| Objective and meaning:Matrix metalloproteinases (MMPs) was initially described as the extracellular matrixsubstrate until its structural and functional properties had been found and then it wasdefined as a member of enzyme family. It is mainly divided into three categories:collagenase; gelatinase and matrilysin. Numerous studies have shown that collagenaseexisted in the saliva and tissues of the periodontitis patients and would increase with theaggravation of inflammation. One of the most notable is the MMP-2which its expressionwould reduce significantly after periodontitis being effective treated. However, the targetedregulation of MMP-2upstream sites has not been reported. The current discovery anddevelopment of miRNAs have opened up new ideas for the study of periodontitis.The miRNAs are composed of21to24nucleotides, and are small non-coding RNAmolecules that existing among eukaryotic cells and regulating cell behavior by inhibitingtranscription and translation of the protein. The miRNAs not only play a part in cellmigration, proliferation, differentiation and other physiological processes, but also closelyrelate to the development of inflammatory diseases. Studies showed that miRNAs hadnegative feedback regulation mechanism of inflammation. For example, the miR-181b inpatients with psoriasis could regulate the inflammatory response by inhibiting theexpression of nuclear factor NF-KB, and inflammatory stimulation might also reduce theexpression of miR-181b. As endogenous small RNAs, miRNAs are likely to play animportant role in regulating the occurrence and development of periodontitis.The peridontal ligament cells (PDLCs) were discovered by Arnold. They come fromthe periodontal tissue, are the main stromal cells of the periodontal connective tissues andplay a key role in the formation and regeneration of the periodontal tissues. Theproliferation and differentiation of the PDLCs play an important part in tissue regenerationand repair process, while the apoptosis of PDLCs is an important pathophysiologicalprocess of periodontal destruction and repairment. We assume that miRNAs could affect the occurrence and development of periodontitisby regulating the expression of MMP-2. In this experiment, we predicted the miRNAswhich could regulate the MMP-2by biological information method and verified therelationship of miRNA in periodontal ligament cells regulating the expression ofMMP-2mRNA by qRT-PCR and Westernblot. Whether the miRNA acting on theMMP-23’UTR target sites was verify by dual luciferase.Research methods and procedures:1. Biological information detection: By DIANA-microT, TargetScan, miRanda, PITAthese four kinds of online prediction software to predict the possible miRNAs that targetedto MMP-23’UTR. Integrating the results of four prediction softwares and chosing thoseranking in front were the principles.2. Cultivation and identification of PDLCs: to collect healthy and non-cariousmaxillary premolars from surgical orthodontics in the outpatient department of stomatologyof Chongqing Xinqiao Hospital, Third Military Medical University. To isolate cells byenzymatic digestion method and culture conventionally; cell growth conditions and theirchanges were observed under an inverted microscope and the cell phenotype of the secondgeneration cells were detected by immunohistochemical method.3. Validation the regulation of miRNAs on MMP-2: PDLCs were transfected bymimics-miRNAs and miRNAs negative control (control group), then to detect theexpression changes of MMP-2mRNA and its protein in cells by qRT-PCR and Western blot.To verify the target genes by dual luciferase. The wild type of MMP-2, its mutant plasmid,miRNA mimics and miRNA inhibitors were co-transfected in PDLCs, negative control andreporter plasmids were co-transfected as the negative control, while GV272was the blankcontrol.Research results:1. Through online prediction software TargetScan, DIANA-microT, miRanda and PITA to predictthe possible miRNAs that targeted to MMP-23’UTR. By integrating the four different softwarealgorithms, the prediction that miR-520h, miR-520g, miR-519d-3p, miR-519c-3p and miR-519d thesefive miRNAs might target to the MMP-23’UTR has made.2. The human PDLCs proliferated rapidly with continuous cultivation, and were colony formed.2to4cell processes could be seen in a single cell. Immunohistochemical method showed that anti-keratin negative and anti-vimentin positive.3. After transfected by mimics-miRNAs, the miRNA expression of human periodontal ligamentcells was increased significantly compared with the control group(P<0.01). After transfected bymiR-520h-mimics, miR-520g-mimics, miR-519d-3p-mimics and miR-519c-3p-mimics, humanperiodontal ligament cells could obviously inhibit the expression level of MMP-2mRNA and itsprotein (P <0.05). However, there were no protein expression changes in MMP-2mRNA or its proteinafter transfected by miR-519d-mimics (P>0.05). The miR-520h-mimics, miR-520g-mimics, miR-519d-3p mimics, miR-519c-3p-mimics and the wild-type of MMP-2transfected respectively withPDLCs would cause a decrease of luciferase activity (P<0.05), while in the mutant plasmid group it didnot change. Luciferase activity did not change after miR-519d-mimics and wild-type plasmid of MMP-2transfected into cells which was consistent with the results of PCR and Western (P<0.05).Conclusions:1. Five MiRNAs miR-520h, miR-520g, miR-519b-3p, miR-519c-3p and miR-519d that predictedby the integrated results of the miRNAs prediction softwares could negatively regulate the expression ofMMP-2.2. After transfected with miR-mimics group, the miR-520h, miR-520g, miR-519b-3p, miR-519c-3pand miR-519d of human periodontal ligament cells showed a high expression.3. The miR-520h, miR-520g, miR-519b-3p and miR-519c-3p in periodontal ligament cells coulddown regulate the expression level of MMP-2mRNA and its protein. And these miRNAs could regulatethe MMP-2expression successfully by direct sequence complementary pairing with MMP-23’UTR andinhibiting the translation of MMP-2m RNA. |
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