Dental erosion in mice with impaired salivary gland function.

Objective: Salivary flow rate exerts an essential impact on the development and progression of dental erosion. In this work, the experimental dental erosion in non-obese diabetic (NOD) mice with reduced salivary flow rate was induced, and the erosive effect of acidic drinks on their dentition was studied.Material and methods: Three acidic drinks (sports drink, cola light drink and sugar containing cola drink) were given to adult NOD mice (groups: N = 11) as the only drink for 6 weeks. Two control groups were included; wild type and NOD control (groups: N = 9). Experimental and control (water) teeth were dissected out and observed by scanning electron microscopy (SEM). Mandibular first molars were subsequently embedded in Epon, ground transversely, observed again by SEM, and the enamel thickness and tooth height were measured.Results: Mandibular molars were considerably more eroded than maxillary molars. The erosive process started at the top of the cusps and subsequently extended in the cervical, mesio-distal, and pulpal direction. Erosive lesions were evident in increased succession from sports drink, cola light to cola drink exposed mandibular molars, with the lingual tooth height being approximately 23%, 26%, and 37% lower, respectively, compared to the control. The lingual enamel was approximately 48% thinner in sports drink molars and 62% thinner in cola light molars. In cola drink molars, the lingual enamel was totally eroded, and significant erosion of dentine was evident.Conclusion: Reduced salivary flow, together with a high consumption of acidic drinks, results in severe erosion of NOD mice molars.

Circadian rhythms and gene expression during mouse molar tooth development.

OBJECTIVES: Incremental markings in dental enamel suggest that the circadian clock may influence the molecular underpinnings orchestrating enamel formation. The aim of this study was to investigate whether the genes and microRNAs (miRNAs) oscillate in a circadian pattern during tooth and enamel development. MATERIAL AND METHODS: Comparative gene and miRNA expression profiling of the first mandibular molar tooth germ isolated at different time-points during the light and night period was performed using microarrays and validated using real-time RT-PCR. Bioinformatic analysis was carried out using Ingenuity Pathway Analysis (IPA), and TargetScan software was used in order to identify computationally predicted miRNA-mRNA target relationships. RESULTS: In total, 439 genes and 32 miRNAs exhibited significantly different (p < 0.05) levels of expression in the light phase compared with the night phase tooth germs. Genes involved in enamel formation, i.e. Amelx, Ambn, Amtn, and Odam, oscillated in a circadian pattern. Furthermore, the circadian clock genes, in particular Clock and Bmal1, oscillated in mouse molar tooth germ during 24-h intervals. The expression of Clock and Bmal1 was inversely correlated with the expression of miR-182 and miR-141, respectively. CONCLUSIONS: MiRNAs, including miR-182 and miR-141, are involved in the control of peripheral circadian rhythms in the developing tooth by regulating the expression of genes coding for circadian transcription factors such as CLOCK and BMAL1. Regulation of circadian rhythms may be important for enamel phenotype, and the morphology of dental enamel may vary between individuals due to differences in circadian profiles.

Expression of Clu and Tgfb1 during murine tooth development: effects of in-vivo transfection with anti-miR-214.

Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-ß1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.

Expression of delta-like 1 homologue and insulin-like growth factor 2 through epigenetic regulation of the genes during development of mouse molar.

Delta-like 1 homolog (Dlk1) and insulin-like growth factor 2 (Igf2) are two of six well-studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non-coding RNAs, encoded by Gene trap locus 2 (Gtl2) and Imprinted maternally expressed transcript (H19), co-located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real-time RT-PCR analysis we showed Dlk1 and Gtl2 to exhibit a time-course of expression during tooth development that was similar to that of Igf2 and H19. Western blot analysis of proteins encoded by Dlk1 and Igf2 suggested that the levels of these proteins reflected those of the corresponding mRNAs. Immunohistochemical studies of DLK1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine-phosphate-guanine (CpG) islands in both Dlk1 and Igf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of Dlk1 and Igf2 and with decreased levels of DLK1 and IGF2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of CpG islands present in these genes.

Transcriptomic and functional studies reveal miR-431-5p as a tumour suppressor in pancreatic ductal adenocarcinoma cells.

