| Objective: Breast cancer is one of the most common malignant tumors in women.According to the global cancer data released by the International Cancer Institute in 2018,the incidence rate of breast cancer ranks the first among all female cancer patients,accounting for 24% of the total number of female tumors.Compared with other types of cancer,although the prognosis of breast cancer is better,there is still 15% of breast cancer patients die.In recent years,the incidence rate and mortality rate of breast cancer are increasing.Breast cancer has become the number one killer of women aged between 40 and 55.At present,the cure rate of early breast cancer could be as high as 80%-90%,while the advanced breast cancer is less than 40%.Triple negative breast cancer(TNBC)is a kind of breast cancer whose immunohistochemical results are negative for estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor-2(HER-2).This kind of breast cancer has special biological behavior and clinicopathological characteristics.Compared with non-triple negative breast cancer,TNBC has the characteristics of small average age of onset,strong invasion,strong metastasis,low survival rate,high mortality and local recurrence rate.At present,the treatment of TNBC is still based on local surgery and assisted by whole body radiotherapy and / or radiotherapy.For TNBC,there is no clear molecular target at present.TNBC under standardized treatment is more prone to distant recurrence,and its prognosis is worse than other subtypes of breast cancer.Although TNBC usually have a high response rate to chemotherapy,they often develop chemotherapy resistance.Therefore,it is very important to elucidate the mechanism of drug resistance,find new molecular targets and develop new strategies.Methods: 1.Bioinformatics method and immunohistochemistry were used to analyze the expression of FZD5 triple negative breast cancer patients and non-triple negative breast cancer patients.The relationship between the expression of FZD5 and the survival time of breast cancer patients was analyzed by the website.2.MDA-MB-231 and Hs-578 t triple negative breast cancer cells were selected.Lentivirus infection(sh Ctrl and sh FZD5-1/2)was used to knock down FZD5 gene in MDA-MB-231 cells to form stable cell lines.Lentivirus infection(OE Ctrl and OE)was used in Hs-578 t cells.The effect of FZD5 on the cell viability was detected by CCK8 method;the effect of FZD5 on the proliferation of triple negative breast cancer cells was detected by cloning assay;the tumor bearing experiment of MDA-MB-231 cells was carried out in nude mice,measuring the size of the tumor to detect the effect of FZD5 on the proliferation in vitro;the tumor formation was immunized the expression of Ki67 and p-H3 was detected by immunohistochemical staining to detect the effect of FZD5 on proliferation in vivo.3.Flow cytometry was used to detect the cell cycle distribution of MDA-MB-231 and Hs-578 t in each group to analyze the effect of FZD5 on the cell cycle distribution of triple negative breast cancer;Western blot was used to detect CDK2,cyclin E2 and cyclin A2 in order to explore the mechanism of FZD5 on cell cycle regulation,the number of EDU positive cells in each group was detected by EDU staining test to explore the effect of FZD5 on the proliferation of triple negative breast cancer cells.4.The expression of γ-H2 AX in the nuclei of MDA-MB-231 and Hs-578 t cells was detected by immunofluorescence assay to explore the effect of FZD5 on DNA damage repair of triple negative breast cancer cells.The m RNA expressions of EXO1,PLK4 and RFC4 were detected by real-time PCR to explore the regulatory mechanism of FZD5 on DNA damage repair in triple negative breast cancer cells Adriamycin(ADR)and Paclitaxel treatment for 48 hours,flow cytometry was used to detect the apoptosis of each group,in order to explore the effect of FZD5 on triple negative breast cancer cell apoptosis.5.Real-time PCR was used to detect the m RNA expression of CD133,EPCAM,ALDH1A2 and POU5F1 in MDA-MB-231 and Hs-578 t cells.Flow cytometry was used to detect the proportion of CD133,EPCAM and ALDH1A2 positive cells in MDA-MB-231 and Hs-578 t cells.Tumor cell spheroidization test was used to detect the ability of each group to form tumor cell spheroids,so as to explore the effect of FZD5 on the stemness characteristics of tumor cells.6.CCLE database was used to analyze the correlation between FOXM1,BRCA1,BIRC5 and FZD5.The correlation between BRCA1,BIRC5 and FOXM1 was analyzed by western blot.The protein expressions of FOXM1,BRCA1,BIRC5 and β-catenin were detected by western blot;the binding of FOXM1 to