3 siRNA以抑制CCN3。将Fra-1 siRNA转染至CCN3受到抑制的PDLFs中，以同时实现对CCN3和Fra-1的抑制。通过定量实时PCR（qRT-PCR）检测CCN3和Fra-1的mRNA表达。通过蛋白质印迹法检测CCN3、Fra-1和Bcl-2的蛋白质表达。采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和溴脱氧尿苷（BrdU）检测法测量PDLFs的细胞生长、活力和增殖情况；使用现成试剂盒检测Caspase-3活性。数据采用SPSS20.0软件包进行分析。
结果显示，在pcDNA.3.1-CCN3转染实验组中，CCN3的mRNA（P<0.05）和蛋白质（P<0.05）表达显著上调。此外，在CCN3 siRNA转染实验组中，CCN3的mRNA（P<0.05）和蛋白质（P<0.05）表达显著降低。在CCN3受到抑制的PDLFs中，细胞生长（P<0.05）、活力、增殖（P<0.05）和Bcl-2（P<0.05）蛋白质表达增加，而Caspase-3活性（P<0.05）降低。然而，CCN3过表达表现出相反的作用。CCN3过表达或抑制可分别显著抑制（P<0.05）或促进（P<0.01）Fra-1的表达。此外，同时抑制CCN3和Fra-1可促进PDLFs凋亡（P<0.01）并抑制增殖（P<0.05）。
结果表明，抑制CCN3可通过上调PDLFs中Fra-1的表达来加速增殖并抑制凋亡。
