| Objective:Breast cancer is one of the most common malignant tumors that seriously threaten the health of our women.The high mortality rate is closely related to the early metastasis of breast cancer.Previous studies have found that mammary epithelial mucin?SMEM?is a specific marker of mammary glands and is closely related to the early metastasis of breast cancer.However,SBEM is expressed and promoted in breast cancer cells in breast cancer cells Invasion and metastasis research reports less.Epithelial interstitial transformation?EMT?is an important biological process in the migration and invasion of malignant cells derived from epithelial cells.It plays an important role in the process of tumor metastasis.The correlation between SBEM gene and EMT process is also to be studied.Chinese medicine that phlegm is the body of the water metabolism of the pathological changes in the formation of pathological products,especially the organs of the phlegm in the meridian,coagulation in the body of the organs,bones,meridians,phlegm is yin,heavy,easy to gather,with The characteristics of viscous unhappy,viscera dysfunction,qi and blood deficiency,Yu Sheng phlegm,wet stop together,plug and pass,the formation of evidence,in addition,the sputum of things,with the gas movements,everywhere,in clinical To study the relationship between SBEM and phlegm in the process of breast cancer metastasis,the relationship between SBEM and phlegm in the process of breast cancer metastasis was analyzed.Methods:1.The flow cytometry was applied to detect SBEM expression in MCF-7,MDA-MB-231 and SK-BR-3 cells.2.SBEM interference and over-expression plasmids were built,and MDA-MB-231 interference and over-expression cell lines were established by Lentivirus stable transfection method.3.The morphology change of stable transfection cells was observed.4.Western blot method was used to detect SBEM protein expression in the interference and over-expression cell lines.5.Scratch experiment and Transwell invasion experiment were conducted to detect migration and invasion ability of breast cancer cells respectively.6.Western blot method was used to detect EMT-related marker protein expression of epithelial and interstitial phenotype.7.Symptomatic score was 0,symptomatic score was 1,and K-mean clustering analysis was used to cluster,and the main symptoms of each group were classified as quantification score,0 asymptomatic,mild 1?POC?,and the related syndromes variables were further studied by using the subject characteristic curve?ROC?.The results were analyzed by the method of principal component analysis,the difference was statistically significant.The amplification efficiency of the target gene and the internal reference gene was similar,and the experimental data were treated with 2-??Ct.Calculate the average Ct value and the?Ct value?Ct=Ct SBEM-Ct GAPDH?for each sample,Calculate 2-??Ct?Ct=Ct target gene-C reference sample?,The value is a relative multiple of the target value relative to the reference value.8.the application of cluster analysis,principal component analysis and factor analysis,and reference standard for evaluation of phlegm dampness constitution score,symptom score of patients with quantization,then the patients were divided into phlegm and non phlegm syndrome group,through the detection of SBEM gene expression in patients with breast cancer,the correlation between Phlegm Dampness Syndrome and SBEM gene expression analysis,and the correlation between phlegm dampness syndrome and tumor metastasis.Results:1.Flow cytometry results showed that the expression level of SBEM protein in MDA-MB-231 cells was higher than that in MCF-7 and SK-BR-3 cells.The expression level of SBEM protein in MDA-MB-231 cells was higher than that in MCF-7 and SK-BR-3 cells.2.After MDA-MB-231 cells was transfected by Lentivirus,no significant change was found of cell morphology in Sh1 and sh2 interference groups and the overexpression groups.No obvious morphology change was found in stable transfection cells.3.Western blot was used to detect the expression of SBEM protein in every group.SBEM expression decreased in sh1 and sh2 interference groups than that in interference control group.SBEM expression increased in overexpression groups than that in overexpression control group.The difference was statistically significant?P<0.05?.The stable transfection cell lines of SBEM interference and overexpression groups were successfully established.SBEM gene was successfully interfered and over-expressed by Western blot detection.4.The cell scratch repair after 8 hours showed that cell damage repair rate of MDA-MB-231interference group in sh1 and sh2 was about 4.8%,6.2%,which was lower than that in interference control group of about 31%?P<0.05?.The cell damage repair rate of MDA-MB-231 overexpression group was about 69.9%,which was higher than that in overexpression control group of about 26.2%?P<0.05?.Scratch experiment showed that the migration ability was weakened when SBEM gene was interfered and migration ability was enhanced when SBEM gene was over-expressed.5.The results of trans-membrane