The lactate receptor HCAR1 (hydroxycarboxylic acid receptor 1) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), where it regulates lactate transport between the cancer cells. Little is known about the underlying cause of high HCAR1 expression in PDAC, and in the present study, we investigated whether HCAR1 could be a target of miRNA regulation. By searching for predicted miRNA candidates in silico, we identified miR-431-5p as a possible regulator of HCAR1. We found miR-431-5p to repress HCAR1 expression through direct binding to the 3' UTR. The endogenous expression of miR-431-5p and HCAR1 was found to be negatively related in the PDAC cell lines BxPC-3, Capan-2, and PANC-1. Overexpression of miR-431-5p inhibited cell proliferation in all the cell lines, and a shift in cell cycle progression and induction of apoptosis were found in the BxPC-3 cells. Transcriptomic analysis of mRNA from BxPC-3 cells revealed numerous differentially expressed genes (DEGs), including HCAR1, in response to miR-431-5p overexpression. A significant proportion of these DEGs was enriched in cancer-related processes and signalling pathways. Among the most significantly repressed DEGs was ATP6V0E1, encoding the integral subunit e of vacuolar ATPase (V-ATPase), an enzyme that is important for cancer cell survival. Small interfering RNA (siRNA)-mediated knockdown of HCAR1 and ATP6V0E1 showed that only knockdown of ATP6V0E1 mimicked the effect of miR-431-5p overexpression on cell viability. Our findings indicate that miR-431-5p acts as a tumour suppressor in PDAC cells, with BxPC-3 cells being most responsive.

Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ.

MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.

Expression of members of the miRNA17-92 cluster during development and in carcinogenesis.

The six microRNAs (miRNA) encoded by the miR-17-92 cluster, also named oncomir-1, have been associated with carcinogenesis and typically exhibit-increased expression in tumors. Despite the well-established role for the miR-17-92 cluster in an oncogenic network, the physiological function of these miRNAs in normal tissues remains unresolved. In order to investigate whether there are similar patterns of miR-17-92 expression during embryogenesis and carcinogenesis, we have preformed a systematic study of the expression in cultured carcinoma cells, cultured primary human keratinocytes (KC), and during development of some murine tissues. Both levels of expression of the primary transcript (pri-miRNA) and levels of expression of the individual members of the cluster were monitored. Irrespectively of tissue examined we found that the level of expression decreased markedly during development. With cultured primary human KCs their levels of expression of some of these microRNAs decreased as the number of cell passages increased. Their levels of expression in cultured carcinoma cells, in contrasts, increased, or remained unchanged, with increasing number of cell passages. The results suggest these microRNAs are involved in the regulation of foetal development and that they may promote proliferation and inhibit differentiation during embryogenesis and carcinogenesis. Additionally, the six microRNAs exhibit variable tissue expression, suggesting selective processing of these microRNAs.

Nuclear and cytoplasmic expression of Met in oral squamous cell carcinoma and in an organotypic oral cancer model.

Met, the hepatocyte growth factor receptor, is important in transducing signals for tumour growth and metastasis. The aim of this study was to examine the pattern of Met expression and its value as a prognostic factor in oral squamous cell carcinomas (OSCCs). The material consisted of 53 OSCCs and five healthy controls from normal oral mucosa supplied with cell lines, 10 organotypic models supplied with oral cancer cells, and three organotypic models supplied with normal keratinocytes. Met protein expression was assessed by immunohistochemistry and western blotting. Met expression was scarce and limited to the basal layer in normal oral mucosa, but was more extensive in the tumours. Cytoplasmic expression of Met was found in the majority of the tumours, and nuclear expression was found in 72%, including a high fraction of the cells located at the invasive front. Organotypic models with normal or malignant oral cells yielded principally similar results as in the mucosa and the cancers, respectively. A smaller amount of Met immunoreactivity was detected, by western blotting, in the nuclear fraction of cultured oral cancer cells. In conclusion, Met was upregulated in OSCCs and was also found in the nucleus. However, Met was not a marker for prognosis in this study.

Gene expression and dental enamel structure in developing mouse incisor.

At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.

Expression of the T-cell-specific adapter protein in oral epithelium.

