Study On The Molecular Mechanism Of The Formation Of Tongue Fur In Gastric Cancer Patients

ObjectiveTraditional Chinese medicine theories believe that tongue fur is resulted from the upgoing spleen wetness steamed by stomach. In this study, malignancy-gastric cancer, the most commonly seen cancer in Nanjing, was selected as the main sample of disease. To study the formation of tongue fur in molecular genetics of gastric cancer, markers of EGF, TGF-a, EGF-R and E-cad (CDH1) were selected for research observation which are related to the formation of tongue fur. The purpose of the study is to reveal the relationship between gene polymorphism of EGF, TGF-?, EGF-R and E-cad (CDH1) and the formation of tongue fur in gastric cancer, to explore the impact of the formation mechanism of tongue fur of gastric cancer at gene level, and to provide a new theoretical basis for Chinese medicine tongue diagnosis in the clinical diagnosis of gastric cancer.MethodsThe study compared two groups of clinical cases, with 258 patients of gastric adenocarcinoma as the case group and 261 healthy people (chosen through physical examinations) without specifically harmful habits as Group B. According to standards set in Diagnostics of TCM ( edited by Wu Chenyu, edition 2011), two groups of people were categorized, based on different types of tongue furs, as pathologic thick fur, pathological thin fur and normal coated tongue. Peripheral blood of the two groups were collected through PCR-LDR to detect EGF, TGF-a, EGF-R and E-cad SNP loci, so as to acquire the SNP genotype and information of allele frequency, and then the expression level of EGF, TGF-a, EGF-R and E-cad were measured in serum by ELISA. According to the associated analysis of the SNP genotype, it was expected through the study to explore the formation of tongue fur of gastric cancer patients and the changes in the molecular genetics based on the expression level of EGF, TGF-a, EGF-R and E-cad, clinical staging of gastric cancer, medical history, tongue fur type, demographic characteristics and lifestyle.Results1. Demographic characteristics of the two groups: 258 Gastric adenocarcinoma patients as Group A,including 180 males with an average age of 60.61±10.83 and 78 females with an average age of 56.14±13.68; 261 healthy people as Group B, including 158 males with an average age of 51.36±13.94, and 103 females with an average age of 50.34±13.35.2. Relationship between gastric cancer and lifestyle: the proportion in group A who do have timely meals is higher than that in Group B (?2=9.124, P=0.010); compared with Group, Group A is higher in the intake of salt (?2=74.409, P<0.001); the smoking rate of Group A is higher than that of Group B (?2=18.019,P<0.001); the frequency of eating soy products in Group A is significantly lower than that Group B (?2= 10.669, P=0.014); the frequency of eating fresh fruit in Group A is significantly lower than that in Group B (x2=59.905, P <0.001); 29.2% of Group A have drinking history compared to 17.3% in Group B (P <0.001).3. In this study, dialectical analysis was conducted on the 258 newly observed cases of gastric cancer based on the eight basic syndrome types. As to the syndrome distribution of TCM,the total number of cases with more than two syndrome types (57.63%) was significantly greater than that with a single one (36.69%); and as to the distribution of single syndrome types, it was more commonly found deficient cold of spleen and stomach (22.11%), yin-insufficiency due to stomach-heat (20%), deficiency of both qi and blood (15.79%), and damp-heat with exuberance of virulence (12.63%). In all syndrome types, another inclusion of asthenia and sthenia (46.90%)is dominant.4. Results from the correlation research between EGF gene of 10 SNP loci and its molecular and the formation of tongue fur of gastric cancer patients. the gene distribution of its exon area of rs2237051 is significantly different. Multivariate logistic regression analysis of rs2237051 reveled that, compared with the AA genotype, AG/GG genotype can increase the risk of gastric cancer(Adjust OR=1.577; 95%CI=1.163-2.138); as to Rs3733625 in 3'UTR region, compared with the AA genotype, AG genotype can reduce the risk of gastric cancer (Adjust OR = 0.714;95% CI=0.512-0.996). Furthermore, in the gastric cancer patients