| The optimal component formula(OCF)is derived from the anti-tumor active ingredients in salvia and ginseng screened in the previous orthogonal experiments of the research group,and its composition is salvia total phenolic acid,panax ginseng total saponin and ginseng polysaccharide,with a ratio of 5 mg/L,10 mg/L,5 mg/L.The previous research results showed that OCF can significantly reduce the migration and invasion ability of lung cancer A549 cells,and has a strong proliferation inhibition and apoptosis induction effect on breast cancer MCF-7 cells.Triplenegative breast cancer is the most malignant and aggressive of breast cancers,accounting for 1217%of all breast cancers.This paper mainly conducts a preliminary study on the combined effects of OCF and cisplatin(DDP)on triple-negative breast cancer.Objective:Based on previous research group studies,to observe the combined effect of OCF and DDP on triple-negative breast cancer cells.Method:1.Identification and screening of differentially expressed genes in triple-negative breast cancer:Using "TNBC" as the search term,a series of mRNA microarray datasets were screened in the GEO database.MRNA sequencing data for breast cancer and normal breast tissue and their corresponding patients were obtained from the TCGGA database.Tumor purity estimation and principal component analysis(PCA)were used to assess the tumor purity of the data samples and distinguish the sample source.Differential expression matrix acquisition and differential expression gene analysis were then performed using the limma software package.These differentially expressed genes are further validated in TCGAs and these differentially expressed genes are clustered and visualized by heat mapping.The above-mentioned differentially expressed genes after verification are analyzed by the plotROC software package to predict their ability to distinguish between normal tissue and TNBC and between TNBC and non-TNBC.2.Prognostic analysis of differentially expressed genes in triple-negative breast cancer:Onefactor risk regression analysis of differentially expressed genes in triple-negative breast cancer was carried out using survival packets in R software,and based on these eligible differentially expressed genes,a multifactorial risk regression model of disease-free interval(DFI)and progression-free interva(PFI)was established.RoC curves were used to verify the prediction accuracy of multivariate risk regression models of DFI and PFI.Finally,the RMS software package was used to integrate clinical information into the multivariate risk regression model,and the accuracy and specificity of the model were calculated by ROC curve analysis.3.QRT-PCR technology was used to detect the expression level of differentially expressed genes in triple-negative breast cancer to verify the results of bioinformatics analysis.4.Molecular docking technology to explore the affinity level of the target gene for the differential expression of danshen,ginseng active ingredient and triple-negative breast cancer:In the molecular docking analysis,we simulated the molecular docking of 1713 chemical components of 261 chemical components of danshen-ginseng,and found that the binding regions of danshen,ginseng active ingredient and prognostic related genes FAM83B,RAM24,KITLG,S100B complexes were more numerous.This shows that the compounds in salvia-ginseng have a high molecular bio affinity with four prognostic-related genes and have high pharmacodynamic activity.5.(1)The inhibition rate of OCF and DDP single drug and combined triple negative breast cancer under different concentration conditions was detected by MTT method,and the drug combination effect was evaluated by Chou-Talalay method,and the triple negative breast cancer cells with the best inhibition of proliferation and the optimal drug concentration combination were screened;(2)Clonal formation experiments detected the effect of OCF and DDP monodrugs and combinations on the proliferation ability of triple negative breast cancer cells;(3)Classical scratch experiments detected the effects of OCF and DDP monodrugs and combinations on the migration capacity of triple-negative breast cancer cells;(4)Transwell experiments detected the effects of OCF and DDP monodrugs and combinations on the migration capacity of triple-negative breast cancer cells;(5)the effects of OCF and DDP monodrugs and combinations on differential expression gene expression of triple-negative breast cancer cells by qRT-PCR method.Result:1.Identification and screening results of differentially expressed genes in triple-negative breast cancer:2 mRNA microarray datasets were screened in the GEO database:GSE115275 and GSE62931.Data from the TCGA and GEO datasets were filtered to include 120 normal tissue samples,176 TNBC samples,and 916 non-TNBC samples.Tumor purity of 1092 breast cancer tissue samples taken from the TCGA database was assessed using tumor purity estimation and principal component analysis(PCA),with tumor purities ranging from 0.159 to 0.994,56.4%of tumor samples with a purity greater than the mean of 0.741,and the selected datasets accurately distinguished between the control groups(normal breast group and non-TNBC group)and the TNBC group.Differential expression gene analysis showed that 2158 differentially expressed genes between TNBC and normal breast tissue were obtained from GSE115275,including 1291 upregulated genes and 867 downregulated genes,and 1170 differentially expressed genes between TNBC and non-TNBC were obtained from GSE62931,including 558 upregulated genes and 612 down-regulated genes.After verification by the TCGA database,a total of 991 differentially