Possible Mechanism Of Crosstalk Between MicroRNA And RUNX2gene In Human Dental Follicle Cells

The gene mutation of RUNX2（RUNT-related transcription factor2,Cbfal） in CCD（cleidocranial dysplasia） patient would lead to delayed eruption of permanent tooth. Dental follicle cells have potency of multi-directional differentiation, regulating osteoblasts and osteoclasts interact activity. Tooth eruption is a complex process, and the mechanism has yet not well clarified. So the dental follicle cells of unerupted teeth are natural human models to study eruption abnormalities. MicroRNA（miRNA）, are the hotspot recently, which have been reported as powerful post-transcriptional regulators of gene expression. It is revealed that there is a crosstalk between TF（transcription factor） and miRNA in human protein interaction network.In the present study, to study the effect of RUNX2and miRNA network on the proliferation and differentiation of HDFCs, the characteristics of DFCs was analyzed and the differential expression of miRNA was identified, which provided new insights into understanding of delayed tooth eruption in CCD patients.The study was divided into three parts described as followings.1.The patient with CCD and identification of RUNX2gene mutationObjective:To study the RUNX2gene mutation in a patient with cleidocranial dysplasia （CCD）.Methods:In this part, a case of CCD was reported.The patient was a21-year-old male, who was referred to our hospital complaining of late eruption of permanent dentition and dental abnormalities. The medical history was collected. The unaffected parents and sibling were included. Radiological examination regarding osseous malformations was taken. And examination was carried out over the oral cavity and the entire body. This patient was characterized by facial abnormalities: wide set eyes, collapsed bridge of nose, deficient development of middle face. The oral examinations revealed retained deciduous teeth, delayed eruption of permanent tooth, supernumerary teeth and impacted teeth. And radiological examinations revealed hypoplastic clavicles, delayed closure of the anterior fontanel, deformity of interphalangeal articulations of the1st phalanges joint. According to the diagnostic criteria, the patient was diagnosed as CCD. His older sister and their parents with no abnormal symptom were also investigated as control. Genomic DNA was extracted from whole blood. And the exons0-7of RUNX2gene was amplified by PCR from genomic DNA, using primers which were reported in our previous study. And DNA sequences were analyzed using BLASTN nucleotide program. RUNX2+/m and RUNX2+/m HDFCs was cultured. Total RNAs from the cultured dental follicle cells were isolated and reverse-transcribed. cDNA sequences of HDFCs were analyzed to confirm the mutation analysis of DNA. And Expression of RUNX2mRNA was examined by real-time reverse-transcription PCR （qRT-PCR）.Results:DNA sequences were analyzed, and a novel insertion mutation of TG in the exon2was identified, the control group did not carry the same mutation. This mutation would result in multilocus peakoverlaps in downstream, and frame-shift mutated the code GAG to TGG in104, then premature termination code TAG in144, therefore the mutation is predicted to yield a amino acids lacking in the C-terminal. And the same mutation was found in dental follicle cells cDNA too （c.309₃10insTG）.Gene expression detected by real-time RT-PCR revealed lower expression of RUNX2mRNA, but there was no statistical difference（P>0.05）.While the healthy people in this family（his older sister and their parents） with no abnormal symptom did not carry the same mutation.Conclusion:This result could further verify that RUNX2mutations were responsible for the development of cleidocranial dysplasia. In the SNP database, the c.309₃10insTG was absent from the SNPs within the homo sapiens RUNX2gene, suggesting that this CCD patient possessed a novel mutation, which supplemented the gene mutation database of CCD.2. miRNA microarray analysis and target gene prediction.Objective:To study the differential expressed miRNA between human RUNX2+/m and RUNX2+/+dental follicle cells, and predict the target genes.Methods:The experimental tooth was an embedded mandibular premolar from the patient with CCD above-mentioned, not rotated and inverted, with complete dental follicle and no deciduous teeth retained above. The control tooth from normal patient matched in age, gender and tooth position. Dental follicles were washed by PBS and cut into1-2mm3small blocks. Explant culture method was used. Dental follicle cells （HDFCs） were cultured in Dulbecco’s modified Eagle’s medium. Nonadherent tissues were removed3days later and medium was changed every3days. Cells were obtained with enzymes, and1:3passaged. The morphology of HDFCs were observed by microscope. And the mesenchymal natures were identified as vimentin-positive and cytokeratin-negative. HDFCs