of intestinal type , as well as for drinking male over 50, the risk effects of rs2237051 and the protective effects of rs3733625 were both statistically significant. As to Rs2237051 in white fur, compared to the wild type of AA , mutation heterozygous AG and mutation homozygous GG can reduce the incidence of gastric cancer (P = 0.034); as to Rs2237051 in thick fur, compared to the wild type of AA,mutation heterozygous AG and mutation homozygous GG can reduce the incidence of gastric cancer(P = 0.045); the other factors including the fur color, fur nature, as well as polymorphic loci were not significantly associated (P> 0.05). For gastric cancer patients who showed decreased white blood cell, lymphocyte and total protein, the proportion of thin fur also declined while the thick fur and stripping fur increased (P <0.001); the expression of EFG in Group A with declined total protein was lower than that in Group B (P <0.01). However, there was no significant difference in the expression of EFG between the two groups when it came to abnormal white blood cell and lymphocyte.5. Results of the correlation research between EGF-R gene of 11 SNP loci and its molecular and the formation of tongue fur of gastric cancer patients. (1) the expression level of EGF-R in serum of Group A is higher than that of Group B (P <0.05), particularly among people with white fur or thin fur who have normal clinical indexes including percentage of lymphocytes,percentage of neutrophils and hemoglobin. No association was found between the expression level of EGF-R and age (P> 0.05); (2) as to exon region of EGF-R gene, there existed only T allele of SNP rs28384375. T allele of SNP rs2072454 can increase the risk of gastric cancer (P =0.05), while the other five SNP have no significant correlation with the risk of gastric cancer. It was found that, by constructing haplotype, ACAGCG and ACAGCA were correlated with gastric cancer significantly(OR= 0.67; 95% Cl = 0.49-0.92; OR = 0.75, 95% Cl = 0.57-1.00); (3) there were no statistical difference between Group A and Group B in the distribution of rs6965469,rs884225, rs884904 and rs763317 allele, genotype of the functional area of EGF-R gene.Haplotypes constructed by rs6965469, rs884225 and rs884904, haplotype of TGA can significantly increase the risk of gastric adenocarcinoma (OR=3.52; 95%CI= 2.34-5.29), while haplotype of TAG can significantly reduce the risk of gastric adenocarcinoma (OR = 0.41; 95%CI = 0.26-0.64; (4) variations of SNP rs1050171 and rs2293347 have impact on the fur color in both groups. Variations of SNP rs17337023, rs1050171 and rs2293347 have impact on the tongue fur nature in both groups. Variations of SNP rs1050171 and rs2293347 have impact on both the fur color and tongue fur nature in both groups, and the influence of SNP rs2293347 is more obvious.6. Results of the correlation research between TGF-agene of 8 SNP loci and its molecular and the formation of tongue fur of gastric cancer patients. The content of TGF-a in serum in Group A was significantly lower than that in Group B (P<0.05); in aspect of fur color, white fur in Group A was significantly lower than that in Group B (P<0.05); in aspect of fur nature, thick fur in Group A was significantly higher than that in Group B (P<0.05); stripping fur in Group A was significantly lower than that in Group B (P<0.05); The content of TGF-a in serum in the gastric adenocarcinoma patients with yellow thick fur was significantly higher than that with the stripping fur (P <0.05).7. The correlation between E-cad(CDH1) gene of 4 SNP loci and its molecular and the formation of tongue fur of gastric cancer patients. There are correlations between types of tongue fur, histological type, SNP of E-cad (CDH1) gene, level of sE-cad and clinical test results. No correlation was found between the level of sE-cad and SNP of E-cad (CDH1) gene.Conclusions1. There is a close relationship between gastric cancer and lifestyle. The incidence of gastric cancer is obviously increasing in the people who have the bad habits of untimely meals, heavy taste, smoking and drinking. Eating soy products and fresh fruit can obviously decrease the risk of gastric cancer.2.Dialectical analysis shows that in the distribution of single syndrome types among the eight basic syndrome types in the gastric cancer