Study And Analysis Of Metabolic Pathways Of NPYIR And TMEM47 Regulating Multidrug Resistance Of Tamoxifen And Paclitaxel In Breast Cancer

Breast cancer ranks first in the incidence and death rate of female malignant tumors,which seriously threatens women's life and health.With the continuous development and progress of science and medical technology,new clinical diagnosis methods,treatment schemes and new anticancer drugs are emerging,which benefit breast cancer patients.However,with the long-term widespread use of chemotherapeutic drugs and endocrine drugs,the proportion of acquired drug resistance of tumor is increasing year by year,especially when tumor cells are resistant to one kind of anti-tumor drugs,they also produce multidrug resistance to anti-tumor drugs with different structures and mechanisms of action,which leads to the reduction or even failure of endocrine therapy or chemotherapy.The reasons of acquired multidrug resistance of tumor patients to therapeutic drugs are very complex.Different cancer cells of the same tumor have different drug resistance,and the same kind of tumor can produce multidrug resistance through different ways.This has become the bottleneck of clinical diagnosis and treatment of breast cancer.Screening molecular markers related to multidrug resistance and exploring the mechanism of multidrug resistance is the key to reverse multidrug resistance in breast cancer.Based on the background,this study aims to explore mechanism of Tamoxifen(TAM)and Paclitaxel(PTX)acquired drug resistance in breast cancer,screen potential molecular markers related to multidrug resistance of breast cancer through detecting and comparing the transcripts information of the in vitro breast cancer drug-resistant cell model and the original cell line by genomics sequencing analysis platform,combined with TCGA(The Cancer Genome Atlas)breast cancer RNA-seq data.And then cell lines with drug resistance related molecular gene knockdown or overexpression were construct to verify the effect of differential expression genes related to multidrug resistance of breast cancer,further explore the generation of multidrug resistance.It can provide molecular targets for drug resistance diagnosis and new drug development of breast cancer,and provide new ideas for improving the therapeutic effect of anti-cancer drugs and improving the prognosis of breast cancer.In this study,human breast cancer cell line MCF-7 was used as the starting cell,high concentration shock method was used to induce cell resistance,CCK-8 method was used to detect the cell viability after TAM and PTX treatment,and the multidrug resistance cell line TAMR/MCF-7 was screened to provide a cell model for subsequent research.Then,the transcripts of the original cells and drug-resistant cell lines were detected by high-throughput sequencing,and the differentially expressed genes(DEGs)were obtained by comparative analysis.The DEGs were annotated and enriched by GO,COG and KEGG databases.Clinical diagnosis and treatment information and RNA-seq data of breast cancer samples in TCGA database were combined to analyze for DEGs in PD samples.Comparing the cell lines transcriptome sequencing data with the RNA-seq data from TCGA,the potential molecular markers related to drug resistance were systematically analyzed.The relationship between DEGs and estrogen receptor(ER)was studied by PPI(protein protein interaction).The relationship between DEGs and disease-free survival(DFS)was analyzed.Then,by using lentiviral plasmid or si RNA transfection strategy mediated by liposome,the expression level of target gene in different cell lines was detected by q RT-PCR and Western blot;the drug sensitivity changes of different cell lines to TAM and PTX were detected by CCK-8 method and annexin V-FITC/PI double staining method to verify the association between the DEGs and multidrug resistance.The transplanted tumor mouse model was constructed,the drug sensitivity of transplanted tumor was detected by biological in vivo imaging technology,and the molecular mechanism of multidrug resistance induced by the DEGs related was explored.Finally,based on the systematic pharmacology database,the PPI network was established,and the best match method was used to identify the cancer-related Active Linked Modules(ALMs),to identify the potential anti-cancer natural products.The anti-tumor and anti-drug effects of new drugs were verified by clinical trials database and in vitro assays.The results showed that the RI(resistance index)of TAMR/MCF-7 to TAM and PTX was2.56 and 12.18 respectively(P<0.001).Compared with the original cell line,there were3276 DEGs in the transcripts of TAMR/MCF-7,of which 1449 were up regulated and1827 were down regulated.Through the functional annotation and pathway enrichment analysis of GO,COG and KEGG databases,42 potential key genes were screened.These genes are widely involved in the regulation of cell proliferation and survival,movement and migration,tumor cell invasion and other biological processes.TCGA data analysis showed that 89 cases of DEGs(log₂FC(fold change)>1,FDR(false discovery rate)<0.01)were obtained between PD(progressive disease)samples and CR(complete response)samples.Further comparative analysis showed that there were 20 common DEGs in TAMR/MCF-7 and PD samples,among which GFRA3(GDNF family receptor alpha 3),NPY1R(neuropeptide Y receptor Y1),PTPRN2(protein tyrosine phosphatase receptor type N2)and GABRP(gamma-aminobutyric acid type A receptor subunit pi)were closely related to ER related pathways,and the survival rate of patients with high expression of NPY1R was significantly higher than that of patients with low expression of NPY1R(P<0.01).In vitro cell experiments showed that the differential expression of NPY1R and TMEM47(transmembrane protein 47)was closely related to the drug sensitivity of MCF-7 cells.Down regulation of NPY1R or up regulation of TMEM47 promoted the viability and proliferation of MCF-7 cells and significantly reduced the apoptosis rate of MCF-7 cells induced by drugs;Down regulation of TMEM47 expression in drug-resistant cells TAMR/MCF-7 can restore the sensitivity of cells to TAM and PTX.NPY1R can negatively regulate the activity of c AMP and regulate the expression of CREB(c AMP responsive element binding protein)and p-CREB through NPY1R-c AMP-PKA axis;TMEM47 further regulates ERK and AKT pathways and affects cell proliferation and apoptosis by regulating the expression of TP53(tumor protein p53)and FYN(FYN proto-oncogene,Src family tyrosine kinase).The experimental results of transplanted tumors in nude mice showed that the transplanted tumors that down regulated NPY1R or up regulated TMEM47 mediated ERK and AKT pathways,promoted tumor proliferation and induced TAM and PTX multidrug resistance.Through the analysis of ALMs module based on PPI network,12 candidate natural products were predicted;It was verified that Fisetin could significantly inhibit the viability of MCF-7 and T47D cells(P<0.01),promote apoptosis,and restore the drug sensitivity of drug-resistant cells TAMR/MCF-7 in cooperation with TAM and PTX to a certain extent.This study confirmed that NPY1R and TMEM47 were significantly differencially expressed in TAMR/MCF-7 or breast cancer PD patients compaired with the drug sensitive group.NPY1R and TMEM47 could regulate cell proliferation,cell cycle and apoptosis through regulating ERK and AKT pathways,and induce multidrug resistance.The findings suggest that NPY1R and TMEM47 may be prognostic markers for breast cancer treatment,and lay a good foundation for further exploring the mechanism of multidrug resistance in breast cancer.Fisetin and Progesterone have significant inhibitory effects on breast cancer cells in vitro(P<0.01),which can synergy with TAM and PTX to restore drug sensitivity of TAMR/MCF-7,and is expected to play an auxiliary role in breast cancer treatment.