Expression and clinical significance of the genes of Hedgehog signaling pathway in sporadic keratocystic odontogenic tumor of the jaw bones

Kong LiYuan Rong等

【Abstract】PURPOSE It was to study the role of genes of Hedgehog signaling pathway in sporadic keratocystic odontogenic tumor （KCOT） of the jaw bones.METHODS Fresh specimens of sporadic KCOT and the same patient 's normal oral mucosa were obtained. Then RNA was extracted. Gene chip was used to detect the genes of Hedgehog signaling pathway. RESULTS Compared to normal oral mucosa， there were five genes of Hedgehog signaling pathway in KCOT changed， including PRKX ，WNT5a，PTCH1 up-regulated. CONCLUSION There were abnormal expressions of genes of Hedgehog pathway in sporadicKCOT. Genes of Hedgehog pathway played roles in sporadic KCOT.

【key words 】 Keratocystic odontogenic tumor； Hedgehog signaling pathway； Oligonudeotide array stecllleuce analysis

【CLC】R722.12 【Document code】B【Article No.】1004-4949（2015）02-0666-01

Keratocystic odontogenic tumors （KCOTs） are benign cystic lesions of the jaws that occur sporadically in isolation or in association with nevoid basal cell carcinoma syndrome （NBCCS）[1，2]. Hedgehog pathway is an osteogenesis-related signaling pathway . During embryonic development ， it regulates the growth and proliferation of progenitor cells and tissue formation[3].Previous studies haverevealed that syndromic KCOTs harbor PTCH1 mutations， but seach about sporadic KCOTs is rare， our studies role of genes of hedgehog signaling pathway in sporadic KCOTs of the jaw bones.

Materials and methods

1.1Subjects and samples

KCOT samples from 2 unrelated Chinese individuals were obtained from the Qingdao University Hospital.All cases in this series were diagnosed as sporadic KCOTs in histopathology from patients with no signs or histories of NBCCS. All samples were newly onset of KCOTs and not recurrent cases. Fresh tissue specimens were collected and stored at Liquid nitrogen for subsequent analysis.

1.2RNA extraction and purification

Total RNA was extracted using TRIZOL Reagent （Cat#15596-018，Life technologies， Carlsbad， CA， US）following the manufacturers instructions and checked for a RIN number to inspect RNA integrity by an Agilent Bioanalyzer 2100 （Agilent technologies， Santa Clara， CA， US）.Qualified total RNA was further purified by RNeasy micro kit （Cat#74004， QIAGEN， GmBH， Germany） and RNase-Free DNase Set （Cat#79254， QIAGEN， GmBH， Germany）.

1.3RNA amplification and labeling

Total RNA were amplified， labeled and purified by using Affymetrix WT Amplication Kit（Cat#902224， Affymetrix， Santa Clara， CA， US） and GeneChip WT Terminal Labeling Kit（Cat#900671， Affymetrix， Santa Clara， CA， US） followed the manufacturers instructions to obtain biotin labeled cDNA.

1.4Array hybridization

Array hybridization and wash was performed using GeneChip· Hybridization， Wash and Stain Kit （Cat#900720， Affymetrix， Santa Clara， CA， US）in Hybridization Oven 645 （Cat#00-0331-220V， Affymetrix， Santa Clara， CA， US）and Fluidics Station 450 （Cat#00-0079， Affymetrix， Santa Clara， CA， US） followed the manufacturers instructions.

1.5Data acquisition

Slides were scanned by GeneChip Scanner 3000 （Cat#00-00212， Affymetrix， Santa Clara， CA， US） and Command Console Software 3.1 （Affymetrix， Santa Clara， CA， US） with default settings. Raw data were normalized by Expression Console.

RESULT

Two samples of genetic test results to compare the application Fold Change program， taking FC <0.5， FC> 2.0 for conditions screening genes in 56 genes in the hedgehog signaling pathway .Fresh specimens of KCOT compared to the same patient 's normal oral mucosa， there were three genes of hedgehog signaling pathway in KCOT changed， including PRKX ，WNT5a，PTCH1 up-regulated.

Discussion

KCOT with high growth potential and propensity for recurrence[4]. Previous studies revealed that greater than 85% of syndromic KCOTs harbor PTCH1 mutations， suggesting that PTCH1 plays a critical role in the pathogenesis of these jaw tumors[5].We used Gene chip to detect the genes of Hedgehog signaling pathway Fresh specimens of KCOTcompared to the same patient 's normal oral mucosa， there were three genes of hedgehog signaling pathway in KCOT changed， including PRKX，WNT5a，PTCH1 up-regulated.

PTCH1as one of Hedgehog pathway target genes，can active hedagehog pathway. Beside hedagehog pathway role in embryogenesis， multiple studies couldshow aberrant activation of the Hh pathway in different human cancers， such as basal cell carcinoma （BCC），medulloblastoma， and different gastrointestinal tumors [6，7]. Especially in pancreatic cancer， evidence is given that Hhacts as a mediator of carcinogenesis and metastasis [8， 9].

PRKX was identified as a novel type-I cAMP-dependent protein kinase gene expressed in multiple developing tissues. PRKX stimulatesendothelial cell proliferation， migration， and vascular-like structure formation . PRKX binds to Pin-1， Magi-1 and Bag-3， which regulate cell proliferation， apoptosis， differentiation and tumorigenesis[10]. Wnt5a regulates a variety of cellular functions， such as proliferation，differentiation， migration， adhesion and polarity. Consistent with the multiple functions of Wnt5a signalling， Wnt5a knockout mice show various phenotypes， including an inability to extend the embryonic anterior-posterior and proximal-distal axes in outgrowth tissues. Thus， many important roles of Wnt5a in developmental processes have been demonstrated. Moreover， recent reports suggest that the postnatal abnormalities in the Wnt5a signalling are involved in various diseases， such as cancer， inflammatory diseases and metabolic disorders [11].

In conclusion， this study provides that abnormal expressions of genes of Hedgehog pathway in sporadic KCOT，including PRKX 、WNT5a、PTCH1 up-regulated. But still need further study to increase the sample size， to verify the regulation of the Hedgehog signaling pathway genes in sporadic KCOT in and screening key regulatory genes， providing a theoretical basis for sporadic KCOT treatment.

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