| ObjectiveTongue coating,an essential part of tongue appearance in traditional Chinese medicine(TCM),is critical to the objectification and modernization of tongue diagnosis.TCM practitioners believe that the nature and location of disease can be differentiated by tongue coating observation.The formation of tongue coating is closely related to proliferation,differentiation,apoptosis and migration of tongue dorsum mucosal epithelial cells.It is generally accepted that equilibrium between proliferation and apoptosis of tongue dorsum mucosal epithelial cells is one of the formation mechanisms for tongue coating.In this study,we collected clinical samples and proceeded cell experiments in vitro to explore the relationship between EGF(epidermal growth factor)and tongue coating in gastrc cancer patients.By investigating the molecular mechanism of tongue coating,our research may help to provide objective basis for tongue diagnosis and microcosmic index for syndrome differentiation.MethodsGastric caner cases and healthy controls were recruited from Jiangsu Hospital of TCM.After an informed consent form was signed by every participant,information of demographic characteristics,living habits,tongue diagnosis and clinical examination parameters were colleted.DNA was extracted from peripheral venous blood,and EGF SNPs(rs11568943,rs2237051,rs11569017 in the non-synonymous exon region,rs3756261,rs11568835,rs4444903 in the promoter region,rs3733625 in the 3’untranslated region)were genotyped by PCR-LDR(polymerase chain reaction-ligation detection reaction).Serum EGF content was analized by ELISA(enzyme-linked immunosorbent assay).Association among EGF SNPs,EGF in serum,living habits,clinical examination parameters,pathological characteristics,and tongue coating formation was explored in our study.To imitate the normal environment of tongue mucosa epithelial cells,tongue squamous cell carcinoma cell line Tca-8113 was cultured in RPMI-1640 without serum and its basic functions,such as cell division,proliferation,survival and migration was investigated.In order to evaluate the effect of EGF or TGF-α on Tca-8113,proliferation rate was tested by MTT assay,and cell cycle was analized by flow cytometric analysis.Apoptosis was assessed by staining with annexin Vpropidium iodide or hoechst 33258.DNA fragmentation was detected by agarose gel electrophoresis.To explore the EGFR signal pathway induced by EGF or TGF-α(transforming growth factor-α)and to investigate molecular mechanism of proliferation-apoptosis balance of tongue coating formation,mRNA and protein expression,including Bcl-2,Bax,Fas,total AKT,NF-κB p50,β-actin and HSP70,were evaluated by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR)and western blot test,respectively.ResultsClinical studies showed that there were statistically significant differences in distributions of tongue coating between gastric cancer(GC)cases and the controls.The percentage of white thick coating was the highest in GC group,while white thin coating was more popular in the controls.For GC patients,the percentages of grey,thick and exfoliative coating were higher than those in the controls.In diffuse type-gastric cancer cases,patients who carried GA/GG genotype of rs2237051 had an increased possibility of thick or exfoliative coating,compared with AA genotype.Patients with diffuse type GC who carried GA/AA genotype of rs4444903 had an increased possibility of thick,exfoliative or grey coating,compared with GG genotype.The thick or exfoliative coating had a more decreasing possibility in patients with diffuse type GC who carried GA/GG genotype of rs3733625 than AA genotype.The serum EGF concentrations in GC cases were obviously higher than those in the controls.GC cases with yellow coating had higher level of serum EGF than the patients with white coating(P>0.05).As to the relationship between tongue coating texture and serum EGF,patients with thin or thick coating had increasing serum EGF level compared with cases with exfoliative coating.The trend of serum EGF expression in the controls in descending order was thick coating,thin coating and exfoliative coating in sequence.There was no obvious correlation between coating color and lifestyle.However,our study showed that GC patients who often ate breakfast had an increased possibility of thick or exfoliative tongue coating,compared with thin coating.And the GC patients who did not prefer salty food had a decreasing possibility of thick or exfoliative tongue coating.As for the controls group,smoking or drinking might increase the probability of thick coating.This study revealed that of 141 GC patients with abnormal serum total protein(TP)level,32(22.7%)patients had yellow coating,while the controls with abnormal serum glucose had high proportion of grey coating.According to tongue coating texture analysis,white blood cell count or lymphocyte percent was associated with thickness of tongue coating for GC patients.For the controls,the level of serum TP,glutamyl transpeptidase,or serum glucose was related to the thickness of tongue coating.After tongue squamous cell carcinoma cell line Tca-8113 was cultivated in RPMI-1640 without serum in 12 hours,MTT assay showed that cell survival rate decreased prominently,while the apoptosis rate of Tca-8113 cells increased,which was detected by staining with annexin V-propidium iodide or hoechst 33258.The flow cytometric analysis revealed that the cells cycle was arrested at G0/G1 phase and the percentage of cells at S phase was decreasing.Real-time FQ-PCR and western blot test suggested that the expression of Fas increased,and the contents of AKT,NF-κB p50,HSP70,and the ratio of Bcl-2/Bax decreased in this study.The experiments in vitro indicated that EGF or TGF-α could promote cell proliferation,increase the proportion of Tca-8113 cells at S phase,stimulate cell division and migration.The expression of Bax and Fas was not inhibited obviously,whereas the level of AKT and NF-κB p50 increased.ConclusionsThe genotypes of rs2237051,rs4444903 and rs3733625 in EGF were related to tongue coating formation for patients with diffuse type GC.The lifestyles(eating breakfast and preference for salty food)of GC cases correlated with thickness of tongue coating,while the living habits,such as smoking and drinking,in the controls were related to thickness of tongue coating.For GC patients,abnormal serum TP level had closely relationship with yellow coating,and the white blood cell count or lymphocyte percent was associated with thickness of tongue coating.For the controls,abnormal serum glucose content could increase the percentage of grey coating,and the level of serum TP,glutamyl transpeptidase,or serum glucose had significant correlation with thickness of tongue coating.Subject with yellow coating had higher level of EGF than the participant with white coating,though there was no statistical significance.GC patients with thin or thick coating had increasing serum EGF contents compared with cases with peeling coating.According to the thickness of tongue coating,the order of serum EGF contents in the controls from high to low was thick coating,thin coating and peeling coating.When tongue squamous cell carcinoma cell line Tca-8113 was cultured in RPMI-1640 without serum,in vitro experiments indicated that the Tca-8113 cells were arrested in G0/G1 phase,apoptosis was mediated by Bcl-2 and Fas,and the apoptosis rate and dead cells increased.EGF or TGF-α could promote cell proliferation,division and migration through PI3K/AKT or NF-κB pathway,which influenced the balance of proliferation and apoptosis. |
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