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Identification of differential microRNA expression during tooth morphogenesis in the heterodont dentition of miniature pigs, SusScrofa.

Li A, Li Y, Song T, Wang F, Liu D, Fan Z, Cheng S, Zhang C, Wang J, He J, Wang S - BMC Dev. Biol. (2015)

Bottom Line: It has been found that microRNAs (miRNAs) play important roles in the regulation of tooth development, and most likely increase the complexity of the genetic network, thus lead to greater complexity of teeth.The hierarchical clustering and principal component analysis results suggest that the miRNA expression was globally altered by types and temporal changes.The results of real-time reverse-transcriptase PCR and in situ hybridization experiments revealed that five representative miRNAs may play important roles during different developmental stages of the incisor, canine, biscuspid, and molar, respectively.

View Article: PubMed Central - PubMed

Affiliation: Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Tian Tan Xi Li No.4, Beijing, 100050, China. drliang@mail.xjtu.edu.cn.

ABSTRACT

Background: It has been found that microRNAs (miRNAs) play important roles in the regulation of tooth development, and most likely increase the complexity of the genetic network, thus lead to greater complexity of teeth. But there has been no research about the key microRNAs associated with tooth morphogenesis based on miRNAs expression profiles. Compared to mice, the pig model has plentiful types of teeth, which is similar with the human dental pattern. Therefore, we used miniature pigs as large-animal models to investigate differentially expressed miRNAs expression during tooth morphogenesis in the early developmental stages of tooth germ.

Results: A custom-designed miRNA microarray with 742 miRNA gene probes was used to analyze the expression profiles of four types of teeth at three stages of tooth development. Of the 591 detectable miRNA transcripts, 212 miRNAs were continuously expressed in all types of tooth germ, but the numbers of miRNA transcript among the four different types of teeth at each embryonic stage were statistically significant differences (p < 0.01). The hierarchical clustering and principal component analysis results suggest that the miRNA expression was globally altered by types and temporal changes. By clustering analysis, we predicted 11 unique miRNA sequences that belong to mir-103 and mir-107, mir-133a and mir-133b, and mir-127 isomiR families. The results of real-time reverse-transcriptase PCR and in situ hybridization experiments revealed that five representative miRNAs may play important roles during different developmental stages of the incisor, canine, biscuspid, and molar, respectively.

Conclusions: The present study indicated that these five miRNAs, including ssc-miR-103 and ssc-miR-107, ssc-miR-133a and ssc-miR-133b, and ssc-miR-127, may play key regulatory roles in different types of teeth during different stages and thus may play critical roles in tooth morphogenesis during early development in miniature pigs.

No MeSH data available.


Validation of miRNAs by real-time RT-PCR. Expression levels of five miRNAs (ssc-miR-103, ssc-miR-107, ssc-miR-127, ssc-miR-133a, and ssc-miR-133b) were detected by real-time RT-PCR and microarray chip. We scaled the raw data from the real-time RT-PCR to make it comparable with the microarray data. Each time point was replicated three times using independently collected samples; the average is shown with the standard deviation. R represents the Pearson correlation coefficient. Expression levels of the five miRNAs were in concordance with the normalized microarray data (R > 0.9). Ssc-miR-103 and ssc-miR-107 expression was slightly lower in premolars (Dpm) than in other types of teeth, ssc-miR-133a and ssc-miR-133b expression was much higher in Dpm than in other types of teeth, and ssc-miR-127 expression gradually increased from the incisor (Di) to the molar (Dm)
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Fig4: Validation of miRNAs by real-time RT-PCR. Expression levels of five miRNAs (ssc-miR-103, ssc-miR-107, ssc-miR-127, ssc-miR-133a, and ssc-miR-133b) were detected by real-time RT-PCR and microarray chip. We scaled the raw data from the real-time RT-PCR to make it comparable with the microarray data. Each time point was replicated three times using independently collected samples; the average is shown with the standard deviation. R represents the Pearson correlation coefficient. Expression levels of the five miRNAs were in concordance with the normalized microarray data (R > 0.9). Ssc-miR-103 and ssc-miR-107 expression was slightly lower in premolars (Dpm) than in other types of teeth, ssc-miR-133a and ssc-miR-133b expression was much higher in Dpm than in other types of teeth, and ssc-miR-127 expression gradually increased from the incisor (Di) to the molar (Dm)

Mentions: The five representative sus-scrofa’s miRNAs described above were validated by real-time RT-PCR using 12 independent samples. Expression levels of all of the selected miRNAs were in concordance with the normalized microarray data (Pearson correlation coefficient >0.9, Fig. 4). In general, the results of the real-time RT-PCR validated the accuracy of the microarray. We also found that expression levels of ssc-miR-103 and ssc-miR-107 were slightly lower in Dpm than in other types of teeth, ssc-miR-133 a and ssc-miR-133b expression levels were much higher in Dpm than in other types of teeth, and ssc-miR-127 expression increased in Di, Dc, Dpm, and Dm, in that order. A schematic diagram (Fig. 4) was created that placed these miRNAs into three possible expression pattern profiles among the three developmental stages.Fig. 4


Identification of differential microRNA expression during tooth morphogenesis in the heterodont dentition of miniature pigs, SusScrofa.