The multifunctional T-cell-specific adapter protein (TSAd) was originally described in T cells but is also expressed in epithelial cells from the respiratory tract and in endothelium. In this study, we found expression of TSAd messenger RNA (mRNA) and protein in both human and murine oral mucosal epithelium as well as in human primary oral keratinocyte cell cultures. In TSAd(-/-) mice, the mucosa and skin appeared macroscopically normal, but severe disturbances were observed in the fine structures of the basal membrane and intercellular epithelial spaces upon analysis using transmission electron microscopy. Oral epithelial cells from TSAd(-/-) mice displayed decreased migration compared with cells from wild-type mice, whereas overexpression of TSAd in a human epithelial cell line resulted in impaired proliferation. This study is the first to show that TSAd is expressed in normal oral mucosa, that it is important for the normal ultrastructural morphology of the epithelium and the basal membrane, and that it is involved in the migration and proliferation of oral keratinocytes.

Differential gene expression profiling of the molar tooth germ in peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and in wild-type mouse: molar tooth phenotype of PPAR-alpha knockout mouse.

Gene expression profiling of the first molar tooth germ at embryonic days (E)17.5 and 18.5, and at postnatal days (P)0, 2, and 6 from peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and from wild-type mouse was carried out using microarrays and validated using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. When comparing expression profiles at each time-point, a total of 1,235 genes showed significantly different expression, 772 of which exhibited significantly decreased expression in tooth germ from knockout mouse. With genes exhibiting significantly decreased levels of expression in tooth germ from PPAR-alpha knockout mouse, bioinformatic analysis using ingenuity pathway analysis yielded significant associations to cellular functions related to cellular growth/proliferation and to networks related to regulation of calcium homeostasis. Using scanning electron microscopy to investigate molars from adult PPAR-alpha knockout mouse, the molar size was found to be slightly reduced, the enamel structure was found to be normal, but cervical molar enamel exhibited evidence suggesting hypomineralization. Although the PPAR-alpha knockout had no significant effect on molar morphology, the results suggest that active PPAR-alpha signaling is required to achieve normal mineralization of molar enamel, most probably through regulation of calcium homeostasis and metabolism of vitamin D. Cyp27b1 was expressed in tooth germ, suggesting that tooth germ can synthesize active vitamin D. Expression of Cyp27b1 was significantly enhanced in postnatal PPAR-alpha knockout tooth germ.

Transcriptomic analysis of MicroRNA expression in enamel-producing cells.

There is little evidence for the involvement of microRNAs (miRNAs) in the regulation of circadian rhythms during enamel development. Few studies have used ameloblast-like cell line LS8 to study the circadian rhythm of gene activities related to enamel formation. However, the transcriptomic analysis of miRNA expression in LS8 cells has not been established yet. In this study, we analyze the oscillations of miRNAs in LS8 cells during one-day cycle of 24 h by next generation deep sequencing. After removal of low quality reads, contaminants, and ligation products, we obtained a high number of clean reads in all 12 samples from four different time points. The length distribution analysis indicated that 77.5% of clean reads were between 21 and 24 nucleotides (nt), of which 35.81% reads exhibited a length of 22 nt. In total, we identified 1471 miRNAs in LS8 cells throughout all four time-points. 1330 (90.41%) miRNAs were identified as known miRNA sequences, whereas 139 (9.59%) were unannotated and classified as novel miRNA sequences. The differential expression analysis showed that 191 known miRNAs exhibited significantly (P-value < 0.01) different levels of expression across three time-points investigated (T6, T12, and T18) compared to T0. Verification of sequencing data using qRT-PCR on six selected miRNAs suggested good correlation between the two methods. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of predicted target genes of differentially expressed miRNAs. The present study shows that miRNAs are highly expressed in LS8 cells and that a significant number of them oscillate during one-day cycle of 24 h. This is the first transcriptomic analysis of miRNAs in ameloblast-like cell line LS8 that can be potentially used to further characterize the epigenetic regulation of miRNAs during enamel formation.

Circadian clock and oral cancer.