BRCA1 and BIRC5 promoters was detected by chromatin immunoprecipitation;XAV939,a β-catenin inhibitor,was given with different concentrations(0,1,10 μM);Hs-578 t cells were selected and transfected with FOXM1 overexpression plasmid,and western blot was used to detect the expression of BRCA1 and BIRC5.The expression of FOXM1,BRCA1 and BIRC5 protein was detected by western blot to explore the mechanism of FZD5-FOXM1.7.In MDA-MB-231 cells,FZD5 was knocked down by lentivirus to form a stable cell line,and then FOXM1 overexpression plasmid was transfected,and FOXM1 overexpression plasmid was transfected into Hs-578 t cells.Cell viability of each group was detected by CCK8 method;cell cycle distribution of each group was detected by flow cytometry;300 n M ADR was given for 48 hours,the expression of γ-H2 AX in the nuclei of cells in each group was detected by immunofluorescence staining,and the effect of FZD5 on the stemness characteristics of cells in each group was detected by tumor cell spheroidization test.8.MDA-MB-231 cells were selected.Lentivirus infection(sh Ctrl and sh Wnt7B-1/2)was used to knock down Wnt7 B gene to form stable cell lines.CCK8 method was used to detect the effect of Wnt7 B on cell viability;flow cytometry was used to detect the effect of Wnt7 B on cell cycle distribution;300 n M ADR was given for 48 hours,the expression of γ-H2 AX in the nuclei of cells was detected by immunofluorescence staining,and the effect of Wnt7 B on the characteristics of cell stemness was detected by tumor cell spheroidization test.Results:1.The results of TCGA database and GEO database showed that FZD5 was highly expressed in triple negative breast cancer,and immunohistochemical staining further confirmed that FZD5 was highly expressed in human triple negative breast cancer.The prognosis of breast cancer patients with high expression of FZD5 is poor.2.The results of CCK8 and clonal assay showed that FZD5 could promote cell viability and clonal formation.The results of tumor bearing mice showed that FZD5 could promote the formation of tumor in nude mice.Immunohistochemistry showed that the number of p-H3 and Ki67 positive cells in tumor tissue was less.3.The results of cell cycle showed that FZD5 promoted the cells from G1 phase to S phase;Western blot results showed that FZD5 could promote the expression of CDK2,cyclin E2 and cyclin A2;EDUresults showed that the number of EDU-positive cells decreased significantly after FZD5 knockdown,on the contrary,the number of EDU-positive cells increased significantly after FZD5 overexpression.4.The results of immunofluorescence showed that FZD5 could promote the expression of γ-H2 AX in nuclear;Real-time PCR results showed that FZD5 could promote the m RNA expression of EXO1,PLK4 and RFC4;The results of apoptosis experiment showed that FZD5 could inhibit the apoptosis of cells induced by ADR and paclitaxel.5.Real-time PCR results showed that FZD5 could promote the m RNA expression of CD133,EPCAM,ALDH1A2 and POU5F1;Flow cytometry showed that FZD5 could promote the expression of CD133,EPCAM and ALDH1;FZD5 could promote the formation of tumor spheroids.6.CCLE database correlation analysis results showed that FZD5 and FOXM1,BRCA1 and FZD5,BRCA1 and FOXM1,BIRC5 and FZD5,BIRC5 and FOXM1 showed significant positive correlation;Western blot results showed that FZD5 could promote the expression of BRCA1 and BIRC5;The results of immunoprecipitation showed that FOXM1 could bind to specific sequences of BRCA1 and BIRC5 promoters;Western blot results showed that FOXM1 could promote the expression of BRCA1 and BIRC5,and FZD5 could promote the expression of activativeβ-catenin,and the expression of FOXM1 was inhibited by XAV939 which is a β-catenin inhibitor.7.FOXM1 could promote cell vitality,promote cells transformation from G1 to S phase,and promote the expression of γ-H2 AX in nucleus,and also could promote the formation of tumor spheroids.8.The results of cell cycle showed that Wnt7 B could promote cells transition from G1 phase to S phase and promote the expression of γ-H2 AX in nucleus,and also could promote the formation of tumor cell spheres.Conclusions: 1.FZD5 can promote G1/S phase transition,DNA replication,DNA damage repair,drug resistance and stemness of triple negative breast cancer.2.Wnt7B/FZD5 regulates FOXM1 expression through Wnt/β-catenin pathway.3.FOXM1,as a downstream molecule of FZD5,can transcriptionally regulate BIRC5 and BRCA1,and then regulate G1/S phase transition,DNA replication,DNA damage repair,drug resistance and dryness of triple negative breast cancer cells. |
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