Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment

doi:10.7518/hxkq.2023.2022409

Abstract

Abstract:

Objective This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis. Methods An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions. Results After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05). Conclusion In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

Key words: human periodontal ligament fibroblasts, Foxp3, lipopolysaccharide, small interfering RNA, perio-dontitis

CLC Number:

R 783.5

Lu Ting, Zhu Jiahao, Yang Shihe, Shen Zhe, Zhong Liangjun. Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment[J]. West China Journal of Stomatology, 2023, 41(3): 269-275.

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引物	序列（5’-3’）
NC	F：UUCUCCGAACGUGUCACGUdTdT R：ACGUGACACGUUCGGAGAAdTdT
Foxp3-si1	F：GGAUGUGAGAAGGUCUUCGAA R：UUCGAAGACCUUCUCACAUCC
Foxp3-si2	F：GCACAUUCCCAGAGUUCCU R：AGGAACUCUGGGAAUGUGC
Foxp3-si3	F：CACUCAAUGAGAUCUACCA R：UGGUAGAUCUCAUUGAGUG

引物	序列（5’-3’）
18s	F：TAGAGGGACAAGTGGCGTTC R：CGCTGAGCCAGTCAGTGT
IL-6	F：CTGGATTCAATGAGGAGACTTGCC R：GGTTGGGTCAGGGGTGGTTA
IL-8	F：ACTCCAAACCTTTCCACC R：CTTCTCCACAACCCTCTG
Foxp3	F：CATCTGTGGCATCATCCGAC R：GTGGAGGAACTCTGGGAATGT

组别	样本量	Foxp3
NC	3	1.001±0.051 42
Foxp3-si1	3	0.592 6±0.081 35
Foxp3-si2	3	0.880 3±0.026 68
Foxp3-si3	3	0.184 4±0.043 35^#

组别	样本量	IL-6	IL-8
NC-LPS	3	1.029±0.280 5	1.013±0.193 8
Foxp3-si3-LPS	3	2.166±0.530 8	1.657±0.314 1
t值		3.282	3.020
P值		0.030 4	0.039 2

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