Effects Of Cancer-associated Fibroblast HMOX1 On Metabolism Of Breast Cancer Cell

Objective:Breast cancer is one of the most common malignant tumors in women in the world.Recurrence and metastasis are the main causes of breast cancer death.Cancer associated fibroblasts(CAFs)are the core components in the microenvironment of tumor,and play an important role in the development of malignant tumors.The results of the previous study group showed that HMOXl was significantly different in the expression of CAFs and normal tissue fibroblasts(NF).The enhancement of aerobic glycolysis is a metabolic adaptation of the rapid growth and proliferation of tumor cells,and is one of the markers of malignant tumor.The purpose of this study is 1.to study the effect of HMOX1 expression in CAF cells on the proliferation,cloning,migration and other malignant biological behaviors of breast cancer cells.2.The effect of HMOX1 expression in CAF cells on glycolysis and mitochondrial function of breast cancer cells.3.To explore the mechanism of HMOX1 expression in CAF cells affecting the metabolism of breast cancer cells.Methods:1.Quantitative real time polymerase chain reaction(qRT-PCR)was used to detect the expression of HMOX1 in CAF and NF.CAF cells with significantly different expression of HMOX1 mRNA were selected for subsequent experiments.The HMOX1 shRNA lentivirus was used to infect CAF with high expression of HMOX1.The knockdown efficiency of HMOX1 shRNA was detected by qRT-PCR and Western blot;HMOX1 shRNA and negative control(NC)fibroblast conditioned medium were collected and used to culture breast cancer cells respectively.Cell proliferation test,scratch test,clone test were performed to observe the effect of HMOX1 knockdown CAF conditioned medium on malignant biological behavior of breast cancer cells.2.The HMOX1 shRNA and NC fibroblast conditioned medium were collected and used to culture breast cancer cells for 12 hours respectively.According to the instructions of Agilent seahorse XFP cell Mito stress test kit and Agilent seahorse XFP glycolic rate assay kit,the effects of HMOX1 knockdown CAF cells on mitochondrial function and glycolysis ability of breast cancer cells were observed.3.The HMOX1 shRNA and NC fibroblast conditioned medium were collected and used to culture breast cancer cells for 12 hours respectively.Western blot was used to detect the protein expression of lactate dehydrogenase A(LDHA),hexokinase 2(HK2)and pyruvate kinase M2 isoform(PKM2)in breast cancer cells under different conditions.4.Spss20.0 software package was used for analysis and statistics,if the experimental data of each group were normal distribution,they were represented by mean ± standard deviation(x±s),and the non normal distribution was represented by median and quartile,the differences between the data were tested by t-test and other test methods according to the data characteristics.Result:1.HMOX1 shRNA significantly decreased the expression of HMOX1 mRNA(P=0.028)and protein(P=0.000)in CAF.2.Compared with NC group,HMOX1 knockdown CAF conditioned medium promoted breast cancer cell proliferation(P<0.05),cloning(P<0.05)and migration(P<0.05).3.The results of metabolic test showed that HMOX1 knockdown CAF conditioned medium promoted the compensatory glycolysis rate and basal proton flow rate of breast cancer cells(all P<0.05);HMOX1 knockdown CAF conditioned medium inhibited the reserve respiration value and maximum respiration value of breast cancer cells(all P<0.05).4.Western blot results showed that compared with NC group,the protein expression of LDHA,PKM2 and HK2 in HMOX1 knockdown CAF conditioned medium was higher(all P<0.05).Conclusion:1.HMOX1 knockdown CAF cells may affect the proliferation,cloning and migration of breast cancer cells by promoting glycolysis and inhibiting mitochondrial function2.HMOX1 knockdown CAF cells may promote the glycolysis ability of breast cancer cells by promoting the expression of key enzymes LDHA,PKM2 and HK2.