Li A, Li Y, Song T, Wang F, Liu D, Fan Z, Cheng S, Zhang C, Wang J, He J, Wang S - BMC Dev. Biol. (2015)

Validation of miRNAs by real-time RT-PCR. Expression levels of five miRNAs (ssc-miR-103, ssc-miR-107, ssc-miR-127, ssc-miR-133a, and ssc-miR-133b) were detected by real-time RT-PCR and microarray chip. We scaled the raw data from the real-time RT-PCR to make it comparable with the microarray data. Each time point was replicated three times using independently collected samples; the average is shown with the standard deviation. R represents the Pearson correlation coefficient. Expression levels of the five miRNAs were in concordance with the normalized microarray data (R > 0.9). Ssc-miR-103 and ssc-miR-107 expression was slightly lower in premolars (Dpm) than in other types of teeth, ssc-miR-133a and ssc-miR-133b expression was much higher in Dpm than in other types of teeth, and ssc-miR-127 expression gradually increased from the incisor (Di) to the molar (Dm)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696248&req=5

Fig4: Validation of miRNAs by real-time RT-PCR. Expression levels of five miRNAs (ssc-miR-103, ssc-miR-107, ssc-miR-127, ssc-miR-133a, and ssc-miR-133b) were detected by real-time RT-PCR and microarray chip. We scaled the raw data from the real-time RT-PCR to make it comparable with the microarray data. Each time point was replicated three times using independently collected samples; the average is shown with the standard deviation. R represents the Pearson correlation coefficient. Expression levels of the five miRNAs were in concordance with the normalized microarray data (R > 0.9). Ssc-miR-103 and ssc-miR-107 expression was slightly lower in premolars (Dpm) than in other types of teeth, ssc-miR-133a and ssc-miR-133b expression was much higher in Dpm than in other types of teeth, and ssc-miR-127 expression gradually increased from the incisor (Di) to the molar (Dm)
Mentions: The five representative sus-scrofa’s miRNAs described above were validated by real-time RT-PCR using 12 independent samples. Expression levels of all of the selected miRNAs were in concordance with the normalized microarray data (Pearson correlation coefficient >0.9, Fig. 4). In general, the results of the real-time RT-PCR validated the accuracy of the microarray. We also found that expression levels of ssc-miR-103 and ssc-miR-107 were slightly lower in Dpm than in other types of teeth, ssc-miR-133 a and ssc-miR-133b expression levels were much higher in Dpm than in other types of teeth, and ssc-miR-127 expression increased in Di, Dc, Dpm, and Dm, in that order. A schematic diagram (Fig. 4) was created that placed these miRNAs into three possible expression pattern profiles among the three developmental stages.Fig. 4

Bottom Line: It has been found that microRNAs (miRNAs) play important roles in the regulation of tooth development, and most likely increase the complexity of the genetic network, thus lead to greater complexity of teeth.The hierarchical clustering and principal component analysis results suggest that the miRNA expression was globally altered by types and temporal changes.The results of real-time reverse-transcriptase PCR and in situ hybridization experiments revealed that five representative miRNAs may play important roles during different developmental stages of the incisor, canine, biscuspid, and molar, respectively.

View Article: PubMed Central - PubMed

Affiliation: Molecular Laboratory for Gene Therapy and Tooth Regeneration, Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Tian Tan Xi Li No.4, Beijing, 100050, China. drliang@mail.xjtu.edu.cn.

ABSTRACT

Background: It has been found that microRNAs (miRNAs) play important roles in the regulation of tooth development, and most likely increase the complexity of the genetic network, thus lead to greater complexity of teeth. But there has been no research about the key microRNAs associated with tooth morphogenesis based on miRNAs expression profiles. Compared to mice, the pig model has plentiful types of teeth, which is similar with the human dental pattern. Therefore, we used miniature pigs as large-animal models to investigate differentially expressed miRNAs expression during tooth morphogenesis in the early developmental stages of tooth germ.

Results: A custom-designed miRNA microarray with 742 miRNA gene probes was used to analyze the expression profiles of four types of teeth at three stages of tooth development. Of the 591 detectable miRNA transcripts, 212 miRNAs were continuously expressed in all types of tooth germ, but the numbers of miRNA transcript among the four different types of teeth at each embryonic stage were statistically significant differences (p < 0.01). The hierarchical clustering and principal component analysis results suggest that the miRNA expression was globally altered by types and temporal changes. By clustering analysis, we predicted 11 unique miRNA sequences that belong to mir-103 and mir-107, mir-133a and mir-133b, and mir-127 isomiR families. The results of real-time reverse-transcriptase PCR and in situ hybridization experiments revealed that five representative miRNAs may play important roles during different developmental stages of the incisor, canine, biscuspid, and molar, respectively.

Conclusions: The present study indicated that these five miRNAs, including ssc-miR-103 and ssc-miR-107, ssc-miR-133a and ssc-miR-133b, and ssc-miR-127, may play key regulatory roles in different types of teeth during different stages and thus may play critical roles in tooth morphogenesis during early development in miniature pigs.

No MeSH data available.