| Periodontitis is one of the most common oral diseases. Tobacco smoking is recognized as one of main risk factors for periodontitis. Nicotine, the active ingredient in tobacco, causes many pathological changes in human periodontal tissues, such as periodontal inflammation, destruction of periodontal tissues, decreases in bone volume, and ultimately the loss of teeth. α7 n ACh R is a predominant subunit of nicotinic acetylcholine receptors(n ACh Rs) and a potent target of the nicotine binding receptor. In previous studies, we demonstrated the functional expression of α7 n ACh R in human periodontal ligament(PDL) cells and rat periodontal tissues. Upregulation of α7 n ACh R upon the administration of nicotine is inhibited by pre-treatment with α-BTX, suggesting that nicotine might induce smoking-related periodontitis via α7 n ACh R. Both tobacco smoking and nicotine have been reported to regulate the occurrence and progression of periodontitis. Many studies have demonstrated that nicotine destroys regeneration of periodontal tissues and aggravates the inflammatory injury in periodontal tissue primarily by inhibiting the cell proliferation and activating inflammatory signal pathways in human PDL cells. However, the mechanism underlying this process is still unclear.Recent evidences suggest that micro RNAs may play an important role in controlling the levels of multiple cell cycle regulators and inflammatory reaction in periodontitis. mi R-30 a is a member of the mi R-30 family, and it is involved in a variety of biological processes, including autophagy, cell cycle, apoptosis, migration and proliferation. Compared to healthy periodontal tissues, Studies have demonstrated that mi R-30 a is highly expressed in periodontal tissues of patients with periodontitis. However, the role of mi R-30 a in periodontitis is still poorly understood. Plenty of studies have demonstrated that mi R-30 a is vital for cell cycle progression and inflammatory reaction recently. In addition, bioinformatics data indicates that mi R-30 a could regulate the expression of CCNE2 and SOCS3, further regulating G1/S transition and JAK2-STAT3 pathway in human periodontal ligament cells. Nevertheless, the relationship among nicotine, mi R-30 a, CCNE2 and SOCS3 in human PDL cells is still not clear.【Aims】 We aimed to identify the effects of nicotine on the expression of mi R-30 a, and further investigating whether mi R-30 a inhibited the cell proliferation and activated JAK2-STAT3 pathway in human periodontal ligament cells by targeting CCNE2 and SOCS3 in vitro. Our results showed for the ?rst time that mi R-30 a plays a key role in smoking associated periodontitis, providing a theoretical basis and new therapeutic targets for the prevention and treatment of periodontitis.【Materials and methods】 1. Periodontal tissues were took from huamn first premolars for orthodontic treatment. Using tissue block method to culture human periodontal ligament cells. Using flow cytometry analysis to identify the cell type. Using micro RNA microarray and quantitative real-time PCR to identify that nicotine could regulate some mi RNAs to participate in the occurrence and development of smoking –related periodontitis. 2. Human periodontal ligament cells were treated with 10-5 M nicotine and/or 10-8M α-BTX and/or mi R-30 a inhibitor. We used MTT and flow cytometry analysis to detect the proliferation and cell cycle distribution of human periodontal ligament cells. Westernblot and real-time PCR to detect mRNA and protein expression of CCNE2. Construction wild type and mutant vectors of CCNE2 3 ’UTR, dual luciferase reporter experiments to verify the direct binding CCNE2 3’UTR region to the mi R-30 a. Design CCNE2 interference RNA to verify the key role in regulating cell proliferation and cell cycle of human periodontal ligament cells. 3. Human periodontal ligament cells were treated with 10-5 M nicotine and/or 10-8 M α-BTX and/or mi R-30 a inhibitor. Western blot and real-time PCR dual luciferase reporter gene assay to detect the expressions of SOCS3, JAK2, p-JAK2, STAT3, p-STAT3, IL-1 beta, IL-6 and TNF-alpha in human periodontal ligament cells. Dual luciferase reporter gene assay to detect the activity of STAT3. Construction wild type and mutant vectors of SOCS3 3 ’UTR, dual luciferase reporter experiments to verify the direct binding SOCS3 3’UTR region to the mi R-30 a. Design SOCS3 interference RNA to verify the key role in regulating JAK2-STAT3 pathway in human periodontal ligament cells. 4. Nicotine can significantly upregulate the expression of pre-mi R-30 a, suggesting that the inhibitory effects of nicotine on mi R-30 a happen at the level of transcription initiation.Therefore, we analyzed promoter sequence of mi R-30 a and found binding sites of transcription factor NF-κB exist in the promoter region of mi R-30 a. Human periodontal ligament cells were treated with 10-5 M nicotine and/or 10-8 M α-BTX and/or 5x10-5 M PDTC and/or 10-7 M Cucurbitacin I. Using quantitative real-time PCR and Western blot experiments to detect expressions of mi R-30 a and NF-κB p65. Dual luciferase reporter gene assay to detect the activity of NF-κB in human periodontal ligament cells.