The circadian clock is comprised of a master component situated in the hypothalamic suprachiasmatic nucleus and subordinate clock genes in almost every cell of the body. The circadian clock genes and their encoded proteins govern the organism to follow the natural signals of time, and adapt to external changes in the environment. The majority of physiological processes in mammals exhibit variable circadian rhythms, which are generated and coordinated by an oscillation in the expression of the clock genes. A number of studies have reported that alteration in the expression level of clock genes is correlated with several pathological conditions, including cancer. However, little is known about the role of clock genes in homeostasis of the oral epithelium and their disturbances in oral carcinogenesis. The present review summarizes the current state of knowledge of the implications of clock genes in oral cancer. It has been demonstrated that the development of oral squamous cell carcinoma undergoes circadian oscillation in relation to tumor volume and proliferation rate. The circadian clock gene period (PER)1 has been associated with oral cancer pathogenesis and it is suggested that changes in the expression of PER1 may exhibit an important role in the development, invasion, and metastasis of oral squamous cell carcinoma. However, its role remains elusive and there is a need for further research in order to understand the underlying mechanisms of the clock genes in oral cancer pathogenesis.

New animal model of extrinsic dental erosion-Erosive effect on the mouse molar teeth.

OBJECTIVE: Consumption of acidic food and drinks is considered as important risk factor for development of dental erosion. There are several in vitro and in situ studies focusing on the risk indicators and preventive treatment, however, the need for a standardized animal model has been emphasised for many years. The aim was to establish an animal model of extrinsic dental erosion, which may serve as a standard for future studies to improve our understanding of the erosion. DESIGN: Two acidic drinks, sports drink and cola drink, were given to young mice for six weeks. Experimental and control (water) molars and incisors were dissected out and observed by scanning electron microscopy (SEM). Mandibular first molars were subsequently ground transversely and observed again by SEM. The tooth height and enamel thickness were measured on the SEM images. RESULTS: The lingual surface of the mandibular molars was most eroded after consumption of acidic drinks. The cola drink exhibited higher erosive effect on mandibular molars compared to sports drink. The lingual tooth height, compared to control, was about 34% and 18% lower in the cola drink and sports drink molars, respectively. Compared to the control molars, the lingual enamel was about 23% thinner in the sports drink molars and totally eroded on the certain lingual areas of the cola drink molars. CONCLUSIONS: This new animal model of extrinsic dental erosion and the presented method with ground molars observed in SEM are suitable for further studies, which will gain deeper insights into the erosive disease.

Regulatory roles of microRNAs in human dental tissues.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that provide an efficient pathway for regulation of gene expression at a post-transcriptional level. Tooth development is regulated by a complex network of cell-cell signaling during all steps of organogenesis. Most of the congenital dental defects in humans are caused by mutations in genes involved in developmental regulatory networks. Whereas the developmental morphological stages of the tooth development already are thoroughly documented, the implicated genetic network is still under investigation. The involvement of miRNAs in the regulation of tooth genetic network was suggested for the first time in 2008. MiRNAs regulate tooth morphogenesis by fine-tuning the signaling networks. Unique groups of miRNAs are expressed in dental epithelium compared with mesenchyme, as well as in molars compared with incisors. The present review focuses on the current state of knowledge on the expression and function of miRNAs in human dental tissues, including teeth and the surrounding structures. Herein, we show that miRNAs exhibit specific roles in human dental tissues and are involved in gingival and periodontal disease, tooth movement and eruption, dental pulp physiology including repair and regeneration, differentiation of dental cells, and enamel mineralization. In light of similarities between the tooth development and other organs originating from the epithelium, further understanding of miRNAs` function in dental tissues may have wide biological relevance.

MicroRNAs: Modulators of Tooth Development.

MicroRNAs (miRNAs) are non-coding RNAs that are involved in various biological pathways by regulating gene expression. Teeth develop via reciprocal and sequential interactions between the epithelium and the ectomesenchyme. The speci.c functions of several genes during tooth development are known, and the involvement of their mutations in the pathogenesis of congenital dental defects has been widely studied. The miRNA pathway is considered to have a significant role in embryogenesis including tooth development. It has been shown that miRNAs regulate morphogenesis of tooth by fine-tuning the signalling networks, however, their precise role in tooth differentiation and morphogenesis is still elusive. The present review focuses on the studies that have used animal models to explore the function of miRNAs in tooth development. Major findings with special emphasis on the miRNA involvement in .ne-tuning and network regulation are presented and discussed. Disturbances in tooth development in the global miRNA processing knockouts mirror the essential fine-tuning guiding appropriate formation of dental hard tissues. However, further investigation of single miRNA function and mutation, including deletion and overexpression, may lead to improved knowledge on development of particular dental defects in humans. In the light of similarities between tooth development and other organs originating from the epithelium, further understanding of miRNAs` function during tooth development may have wide biological relevance.

