Effects Of Modification Of Sijunzi Decoction On Up-regulating The Expression Of NPPA In The Treatment Of Breast Cancer

Objective This study aims to explore whether NPPA has a potential role in the occurrence and development of breast cancer,and whether the Modification of Sijunzi Decoction can play a role in reducing toxicity and enhancing efficacy in the treatment of breast cancer by regulating the expression of NPPA.Methods In the first part,Obtain mRNA sequencing data,molecular classification,immunohistochemical(IHC)staining data and clinical information of breast cancer patients and other cancer types from TCGA-BRCA database,Human Protein Atlas,IST Online,GEO database and GEPIA.Obtain expression patterns of normal breast tissue and adjacent tissues from the TCGA database,GEO database(GES65261),and GTEx database.Kaplan Meier analysis was used to compare the total survival(OS),disease specific survival(DSS),disease free interval(DFI)and progression free interval(PFI)of breast cancer patients.Graphpad,SPSS and GEPIA were used for Kaplan Meier analysis to calculate the log rank significance of breast cancer patients in each group.SPSS software was used for Kaplan Meier analysis and Graphpad visualization.ER status,PR status,postoperative radiotherapy status and PAM50 subtype of breast cancer patients were stratified.Multivariate Cox regression analysis was conducted using SPSS software.The threshold value P<0.05 was used for positive stepwise inclusion,and P>0.10 was used to obtain independent factors affecting the prognosis of breast cancer patients.MeV software is used for heat map and Hierarchical clustering.Construct specific target shRNA lentiviral vector,transfect cells and culture them,carry out breast cancer cell proliferation test and breast cancer cell invasion and migration test,establish nude mice xenotransplantation model with MCF-7 breast cancer cell line,and detect the effect of NPPA knock-down on breast cancer cell proliferation and metastasis.In the second part,50 BALB/c mice were selected as the research object,8 mice were randomly selected as the blank control group(Blank group),and the remaining 42 mice were used to construct the breast cancer model.The mice that successfully established the model were randomly divided into four groups,8 in each group:model group(Model),Modification of Sijunzi Decoction group(MOSD),docetaxel group(DTX),and combination of traditional Chinese and western medicine group(DTX+MOSD).The mice were treated according to the corresponding groups for 21 days.Measure the changes in tumor size and body weight of mice every three days.On the 22nd day,after anesthesia in the mice,① remove the eyeballs and take blood for routine blood testing.②The tumor tissue was stripped,weighed and photographed.Three samples were taken from each group and fixed with 4%Paraformaldehyde for HE staining and immunohistochemical detection of NPPA protein.Three samples were taken from each group and stored with RNAsolid tissue RNA stable preservation solution.The level of NPPA mRNA was measured by qPCR.③ Peel lung tissue,rinse with PBS,fix with Bouin’s fixative for 24 hours,and perform HE staining to count the number of lung metastases.In the third part,retrospectively collected 62 cases of breast cancer patients with neoadjuvant chemotherapy,and divided them into two groups according to different chemotherapy schemes,named the western medicine group(Control group,30 cases)and the western medicine+Modification of Sijunzi Decoction group(MOSD group,32 cases).Observe the tumor control rate of patients after 6-8 cycles of treatment,and observe the common adverse reactions,including leukopenia,thrombocytopenia,Transaminase increase,anorexia,diarrhea,etc.Results In the first part,(1)The TCGA database and GSEA website conducted SC3 clustering analysis on the expression of extracellular matrix related genes.Perform Kaplan Meier analysis to compare the total survival(OS),disease specific survival(DSS),disease free interval(DFI),and progression free interval(PFI)of all cluster patients.When breast cancer patients were divided into two groups,DSS,DFI and PFI were significantly different.To further understand the significance of clustering,the pathological staging,AJCC staging,radiotherapy,PR status,PAM50 status,new tumor event status,new tumor anatomical location and new tumor event type of breast cancer patients in these two clusters were marked.There were no differences in pathological staging,AJCC staging,postoperative treatment,and molecular subtypes between the two clusters of patients,but there were differences in tumor recurrence rates.Further bioinformatics analysis shows that new tumor events after initial treatment are the most common distant metastasis,with the most common site of metastasis being the bone.The most significant path changes were observed in cluster 1 and cluster 2 through single sample gene enrichment analysis(ssGSEA).Pathways related to tumor recurrence,including telomerase regulation,SMAD2/3/4 heterotrimer regulated transcription,and NIMA kinase activation,were significantly activated,while pathways involved in DNA damage repair,such as the recognition and association of DNA glycosylases and the cleavage of damaged purine pathways,were inhibited.