at passage3to5were collected for further experiments. RUNX2+/m and RUNX2+/+HDFCs at passage5were used for miRNA microarray analysis. Total RNAs from the cultured dental follicle cells were isolated and reverse-transcribed. RNA ladder was detected by agarose gel electrophoresis.. TaqMan（?） Array Human MicroRNA A+B Cards Set v3.0were used for miRNA expression analysis. The evaluation of relative differences between dental follicle cells with RUNX2mutation and RUNX2+/+dental follicle cells was carried out by the comparative cycle threshold （CT）. Data analysis were performed using Average Delta Ct protocols. Potential targets of differentially expressed miRNA were searched using the miRNA target prediction application miRGen. GO function and KEGG pathway enrichments were performed using DAVID bioinformatics resources. Changes in miRNA expression profiles were indicated by miRNA microarray analysis.Results:Compared with the normal dental follicle cells, there were69miRNA more than2-fold up-regulated and54miRNA more than2-fold down-regulated in the dental follicle cells with RUNX2mutation. There were41miRNA with target genes by miRNA target forecast tool analysis. The total target genes was2718, eight of which related to tooth eruption or osteoblast/osteoclast differentiation. Gene Ontology （GO） function and KEGG pathway enrichments in DAVID bioinformatics resources were used to analyze functional annotations.The result of GO functions enrichments analysis revealed that the main proteins function of the target genes were cellular components and process, the binding of signal and its receptor. Though the enriched pathways indicated by KEGG were pathways in cancer and MAPK signaling pathway, only4.6%-2.2%target genes were involved in. We though it was not enough to identify the relationship between the target genes and the pathway, so these result of KEGG pathway was not further discussed.Conclusion:The results of this part suggested that RUNX2mutation have some influence on miRNA expression, further study was necessary for the expression and function of these miRNA. 3. Expression of miR-146a and miR-335in HDFCs and functional analysis of miR-146a.Objective:To study the differential expression of miR-146a and miR-335between RUNX2+/m and RUNX2+/+dental follicle cells, and to analysis the function of miR-146a.Methods:In the above differently expressed miRNA, miR-146a and miR-335were more than10-fold down-regulated （miR-146a was16.96-fold down-regulated, miR-335was10.72-fold down-regulated）, the target gene prediction results were similar, and it was reported that these two miRNAs are related to RUNX2expression or osteogenic differentiation, so miR-146a and miR-335were chosen for further experiment. The miRNA in-situ hybridization was performed to confirm the expression of miR-146a and miR-335in RUNX2+/m and RUNX2+/+HDFCs. Cell slides at cell passage3were fixed and in situ hybridized with5’-DIG-labeled miRCURY detection probe pecifically targeting miR-146a and miR-335. Positive signals were visualized. To semi-quantitative analysis, the cell slides of RUNX2+/m and RUNX2+/+HDFCs were observed and pictured with light microscope, and the optical density value was measured. Then, RUNX2+/+HDFCs at passage5were transfected with miR-146a mimics and miR-146a inhibitor （5’-FAM labeled）. Normal RUNX2+I+HDFCs served as control. Twenty-four hours after transfection, HDFCs were digested and RNA was extracted, And quantitative real-time reverse-transcription PCR （qRT-PCR） was performed to detect the expression level of genes involved in tooth eruption and osteoblast/osteoclast differentiation, such as RUNX2, CSF-1, OPG.Results:The expression of both miR-146a and miR-335decreased in RUNX2+/m HDFCs. And there was a significant difference in the semi-quantitative analysis（P<0.05）. This result of miRNA in-situ hybridization was consistent with the above microarray analysis. It suggested that the expression of miR-146a and miR-335were related to RUNX2mutation. Quantitative RT-PCR analysis showed that over-expression of miR-146a may down-regulate the expression of RUNX2, CSF-1, OPG. There were marked differences in relative expression level of RUNX2and CSF-1among three groups（miR-146a mimic, control and miR-146a inhibitor, P<0.05）. OPG tended to decline after over-expression of miR-146a. Although there was no statistical difference between miR-146a mimic and control group（P>0.05）, but the difference was significant between control and miR-146a inhibitor transfected HDFCs（P<0.05）.Conclusion:RUNX2mutation affect the expression of miR-146a and miR-335, and over-expression of miR-146a down-regulate the expression of RUNX2,CSF-1,OPG There might be a crosstalk between RUNX2and relative miRNA.This study showed the effect of RUNX2mutation on miRNA expression, and the crosstalk between RUNX2and miRNA may be the mechanism that why the characteristic of dental follicle cells were regulated by RUNX2gene.