【Results】 1. After culturing for six days, fibroblast like cells appeared around tissue block. When cells aggregated to 90%, we producted routine cell passage. Flow cytometry analysis indicate that high expression of the mesenchymal cell phenotype: CD29, CD105 and low expression phenotype: CD31, CD14. We found that 20 upregulated micro RNAs and 16 downregulated micro RNAs after stimulating with nicotine through micro RNAmicroarray in human periodontal ligament cells. In addition, we further confirmed 2 upregulated mi RNAs(mi R-30 a and let-7f) and 2 downregulated mi RNAs(mi R-21 and let-7e) by quantitative real-time PCR. We found that nicotine could upregulate the expression of mi R-30 a in a time dependent manner, while let-7f, mi R-21 and let-7e showed no obvious rules in expressions. We chose mi R-30 a to product the in-depth study to verify its roles in the development of periodontitis. 2. We observed an inhibitory proliferation rate of human PDL cells treated with 10-5M nicotine compared to the control, mi R-30 a inhibitor could partially inhibited these effects caused by nicotine. Flow cytometry analysis indicated that nicotine could induce G1 arrest in human periodontal cells. We used q RT-PCR and western blot to examine the effects of nicotine on m RNA and protein expression of CCNE2 in human PDL cells, and we found that nicotine significantly inhibits m RNA expression and protein expression of CCNE2 compared to the control group. Transfected the human PDL cells with mi R-30 a inhibitor could significantly inhibit the expression of mi R-30 a in periodontal ligament cells. Transfected the human PDL cells with mi R-30 a inhibitor, then treated with nicotine. We found that down-regulation of mi R-30 a expression may regulate nicotine –activated inhibited expression of CCNE2, proliferation and cell cycle of human periodontal ligament cells. Dual luciferase reporter gene experiments confirmed that mi R-30 a can directly bind to the CCNE2 3 ’UTR region, then regulates its expression. We found that CCNE2 play a key role in regulating cell proliferation and cell cycle of human periodontal ligament cells through CCNE2 interference RNA experiment. 3. Nicotine could activate JAK2-STAT3 signal pathway in human periodontal ligament cells: we found that nicotine significantly upregulate protein expression of phosphorylated JAK2 and STAT3, and activated the STAT3 activity through dual luciferase reporter gene assay. In addition, nicotine could also inhibit the expressions of IL-1 beta, IL-6 and TNF-α. Concentration gradient experiments of Cucurbitacin I, STAT3 inhibitor, demonstrated that 10-6 M Cucurbitacin I can significantly inhibit the phosphorylation STAT3 and STAT3 activity, and 10-6 M Cucurbitacin I showed lower toxicity compared with 10-5 M Cucurbitacin I, so we chose 10-6 M Cucurbitacin I for the follow-upexperiments. These results suggest that nicotine could participate in smoking associated periodontitis inflammatory injury process through the JAK2-STAT3 pathway. Transfected the human PDL cells with mi R-30 a inhibitor, then treated with nicotine. We found that down-regulation of mi R-30 a expression may regulate nicotine –activated JAK2-STAT3 pathway, upregulated expressions of IL-1 beta, IL-6 and TNF-α in human periodontal ligament cells. Dual luciferase reporter gene experiments confirmed that mi R-30 a can directly bind to the SOCS3 3’ UTR region, then regulates its expression. We found that SOCS3 play a key role in regulating JAK2-STAT3 pathway in human periodontal ligament cells through SOCS3 interference RNA experiment. 4. We found that binding sites of transcription factor NF-κB exist in the promoter region of mi R-30 a through analysis of the DNA region upstream of the transcriptional start site of the 5000 bp to the downstream of 1000 bp. When PDTC inhibits the expression of NF-κB, the expression level of mi R-30 a is inhibited, suggesting that NF-κB may regulate mi R-30 a expression through binding the promoter region of mi R-30 a. Cucurbitacin I can significantly reduce the expression of NF-κB subunit p65 and NF-κB activity, suggesting that STAT3 may regulate activity of NF-κB.【Conclusions】 1. We cultured and identified human periodontal ligament cells in vitro successfully. The micro RNA microarray and quantitative real-time PCR indicate that nicotine may participate in the occurrence and development of smoking –related periodontitis through upregulating mi R-30 a expression. 2. Nicotine –upregulated mi R-30 a may regulate cell proliferation and cell cycle of periodontal ligament cells by directly targeting CCNE2, thereby affecting the repair process of smoking-related periodontitis. 3. Nicotine –upregulated mi R-30 a may regulate JAK2-STAT3 pathway in human periodontal ligament cells by directly targeting SOCS3, thereby aggravating inflammatory destruction of periodontal tissue. 4. Nicotine may regulate the expression of mi R-30 a by activating NF-κB, and STAT3 couldregulate the activity of NF-κB, thus constituting a NF-κB-mi R-30a-STAT3 feedback loop to participate in the development of smoking-related periodontitis. |
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