cell number after 24 hours were shown.There were significant difference among sh1,sh2 MDA-MB-231 interference and interference control group.There were significant difference between MDA-MB-231 overexpression and overexpression control group?P<0.05?.Transwell invasion experiment showed that cell invasion ability was weakened when SBEM gene was interfered and cell invasion ability was enhanced when SBEM gene was over-expressed.6.No obvious difference of EMT-related marker protein was observed after SBEM gene was interfered or over-expressed in Western blot detection.7.The expression of SBEM gene was correlated with ER,PR,lymph node metastasis and Ki67 and P53 expression?P<0.05?.SBEM gene expression was correlated with age,Her-2There was no correlation between them?P>0.05?.8.Three replicates of the fluorescent quantitative PCR were done to analysis of FQ-PCR reaction system.The amplification curve showed that the curve fitting was good with close Ct values and the PCR reaction system was stable.The system was found reproducible and reliable.The contrast of amplification efficiency of target gene and reference gene:The correlation coefficient of SBEM gene initial copy number with Ct value real-time fluorescence quantitative was 0.99794,the amplification efficiency was 102%and the slope of curve was-3.312.The correlation coefficient of GAPDH gene initial copy number with Ct value real-time fluorescence quantitative was 0.99986,the amplification efficiency was 104%and the slope of curve was-3.219.The difference of the two absolute slope values was less than 0.1,indicating that amplification efficiency was similar between SBEM gene and GAPDH gene.2-Delta Ct method could be applied for relative quantitative analysis in the experimental results.The dissolution curve of fluorescent quantitative PCR showed a single Melting peak without primer dimer and non-specific fluorescent signals.9.A total of 19 patients with the first category,principal component analysis:a breast lump pain?0.841?,chest tightness?0.839?,god fatigue strength?0.739?.A total of 26 patients with the second category,principal component analysis is as follows:nausea and vomiting?0.874?,loss of appetite?0.864?,insomnia?0.847?,short of breath?0.775?,diarrhea?0.751?,god fatigue strength?0.736?.A total of 18 patients with class iii,the main ingredients for:dry mouth?0.812?,mammary bump pain?0.811?,constipation?0.787?,loss of appetite?0.731?,dizziness tinnitus?0.722?.A total of 17 patients with the fourth class,main component is:chills?0.841?,diarrhea?0.792?,lumbar debility?0.785?,mammary bump pain?0.729?.The first kind of tongue in patients with light,red moss thin,veins thin as its main clinical symptoms.The second category of patients with red tongue moss greasy,pulse string as the main clinical symptoms.The third type of patients with tongue color sink or less moss and dark purple veins thin,class 4 patients with tongue red,moss,peeling pulse acerbity.According to patients'symptoms,and refer to the TCM syndrome factor analysis and the standard of clinical diagnosis and treatment of traditional Chinese medicine,the second type of syndrome of phlegm syndrome patients wet syndrome patients.10.Four groups of patients with quantification score a non-normal distribution,K-S inspection,the Z value is 0.271,P values?sig 2-tailed?=0.001<0.05,for the non-normal distribution,spearson correlation coefficient=0.518?P=0.002?,a moderate,phlegm dampness?+/-?and SBEM gene expression in patients with syndrome,phlegm dampness syndromes syndrome patients delta Ct value is?4.26±1.47?,delta Ct value of phlegm dampness syndromes syndrome patients?6.87±1.78?,significant difference,phlegm dampness syndromes syndrome SBEM gene expression is 6.105 times of phlegm dampness syndromes syndrome,phlegm damp syndrome associated with high SBEM gene expression.Wet phlegm syndrome group and SBEM ROC curve analysis showed that gene expression of phlegm dampness syndromes syndrome SBEM high gene expression,the area under the curve(0.759,P=0.029,Sensitivity Sensitivity=0.8,1-Specifcity=0.688 specific degree,phlegm dampness syndromes syndrome with correlation analysis,the area under the curve is 0.788,P=0.018,Sensitivity Sensitivity=0.824,1-Specifcity=0.556 specific degrees.Conclusions:SBEM protein expression is much higher in triple negative breast cancer cell line MDA-MB-231 cells.The interference of SBEM gene can reduce migration and invasion ability of MDA-MB-231 cells,and the over-expression of SBEM gene can increase migration and invasion ability of MDA-MB-231 cells.SBEM gene changes the migration and invasion ability of MDA-MB-231 cells through none-EMT pathway.High SBEM gene expression and phlegm dampness syndromes have high correlation between syndrome?P<0.05?;Breast cancer metastasis in patients with phlegm dampness syndromes syndrome and other card syndrome significantly?P<0.05?;In clinical treatment,pay attention to the law of syndrome patients,clear card syndrome characteristics,improve symptoms,improve patient quality of life in patients with. |
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