Anti-proliferative Properties of miR-20b and miR-363 from the miR-106a-363 Cluster on Human Carcinoma Cells.

BACKGROUND: The miR-106a-363 cluster, encoding six miRNAs (miR- 106a, miR-18b, miR-20b, miR-19b-2, miR-92-2 and miR-363), has been shown to be overexpressed in various tumours. In oral carcinoma cells, however only miR- 106a was detectable from this cluster. We have investigated how effects of transfection of oral carcinoma cells with a non-expressed member of this cluster affect mRNA transcriptomes and cellular selected functions. METHODS: Investigate effects of miR-20b and miR-363 mimics on cellular respiration, glycolysis and mobility. Effects on mRNA transcriptomes were monitored using microarrays. RESULTS: The studies show that in oral carcinoma cells transfected with miR-20b -, or miR-363-3p or miR-363-5p mimic different mRNAs were differentially expressed. Nevertheless, bioinformatics analysis suggested significant associations of differentially expressed genes to inhibition of cellular proliferation, cell cycle and cellular migration. These results were also experimentally confirmed. CONCLUSIONS: Transfection of miRNA mimics for unexpressed members of the miR-106a-363 cluster (miR20b, miR-363-3p and miR-363-5p) exhibit an anti-proliferative effect on oral carcinoma cells, although likely mediated by different regulatory mechanisms.

The Three Paralogous MicroRNA Clusters in Development and Disease, miR-17-92, miR-106a-363, and miR-106b-25.

MicroRNAs (miRNAs) form a class of noncoding RNA genes whose products are small single-stranded RNAs that are involved in the regulation of translation and degradation of mRNAs. There is a fine balance between deregulation of normal developmental programs and tumor genesis. An increasing body of evidence suggests that altered expression of miRNAs is entailed in the pathogenesis of human cancers. Studies in mouse and human cells have identified the miR-17-92 cluster as a potential oncogene. The miR-17-92 cluster is often amplified or overexpressed in human cancers and has recently emerged as the prototypical oncogenic polycistron miRNA. The functional analysis of miR-17-92 is intricate by the existence of two paralogues: miR-106a-363 and miR-106b-25. During early evolution of vertebrates, it is likely that the three clusters commenced via a series of duplication and deletion occurrences. As miR-106a-363 and miR-106b-25 contain miRNAs that are very similar, and in some cases identical, to those encoded by miR-17-92, it is feasible that they regulate a similar set of genes and have overlapping functions. Further understanding of these three clusters and their functions will increase our knowledge about cancer progression. The present review discusses the characteristics and functions of these three miRNA clusters.

An investigation into anti-proliferative effects of microRNAs encoded by the miR-106a-363 cluster on human carcinoma cells and keratinocytes using microarray profiling of miRNA transcriptomes.

Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40-50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92-or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed-about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to "Cellular Growth and Proliferation" and "Cell Cycle." Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants.

Nerve growth factor beta/pro-nerve growth factor and their receptors in normal human oral mucosa.

Nerve growth factor beta (NGF-beta) and its precursor proNGF are important for the differentiation and survival of neurons and dermal keratinocytes. The aim of this study was to determine the role that NGF might play in the differentiation and wound healing of oral mucosa. Cultured normal human oral mucosal keratinocytes expressed mRNA for NGF-beta/proNGF and for their receptors TrkA and p75(NTR). Lysates from cultured oral mucosal keratinocytes did not contain detectable amounts of mature 14-kDa NGF-beta but did contain several NGF proforms with molecular weights between 32 and 114 kDa. Culture medium from oral mucosal keratinocytes contained 75 kDa proNGF. The addition of NGF-beta significantly enhanced the proliferation of oral mucosal keratinocyte cultures and in vitro scratch closure. Immunostaining of biopsies from normal oral mucosa showed the presence of proNGF in all epithelial layers. NGF staining was observed in the granular and upper spinous cell layers. TrkA immunoreactivity was detected in basal and parabasal cells, with weak to moderate staining in spinous and granular cell layers. p75(NTR) staining was seen in basal cell layers. These findings indicate that NGF-beta/proNGF have mitogenic and motogenic effects on oral mucosal keratinocytes and therefore may aid in the healing of oral wounds. Differential expression of NGF and NGF receptors throughout the epithelium suggests a role in epithelial differentiation.