| As one of the most common malignant tumors,liver cancer has the characteristics of poor prognosis,high recurrence rate and high metastasis.As a structural component of cells and a major energy storage substance for energy metabolism,lipids play an indispensable role in the growth and proliferation of cells.A common biological feature of malignant tumors is metabolic reprogramming.The liver is the main organ of lipid metabolism.The changes of lipid metabolism have an important impact on the occurrence and development of liver cancer.Existing studies have shown that the rate of fatty acid and cholesterol synthesis in liver cancer tissues is significantly increased compared to normal tissues.It indicates that the lipid metabolism pathway in liver cancer has changed from the equilibrium state of normal cells.Therefore,it can be an effective target for anti-liver cancer treatment.As a natural plant flavonoid,apigenin has attracted attention from researchers due to its low inherent toxicity and its different effects on cancer cells and normal cells.At present,it is found that apigenin can achieve anti-cancer effects by regulating tumor cell cycle,inducing apoptosis and autophagy,inhibiting invasion and metastasis,but less research about metabolism affecting tumors.Epigenetic modification can lead to silence or overexpression the related genes,and then play an important role in tumor proliferation,apoptosis,autophagy,and metastasis.Researchers have studied the relationship between natural drugs and epigenetics,and found that apigenin can regulate epigenetic related enzymes and then affect the occurrence and development of tumors.Histone lysine-specific demethylase 1A(KDM1A),the first histone demethylase to be found,is located in the nucleus and consists of 852 amino acids that composed three regions,its catalytic mechanism starts by transferring hydrides from methylated lysine to FAD cofactors,generates unstable imine intermediates,and releases formaldehyde after hydrolysis.Studies have shown that KDM1 A can regulate the metabolic process of tumor cells,and then meet a series of characteristics such as malignant tumor proliferation.Researchers have studied the relationship between natural drugs and epigenetics,and found that apigenin can regulate epigenetic related enzymes and then affect the occurrence and development of tumors.In this study,apigenin was used to treat liver cancer cell lines(MHCC-97 H,HepG-2)to detect the effect of apigenin on the biological characteristics of liver cancer cells.We through the transcriptome to analyze the molecular mechanism of apigenin affecting the proliferation of liver cancer,we paid the attention to the effect on the lipid metabolism pathway of liver cancer cells,and screen out the epigenetic factor histone demethylase KDM1 A,which has a huge influence on lipid metabolism pathway.We undertook knock-down of KDM1 A in liver cancer cells and to detecte the effect of KDM1 A on the proliferation and apoptpsis of liver cancer cells and the lipid metabolism pathway in order to clarify the mechanism of apigenin’s inhibitory effect on liver cancer cells.Through the research,we got the following results:1.Apigenin influences biological characteristics of liver cancer cells.Liver cancer cells were treated with different concentrations of apigenin,and the effects of apigenin on the proliferation of liver cancer cells were tested by MTT and BrdU experiments.The results showed that apigenin inhibited the proliferation of liver cancer cells;and analyzed liver cancer cell by flow cytometry,the apoptosis results showed that apigenin can induce apoptosis significantly.Western blot(WB)experiments also proved that apigenin can increase the expression level of active apoptotic proteins and reduce the expression level of inactive apoptotic proteins.To determine whether apigenin affect the metastasis and invasion ability of liver cancer cells,we performed Transwell assays and wound healing assays.We found apigenin can reduce the invasion and metastasis of liver cancer cells.We used soft agar experiments to test the effect of apigenin on the tumorigenicity of liver cancer cells in vitro.The results show that apigenin can reduce the colony forming ability of liver cancer cells significantly.2.Apigenin regulates lipid metabolism in liver cancer cells.We performed the analysis of the mRNA microarray expression of apigenin-treated HepG-2,compared with control group.It was found that the pathways related to lipid metabolism were decreased significantly.In order to further verify the effect of apigenin on liver cancer cell lipid metabolism,we used RT-qPCR and WB experiments to detect the mRNA and protein levels of lipid metabolism-related enzymes.The results of RT-qPCR experiments showed that the concentration of apigenin is 100 μM can significantly inhibite the mRNA expression levels of SREBP1,SREBP2,HMGCR,SQLE,and FSAN in MHCC-97 H.Apigenin significantly inhibited the mRNA expression of SREBP1,SREBP2,FASN,HMGCR,HMGCS1,and SQLE in HepG-2 in the concentration-dependent effect.The results of WB experiments showed that the levels of the corresponding proteins such as FASN,SQLE,HMGCS1,and HMGCR were not statistically significant when the concentration of apigenin was 50 μM.Because the reduction of lipid metabolism pathways will reduce the lipid content in cells,author examined the changes of lipids in liver cancer cells after apigenin treating by oil red staining experiments.The results showed that apigenin can reduce the lipid content in liver cancer cells.3.Apigenin regulates lipid metabolism of liver cancer cells by down-regulating KDM1ABy analyzing the database,we found that the expression level of KDM1 A was correlated with the prognosis,progression and malignancy of liver cancer patients.Moreover,the expression level of KDM1 A in clinical liver cancer patients was higher than that in normal people.In order to study the molecular mechanism of apigenin in regulating lipid metabolism of liver cancer cells,we examined the expression of KDM1 A in apigenin-treated liver cancer cells by RT-qPCR and WB experiments.The results showed that apigenin could significantly down-regulate the mRNA and protein levels of KDM1 A in liver cancer cells;in order to verify the role of KDM1 A,we treated liver cancer cells with the inhibitor of KDM1 A ORY-1001 and construced liver cancer cell lines interfering with sh-KDM1 A.The proliferation ability of liver cancer cells after down-regulating KDM1 A was measured by MTT and BrdU experiments.The experimental results showed that the proliferation of liver cancer cells was significantly inhibited after down-regulating KDM1 A.The apoptosis of cells after down-regulation of KDM1 A was detected by flow cytometry.The results showed that down-regulation of KDM1 A significantly induced cell apoptosis.In order to detect the changes in lipid metabolism after interference with KDM1 A,we examined the changes in lipid metabolism pathways by RT-qPCR and WB experiments.The experimental results show that interference with KDM1 A can significantly inhibit the mRNA and protein level of SREBP1,SREBP2,FASN,HMGCR,HMGCS1 and SQLE.In order to further clarify the relationship between apigenin and KDM1 A,we constructed a KDM1 A overexpression vector and treated liver cancer cells with apigenin after the overexpression of KDM1 A.The results showed that KDM1 A overexpression could resist proliferation inhibition and metabolic pathway down-regulation caused by apigenin to some extent.In conclusion,apigenin can regulate the lipid metabolism of liver cancer cells by targeting KDM1 A,thus inhibiting the proliferation and growth of liver cancer cells. |
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