Differentially Expressed Genes And EGF-R Related Signal Pathways In Common Tongue Coating

ObjectiveGlossoscopy plays a key role in diagnostics of traditional Chinese medicine(TCM) and is regarded as an important method in TCM.Thus,the tongue picture has been investigated in clinical and experimental studies.Tongue coating is an important content of glossocopy.The mechanism and objectivity of tongue coating is becoming one of the key problems in the modernizational study of TCM.Histogenetic study of tongue coating showed that the formation of tongue coating was closely related to the epidermis of tongue dorsum.Many studies revealed that the formation of tongue coating was closely related to apoptosis-related genes.Therefore,we collected clinical samples and laboratory animals to investigate the mechanism of the formation of tongue coating,cDNA microarray analysis and real-time PCR were used to identify novel genes in lingual epithelial cells to gain new insights on the molecular biological mechanism of tongue coatings.Cell culture in vitro, flow cytometric analysis,MTT method and immunohistochemistry were employed to investigate the cell cycle distribution and apoptosis,cell proliferation and expressed level of several key molecules in signal pathways.MethodsClinical samples were obtained from 22 patients with histopathologically defined tongue dorsum carcinoma and need surgical removal.The total 22 selected typical samples were separated into 5 groups:5 white thin coatings 5 white thick coatings,5 yellow thick coatings,4 yellow thin coatings,and 3 non-coatings.Four fetal samples covered with white thin coating were regarded as the control.Five cDNA microarrays containing 4096 human cDNA clones were used to estimate the differences in gene expression in the lingual tissues.The epithelia obtained from healthy rats,mice and rabbits were investigated with normal epithelium adjacent to lingual squamous cell carcinoma as control.DispaseⅡand trypsin were combined to digest the lingual dorsum epidermis and then,the epithelia were incubated in RPMI-1640 under different period of time.Flow cytometric analysis was used to analyze apoptosis and cell cycle changes.In addition,the effect of EGF on cell cycle and apoptosis in the lingual dorsal epithelial cells obtained from rats,mice and rabbits was investigated in vitro.To investigate the effect of EGF and TGF-αon modulating the formating of tongue coating,MTT method was used to evaluate the activities of cell proliferation,and flow cytometric analysis to analyze the cell cycle distribution and apoptosis in the lingual dorsal epithelial cells obtained from rats in K-SFM.And immunohistochemistry was employed to investigate the expressed level of several key molecules in signal pathways,such as CK4, p38-MAP kinase,c-fos,c-myc and NF-κB.ResultsThe differential expression in the five kinds of common tongue coatings vs.the fetal white thin coating from the blast analyses revealed that 60 synchronous differentially expressed genes in five kinds of common tongue coatings were identified in cDNA microarray results,of which 21 genes were up-regulated and 39 genes were down-regulated. They were all correlated with proliferation,differentiation and apoptosis of the lingual mucous membrane epithelia.The characteristic of apoptosis and cell cycle changes in the lingual dorsum epithelia from rats,mice,rabbits and human were compared,and the results showed that rats could be the ideal model animal to investigate the apoptosis of tongue coatings.The results of cell cycle distribution and apoptosis in 10%RPMI-1640 revealed that EGF could inhibit the apoptosis of the epithelia from mice and rabbits,but the effect of anti-apoptosis didn't show dose-dependent.In contrast,EGF and TGF-αcould promote the apoptosis of the epithelia from rats in 10%RPMI-1640.EGF and TGF-αcould decrease the activity of cell proliferation dose-dependently in K-SFM,consistenting with apoptosis change,which may be attribute to cell cycle change. The results of immunohistochemistry showed that EGF and TGF-αcould affect the expressed level of CK4,p38-MAP kinase,c-fos,c-myc and NF-κB.EGF and TGF-αcould inhibit the expressed level of CK4.It was noted that both 1μg/L and 10μg/L EGF and TGF-αmay significantly inhibit the expressed level of CK4,but the effect of moderate concentration(5μg/L) was slight.In addition,we foud that EGF and TGF-αcould increase the expressed level of p38-MAP kinase,c-myc and c-fos,but decrease the expressed level of NF-κB p50 and p65.The tendency of the effect on p38-MAP kinase,c-myc,c-fos and NF-κB was similar to that in EGF and TGF-αwhereas the intensity of the effect was different.The effect of EGF on p38-MAP kinase and c-myc was stronger than that of TGF-α,on the contrary,the effect of EGF on c-fos and NF-κB was weaker than that of TGF-α.ConclusionsThe result showed that EGF and TGF-αwere the two key molecules in the formation of tongue coating.EGF and TGF-αcould competitively integrate the same EGF-receptor (EGF-R),but they had different effect on the cell cycle distribution and apoptosis, indicating that EGF and TGF-αmay play distinct roles in the formation of tongue coating. Therefore,the advancement of investigation on the signal pathways of EGF and TGF-αwas very important to understand the molecular mechanism of the formation of tongue coating.The lingual dorsum epithelia obtained from healthy rats was used as the cell model to investigated that the expressed levels of CK4,p38-MAP kinase,c-fos,c-myc and NF-κB were affected by EGF and TGF-α.The cell immunohistochemistry result also evidenced that EGF and TGF-αmay play distinct roles in the formation of tongue coating.Thereby, we infered that EGF and TGF-αwere the two key molecules in the formation of tongue coating,but they play distinct roles in the formation of tongue coating of healthy and various diseases populations.EGF and TGF-αmay attach themselves to multifarious signal pathways to modulating the formation of tongue coating.