(2)Calculate the mRNA level of all ECM genes in Ju 1/2 breast cancer patients,and the genes with the most changes are used as potential gene markers for breast cancer patients.In SC3 method,49 genes between cluster 1/2 are further selected as ECM related marker genes.Through hierarchical clustering,ECM labeled genes are further divided into three different levels based on their expression patterns,namely low,medium,and high ECM groups.Kaplan Meier analysis shows that patients with lower ECM levels have impaired DSS and a trend towards reducing PFI in cancer patients.The multivariate Cox regression model showed that PR status,pathological staging,ADAMTS13,NPPA expression were correlated with DFI;Pathological staging and expression of SEMA3B and GDF15 are correlated with DSS;PR status,pathological stage and expression of EDIL3 and MFAP4 in breast cancer patients are related to PFI,while pathological stage and expression of SEMA3B,COL17A1,EDIL3 and GDF15 are related to OS.Calculate the risk scores for OS,DSS,DFI,and PFI,and display the importance of these risk scores.To evaluate the prognostic value of all these independent genes,the results showed that only SEMA3B and NPPA could significantly predict the DFI,PFI and DSS of breast cancer patients.The biological function of SEMA3B has been proposed,so we try to reveal the role of NPPA in breast cancer.(3)The expression model of NPPA in normal and malignant tumor tissues was established through IST Online tool.NPPA was highly expressed in normal Endocrine system,myeloma lung carcinoid,glioma and normal breast tissues,but significantly reduced in breast cancer tissues.The expression of NPPA is inhibited in various tumor tissues and only overexpressed in low-grade gliomas.In TCGA and GSE65216 databases,NPPA is reduced in breast cancer.NPPA immunohistochemical staining of normal and breast cancer tissues was collected from Human Protein Atlas database,and the results showed that the level of NPPA in breast cancer tissues decreased.(4)According to the proliferative potential of breast cancer patients,they were further divided into three groups.The expression level of NPPA gradually decreased in patients with higher proliferative potential.After establishing four breast cancer cell lines and stably knocking down NPPA,the proliferation potential of four different breast cancer cells was significantly increased,regardless of their subtypes or ER status.Transwell experiments were conducted on MCF-7 and MDA-MB-231 cells.The results showed that knockdown of NPPA could significantly enhance the migration and invasion of breast cancer cells.The results of the nude mouse xenograft model showed that compared with the control group,the proliferation ability of MCF-7 cells with NPPA knockdown was enhanced.In the second part,Although the Modification of Sijunzi Decoction alone could not inhibit tumor growth,it could assist docetaxel to inhibit tumor growth,which showed that the tumor in the combination group was smaller than that in the western medicine group(P<0.05).Compared with the blank control group,the model group and various drug intervention groups showed weight loss,but there was no statistical difference.Compared with the blank control group,the number of red blood cells and hemoglobin levels in the model group mice decreased(P<0.001,P<0.05);The number of white blood cells and neutrophils increased(P<0.001,P<0.001);Although the number of platelets was also increased,there was no statistical difference.Compared with the model group,the docetaxel group reduced the number of white blood cells,neutrophils and platelets.The three types of numbers in the addition and subtraction group of Modification of Sijunzi Decoction remained basically unchanged.Although the combination group of traditional Chinese medicine and western medicine decreased,it did not decrease as much as the western medicine group.These changes indicated that Modification of Sijunzi Decoctio could reduce the decrease of leukocytes,neutrophils and platelets caused by docetaxel.Compared with the model group,the docetaxel group and the combination of docetaxel and Modification of Sijunzi Decoction group had fewer lung metastases,while the combination of Chinese and Western medicine group had the least.The combination of Chinese and Western medicine can increase the expression levels of NPPA mRNA and protein in tumor tissue,suggesting that it may be the reason for inhibiting lung metastasis.In the third part,The decrease rate of white blood cells and platelets in the Modification of Sijunzi Decoction combined chemotherapy group was lower than that in the chemotherapy alone group(P<0.05),and the increase rate of Transaminase was lower than that in the control group(P<0.05).At the same time,the tumor control rate of the combination group was higher than that of the simple chemotherapy group,with a statistically significant difference(P<0.05).The grade of diarrhea and anorexia in the Modification of Sijunzi Decoction combined chemotherapy group was significantly lower than that in the chemotherapy alone group(P<0.05).Conclusions 1.The expression level of NPPA in breast cancer tissues and cells is reduced,and the down-regulation of NPPA results in the enhancement of proliferation,migration and invasion of breast cancer cells in vivo and in vitro.2.The combination of Modification of Sijunzi Decoction and chemotherapy drugs can inhibit the tumor growth by up regulating the expression of NPPA,reduce the the decrease of leukopenia,neutropenia and thrombocytopenia caused by chemotherapy drugs,and inhibit lung metastasis of tumors.3.The combination of Modification of Sijunzi Decoction and chemotherapy drugs plays a role in reducing toxicity and enhancing efficacy in the clinical treatment of breast cancer.