MicroRNA Targets Dentin Sialophosphoprotein Gene In Odontoblast Differentiation

Tooth is a special mineralized organ composed of the enamel, dentin, cementum and pulp, of which dentin is the main part of the teeth. Dentin originated in dental epithelial -mesenchymal interactions, which induced odontoblast differentiation and maturation, the organic synthesis and secretion of extracellular matrix, and hydroxyapatite crystal deposition, mineralization begins to form. In the mineralization process, extracellular matrix plays a very important role. One of the most critical is the dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), which comes from the same gene-dentin sialophosphoprotein gene (DSPP). Over the years, scholars have made a lot of research on DSPP gene and its encoded protein. The regulation of DSPP gene has always been the focus of research.In recent years, researchers found that those non-coding RNA which were not taken seriously in the past played a significant role in the organisms life activities. They have a huge role in regulating transcription and translation of coding RNA, setting off a wave of non-coding RNA research. microRNA (miRNA) that is a kind of non-coding RNA. miRNAs are a class of about 22 nucleotides (nt) endogenous, non-coding single-stranded RNA molecules, they are widely present in from plants, nematodes to human cells. To date, plants and animals have been found thousands of miRNAs. Currently in different species genes encoded miRNA account for about 1% of genes encoded protein. MiRNAs lead to the targeted mRNA degradation or translation inhibition by targeting mRNA with the 3'UTR, and thus as an important class of regulatory molecules involved in regulating the growth, development, aging and death of the organism. Under normal physiological conditions, expression of miRNAs in vivo have a strict organizational and developmental stage-specific. MiRNA expression of different tissues and cells in different stages of development were significantly different. In the field of oral and maxillofacial development, Jevnaker and colleagues were the first to use microarray studying miRNAs expression in rat teeth and salivary gland, and found miRNAs expression were changing in different stages of tooth development,some of which was tissue-specific. At the same time, bioinformatics analysis suggested these miRNAs were necessary for tooth development and even oral and maxillofacial tissues and organs developmental.Based on previous research, to study more comprehensivly, in-depth of miRNA regulating DSPP, and its role in odontoblast differentiation, our research uses bioinformatics software to predict miRNAs targeting DSPP gene, combined with dual luciferase reporter gene system and real-time quantitative PCR method to select and validat miRNAs targeting DSPP gene, and then select one of the miRNAs——mir586 to do further research. The same methods are bioinformatics to predict binding sites between mir586 and DSPP and the dual luciferase reporter gene system for validation. Finally, lentiviral vector of mir586 overexpression and mir586 suppression were transfected into human dental pulp cells to discover the effect of mir586 on odontoblast differentiation.The paper includes the following three parts:PartⅠ:selection and validation of microRNAs Targeting DSPP geneFirstly, we use Targetscan, mirBase, microRNA.org and mirGen-miRanda four different bioinformatics software to predict miRNAs targeting DSPP gene, respectively, got 17,15,10,10 miRNAs. Then ten miRNAs were chosen based upon their targeted prediction by more than one program. In order to further select and validat these miRNAs regulating DSPP gene in the odontoblast, the study used a dual luciferase reporter gene system and quantitative PCR methods. The results showed mir885-5p, mir32, mir586 can inhibit the activity of luciferase, which can regulate DSPP gene. During dental pulp cells differentiation to odontoblast, when the DSPP gene expression increased, mir885-5p, mir32 and mir586 expression on day 10-14 have decreased. Combination of literature and the results of this chapter, we chose has-mir586 that strongestly inhibite luciferase activity and express more significantly as a further study to investigate the relationship with DSPP gene, and its effect on odontoblast differentiation.PartⅡ:analysis of the binding sites between mir586 and DSPP geneFirstly, Targetscan and MicroRNA.org were used to predict the binding sites between mir586 and DSPP gene. Both show the same binding site (AAUGCAUG). To further confirm the mir586 regulating DSPP gene through this binding site, mutations of the binding site were constructed. Normal DSPP 3 'UTR expression vector and mutant DSPP 3'UTR expression vector were used in the dual luciferase reporter gene system to detect luciferase activity. The results showed that mir586 regulate DSPP 3 'UTR through this binding site, thereby inhibiting the luciferase activity.But mir586 can not combine to mutant DSPP 3' UTR, which proved mir586 combined to sequence of 190～196 of DSPP 3 'UTR (AAUGCAUG).PartⅢ:the function of mir586This part mainly includes two aspects:1. Get mir586 overexpression lentivirus and mir586 inhibition lentivirus.First, genomic DNA was extracted from normal human