patients, deficient cold of spleen and stomach,yin-insufficiency due to stomach-heat, deficiency of both qi and blood, damp-heat with exuberance of virulence are prevalent; Among the mixed syndromes, blood type drug resistance and incoordination between liver and stomach were most commonly found. Accompanied symptoms and inclusion of asthenia and sthenia are the main forms of the syndrome type of gastric cancer.3.There is a new finding that EGF gene SNP loci - exon region rs2237051 is associated with the risk of gastric cancer. AG / GG genotype may increase the risk of gastric cancer. It is presumed that changes of genotype have effect on the structure, function or secretion level of EGF, which is worthy of further study. As to the rs3733625 in 3'UTR region, AG genotype has a protective effect. Changes of genotype of rs2237051, which are based on the performance of coated tongue, are closely related with the incidence of gastric cancer. The expression of EGF in serum of Group A was significantly higher than that of Group B. The expression of EGF and gastric cancer have positive related. The content of EGF in serum in the patients with white thin fur and white thick fur is obviously higher than that in Group B. For the expression of EGF in the gastric cancer patients: yellow thin fur > yellow thick fur > stripping fur. The expression of EGF has no linear relationship with the thickness of tongue fur. Therefore, it was assumed that the content of EGF in serum is not the only factor that affect the formation of tongue fur. Many factors such as sojourn microbial,physical condition, clinical treatment and other cytokines(TGF) may also cause the changes of tongue fur. The decline in the number of immune cells and changes in the liver and kidney function of the gastric cancer patients may cast an impact on the formation of tongue fur. The percentage of stripping fur and thick fur is higher in patients with abnormal test results, and the expression of EGF in serum in the Group A is lower than that in Group B (low total protein ),or with no obvious difference ( abnormal in WBC and Lymphocytes ).4.The study showed that: (1) the expression level of EGF-R in serum in Group A is increasing and has no association with age; in the exon region of EGF-R gene, T genotype of SNP rs28384375 may increase the risk of gastric cancer, and the other five SNP have no significant correlation with the risk of gastric cancer. (2) by constructing haplotype, ACAGCG and ACAGCA were significantly associated with gastric cancer; (3) there are no statistical difference between Group A and Group B in the distribution of rs6965469, rs884225, rs884904 and rs763317 allele, genotype of the functional area of EGF-R gene. Meaningfully, among haplotypes constructed by rs6965469, rs884225 and rs884904, haplotype of TGA can significantly increase the risk of gastric adenocarcinoma, while haplotype of TAG can significantly reduce the risk of gastric adenocarcinoma; (4) variations of SNP rs1050171 and rs2293347 have impact on the fur color in the gastric cancer patients and Group B. Variations of SNP rs1 7337023, rs1050171 and rs2293347 have impact on the tongue fur nature in both groups.Variations of SNP rs1050171 and rs2293347 have impact on both the fur color and tongue fur nature in both groups, and the influence of SNP rs2293347 is more obvious.5.Changes of the expression level of TGF-a in serum are closely related to the occurrence and development of gastric adenocarcinoma; tongue fur types of gastric cancer patients are correlated with the expression level of TGF-a in serum; there is no correlation between TGF-a gene of 8 SNP and the occurrence of gastric cancer and tongue fur types.6.There are correlations between the tongue fur type, histological type, SNP of E-cad(CDH1) gene, level of sE-cad and the clinical test results. There is no correlation between level of sE-cad and SNP of E-cad (CDH1) gene.By studying the EGF, EGF-R, TGF-a and E-cad (CDH1) gene locus and its molecular expression in serum, the above mentioned results reveal the mechanism in molecular genetics of the formation of tongue fur of gastric cancer patient. At the same time, it is discovered the scientific value of TCM tongue dialectical in malignant-gastric cancer, which provides basis for future study of translational medicine.