expressed genes(482 upregulated genes and 509 down-regulated genes)were screened out compared with normal breast tissues,and 997 differentially expressed genes(491 upregulated genes,506 down-regulated genes)were obtained between TNBC and non-TNBC.Taking the intersection of the two differentially expressed genes,a total of 125 common differentially expressed genes are obtained,which may play a key role in TNBC.The plotROC software package analyzes the above validated differentially expressed genes,of which 121 differentially expressed genes can accurately distinguish between normal breast tissue samples and TNBC samples,and 109 of which differentially expressed genes can accurately distinguish between non-TNBC samples and TNBC samples.The intersection of the above 121 differentially expressed genes and 109 differentially expressed genes was taken,and finally 105 TNBC differentially expressed genes that can be identified in the TNBC sample and the control group were obtained.Ranked by normal tissue and TNBC tissue AUC values,the top 5 genes were AURKA,ADAM33,CDCA8,CDCA3,and NUF2.2.Prognostic analysis results of differentially expressed genes in triple-negative breast cancer:a one-way risk regression model was constructed,and the DFI and PFI of differentially expressed genes were analyzed according to the ROC analysis results,and finally 9 heterogeneously related genes were considered to be factors affecting the prognosis(P<0.05),of which 4 genes(FAM83B,KITLG,CFD and RBM24)were significantly correlated with the DFI of TNBC patients(n=109),and 5 genes(FAM83B,EXO1,S100B,TYMS,and CFD)were significantly associated with PFI in patients with TNBC(n=120).Multifactorial risk regression analysis was performed on the above 9 genes,and patients were divided into high-risk groups and low-risk groups according to the median risk score of DFI,and the results showed that the number of deaths increased with the increase of risk scores.In the high-risk group of DFI models,KITLG and RBM24 were upregulated genes,and FAM83B was a downregulated gene.In the high-risk group of PFI models,FAM83B and S100B were down-regulated genes.The predictive performance of the ROC curve evaluation DFI and PFI multivariate risk regression models shows that their prediction performance is good.3.QRT-PCR technology detected the expression level of differentially expressed genes in triple-negative breast cancer:Compared with normal tissues,the expression level of FAM83B gene in triple-negative breast cancer showed a downward adjustment,and the expression level of FAM83B in non-triple-negative breast cancer showed an upregulation.KITLG expression in both triple-negative and non-triple-negative breast cancers was significantly upregulated,consistent with bioinformatics results.4.Molecular docking technology to explore the affinity level of the target gene for the differential expression of danshen,ginseng active ingredient and triple-negative breast cancer:In the molecular docking analysis,we simulated the molecular docking of 1713 chemical components of 261 chemical components of danshen-ginseng,and found that the binding regions of danshen,ginseng active ingredient and prognostic related genes FAM83B,RAM24,KITLG,S100B complexes were more numerous.This shows that the compounds in salvia-ginseng have a high molecular bio affinity with four prognostic-related genes and have high pharmacodynamic activity.5.(1)MTT experimental results:OCF has a more obvious inhibitory effect on most triplenegative breast cancer cells,and the inhibitory effect is positively correlated with the drug concentration,while the toxicity to normal breast cells is small.After 48h of action,different concentrations of DDP showed strong inhibitory effect on triple-negative breast cancer cells,and showed concentration-dependent tolerance.The drug combination effect was evaluated by ChouTalalay method,and the triple-negative breast cancer cells with the best inhibition of proliferation were selected as MDA-MB-231 and 4T1,and the optimal drug concentration combination was 0.5OCF+1ug/mlDDP;(2)Clonal formation experimental results:Compared with the control group,the number of clones of triple-negative breast cancer cells in the single drug group and the combined group was significantly reduced,and the monotherapy group was statistically significant compared with the joint group at the same drug concentration;(3)Classical scratch experimental results:In terms of MDA-MB-231,compared with the control group,the cell migration rates of OCF and DDP single drug and 24h and 48h in the combined group were significantly reduced,which showed that each group could significantly inhibit the migration capacity of MDA-MB-231,but compared with the combination group,the cell mobility of OCF and DDP single drug group was not significantly reduced,and in the case of 4T1,after 48h of action,each group could significantly inhibit the migration capacity of 4T1.The ability of the combination drug group to inhibit the migration of 4T1 is stronger than that of the single drug group;(4)The transwell experimental results:OCF and DDP,whether single drug or combination of drugs,can significantly inhibit the migration ability of triple-negative breast cancer cells,and the combination of OCF and DDP can enhance the effect of single drug inhibition of their migration ability.Conclusion:The combination of OCF and DDP alone and triple-negative breast cancer cells MDA-MB-231 and 4T1 can inhibit the proliferation and migration of 4T1,but the synergistic effect of the two drugs under the combination of 0.5OCF+1ug/ml for 48 hours is unclear. |
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