whole blood. Sequences were designed for mir586 over-expression and mir586 suppression (anti-mir586).Sequence of has-mir-586 were got from NCBI, and designed mir586 overexpression sequence contains the has-mir-586 mature sequence and 200 base pairs at each end. According to the literature, take mir586 sequence 3 times in series as anti-mir586 sequence. Then, the extracted genomic DNA as template normal, mir586 gene fragment was amplified by PCR. Anti-mir586 primers as a template polymer to make it a double-stranded anti-mir586 gene. Two gene fragments were connected with pLVX-shRNA1 respectively and then transferred into competent DH5 a bacteria. Positive clones were picked out and then plasmids were extracted and testified. With Lenti-X TM HTX Packaging System, two lentiviral vector and packaging vector Lenti-X TM HTX Packaging Mix, and the transfection reagent Xfect transfection were transfected into 293T cells. Then viral supernatant was collected, filtered, concentrate, packaging. Titer of virus were about 5×106 IFU/ml. Finally, lentivirus transfected into human dental pulp cells and use the appropriate concentration of G418 to select stably transfected cells. Real time quantitative PCR method were determined the expression of mir586 among the mir586 overexpression group, anti-mir586 group and negative control group, The result showed that expression of mir586 in mir586 overexpression is as 6 times as that of the negative control group and anti-mir586 group decreased by about 4 times, so expression of mir586 in anti-mir586 group is very weak.2. During human dental pulp cells cultured in a mineralizing medium differentiation to the odontoblast-like cell, the effect of mir586 on cell proliferation and differentiationSamples were harvested at 0,3,7,10,14 or 21 days of differentiation to detect indicators.Cell proliferation was assessed by MTT method and found that the normal dental pulp cells cultured in a mineralizing medium grew quickly with the increase of cells. On day 7, cells gradually differentiation to odontoblast-like cells grew logarithmicly, so that cells were multiple layer growth and mineralized nodules increased. After 14 days the cell density was too high, cell death occurred. The cells of anti-mir586 group grew slowly at the beginning, after 3 days the growth rate were markedly accelerated, to 10 days, the growth rate began to slow down,perhaps because of excessive growth. The number of cells in mir586 overexpression group was increasing,while the growth rate was slower compared to control group. Overall, in the differentiation process, mir586 little effect on cell proliferation. When mir586 expression inhibited, to a certain extent, promote cell growth.The differentiation of dental pulp cells were evaluated by alkaline phosphatase activity. In normal group, alkaline phosphatase activity were gradually increased with differentiation. ALP activity of cells in mir586 overexpression group changed little, while ALP activity of cells in anti-mir586 group increased significantly from day 7 and to 14 days in mineralization it induced peak. DMP1 and DSPP proteins encoded by two genes is a very important non-collagen matrix proteins during tooth development, which is considered the odontoblast specific factors. Dental pulp cells cultured in the mineralizing medium can differentiate to odontoblast-like cells. DMP1 gene expresses earlier than DSPP, and with the differentiation DSPP expression increased gradually. From day 7 there were a large number of DMP1 expression and on day 14 it reached peak.When mir586 was overexpressed, DSPP and DMP1 expression were reduced. When mir586 expression was inhibited, DSPP expression was significantly more than the control group from day 7, but DMP1 expression is no significant difference compare to the control group. The expression of DSP was detected by Western blotting. DSP protein bands can be seen clearly in the control group on day 10, which appeared in the anti-mir586 group on day 7.Compared with the control group, protein bands was significantly reduced in mir586 overexpression group from day 10, which indicate that the DSP protein is degraded, resulting in reduced expression.In general, when mir586 expression is suppressed, mir586 can promote the dental pulp cells differentiating to odontoblast-like cells. When mir586 is overexpressed, the expression of dentin-specific DSPP gene decreased, DSP expression is delayed and decreased, which greatly slowed the process of differentiation.In summary, this study predicted some miRNAs targeting DSPP by bioinformatics methods. Applying to the dual luciferase reporter gene system and real-time fluorescence quantitative PCR method, we focus on the binding site between mir586 and DSPP. Mir586 overexpression lentivirus and mir586 suppression lentivirus were used to stably transfect to human pulp cells and to detect the effect of mir586 on cell proliferation and differentiation during the differentiation of pulp cells. This is a preliminary study of the miRNAs effect on the process of odontoblast differentiation, and on specific factors of dentin-DSPP and its proteins, which is conducive to extend research of variety of miRNAs in dentinogenesis and to discover new regulatory factors and regulatory pathways.