Mary S. Kalamaki1, Aikaterini El Doufexi*2, Maria Zempila3 and Antonis Konstantinidis4
Received: March 18, 2021 Published: April 5, 2021
Corresponding author: Aikaterini El Doufexi, Associate Faculty, Department of Preventive Dentistry, Periodontology and Dental Implant Biology, Aristotle University of Thessaloniki, Greece
Gingival phenotype; Prx; Periodontal surgery; Transcription factor
In addition to their role in development Prx genes have important roles in cell proliferation and migration . Smooth muscle cells transfected with rat Prx1 showed increased proliferation . Moreover, treatment of fibroblasts in vitro with morpholino oligos designed to specifically inhibit Prx1 and Prx2 protein expression resulted in downregulation of Tenascin-C expression and reduction in cell migration towards fibronectin . Specifically, the healing process after surgical manipulation in the oral mucosa, is determined in a high degree by the gingival phenotype. Assuming that the gingival phenotype might be regulated by gene expression, the aim of our study was to investigate if there is an association between the presence and the levels of Prx1 and Prx2 mRNA and the phenotype of human gingivae.
Subjects included in the study: Twenty two patients in need
of crown lengthening were included in the study, ten with thick and
twelve with thin gingival phenotype. The assessment of the gingival
phenotype was done based on the clinical characteristics of the
Surgical Procedures: All surgeries were performed by postgraduate students at the Department of Periodontology and Biology of Dental Implants at the Aristotle University of Thessaloniki. All patients were required to sign a consent form before.
Tissue samples: Connective tissue fragments 1mm x 1mm were collected from the buccal flap during the surgical procedure and immediately placed in sterile tubes containing 1mL of RNA later (Sigma). Samples were maintained in RNA later at -80°C until further processing.
RNA isolation-Reverse Transcription: Total RNA was isolated from tissue samples using the NucleoSpin RNA Kit (Macherey Nagel) following the manufacturer’s protocol. The concentration and quality of RNA in each sample was determined using a SmartSpec Plus spectrophotometer (Biorad). 200ng of RNA were reversed transcribed to cDNA using the PrimeScript II 1st strand cDNA synthesis kit (Takara) using oligo-dT primer. cDNA samples were stored at -20°C until further processing.
PCR Analysis for Prx1 and Prx2 mRNA Expression: The presence and levels of Prx1 and Prx2 mRNA expression were examined by semiquantitative PCR analysis. Two microliters of the cDNA were directly used as a template for the PCR in a total reaction volume of 25μL. The reaction mixture contained 12.5μL OneTaq Hot Start 2x Master Mix with standard buffer (New England Biolabs), 1μL of each primer (10μM), 2μL cDNA template, and PCR grade water. For detecting the presence of Prx1 primers Prx1F 5’-TCCCTCCTCAAATCCTAC-3’ and Prx1R 5’-ACTATATTCCTTGGCCTTC-3’  were used.
For Prx2 detection primers Prx2F 5’-AGCACAGTGCCACCCTACA-3’ and Prx2R 5’-CTTGGCAGGCTCTTCCACC-3’ . Expression of Gadph was used as an endogenous control. The primers for Gadph detection were: Forward 5’-TGG TAT CGT GGA AGG ACT CATGAC-3’ and Reverse: 5′-ATG CCA GTG AGC TTC CCGTTC AG-3′ .
PCR conditions were as follows: initial denaturation for 5 minutes at 94°C, then 25 cycles of denaturation at 94°C for 30s, annealing at 48°C for Prx1, 52°C for Prx2 and 48°C for Gadph for 30s, extension for 45s at 68°C and final extension at 68°C for 5 min. PCR products were visualized in a 2% agarose gel using Midori green Advance (Nippon Genetics) DNA/RNA stain and a FastGene LED Transilluminator (Nippon). Semi-quantification of the PCR products was done by using the ImageJ (https://imagej.nih.gov/) analysis program. All experiments were repeated twice to ensure statistical accuracy.
Statistical analysis: The differences between thin and thick phenotypes were analyzed using t-test. Inter-group differences were analyzed by t-test and Mann-Whitney test for independent samples. A p- value <0.05 was considered statistically significant.
PCR Analysis for Prx1 mRNA Expression: PCR analysis showed Prx1 mRNA expression in higher levels in thick gingival phenotypes compared to thin. In addition, both Prx1a and Prx1b alternatively spliced isoforms were detected and Prx1a was more abundant in most of the samples compared to Prx1b. In thin gingival phenotypes Prx1a was either expressed in different levels or completely absent, fact that indicates that thin gingival phenotype might be regulated by other genes’ expression (Figure 1).
Figure 1: Paired related homeobox 1a and b gene isoform expression. The single arrow indicates the absence of Paired related homeobox 1 Paired related homeobox expression in a sample with thick gingival phenotype and the double arrow indicates the presence and abundance of both isoforms of Paired related homeobox 1 gene.
PCR Analysis for Prx2 mRNA Expression: PCR analysis showed that Prx2 mRNA expression was absent in thick gingival phenotypes except for one sample. On the contrary, Prx2 was highly expressed in all samples with thin gingival phenotype (Figure 2).
Figure 1: Paired related homeobox 2 gene expression. The single arrows indicate the high levels of Paired related homeobox 2 gene expression in samples with thin gingival phenotypes.
Recent studies determine the gingival phenotype based only on clinical characteristics of the gingiva. Placing the periodontal probe in the gingival sulcus can give the appearance of transparency of the gingiva and this biotype is characterized as ‘thin’. If the gingiva is not transparent when placing the probe, the gingiva is characterized as ‘thick’ [4,5]. In this study we aimed to associate the expression of Prx genes with the gingival phenotype. Our results indicate that Prx2 is expressed in thin phenotypes, whereas Prx1 is expressed in thick phenotypes. It is well established that Prx genes play an important role in craniofacial development. Our study indicates that Prx genes are not only expressed in tissues of ectomesenchymal origin, but they might also play an important role in determining the gingival phenotype at a molecular level. It is interesting t that Prx2 was almost exclusively expressed in thin phenotypes and in the same samples Prx1 was absent, implying opposing roles of the two genes in adult gingival tissues. Overall, our study indicates a strong association of Prx1 and Prx2 gene expression with the gingival phenotype. It is possible that Prx gene expression can be used as a molecular screening test to define more accurately the gingival phenotype.
Apostolos S. Angelidis, D.V.M., M.P.V.M., Ph.D. Laboratory of Milk Hygiene and Technology Department of Food Hygiene and Technology School of Veterinary Medicine Aristotle University of Thessaloniki, Greece.
Bio chemistryUniversity of Texas Medical Branch, USA
Department of Criminal JusticeLiberty University, USA
Department of PsychiatryUniversity of Kentucky, USA
Department of MedicineGally International Biomedical Research & Consulting LLC, USA
Department of Urbanisation and AgriculturalMontreal university, USA
Oral & Maxillofacial PathologyNew York University, USA
Gastroenterology and HepatologyUniversity of Alabama, UK
Department of MedicineUniversities of Bradford, UK
OncologyCirculogene Theranostics, England
Radiation ChemistryNational University of Mexico, USA
Analytical ChemistryWentworth Institute of Technology, USA
Minimally Invasive SurgeryMercer University school of Medicine, USA
Pediatric DentistryUniversity of Athens , Greece
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