FPN1 Knockdown Supports Hepatocellular Carcinoma Malignant Progression Through Activating P-AKT,p-STAT3 Pathways

Objectives: Hepatocellular carcinoma(HCC)is the fifth most common tumor in the word,accounting for 9.1% of all cancer deaths worldwide.The mortality of HCC in China was more than 45% of all over the world.Ferroportin1(FPN1)was the only protein on cell menbrane to transport iron out of cells.Some studies had shown FPN1 was closely related to tumor development.The purpose of this study is to explore the correlation between FPN1 and prognosis of HCC patients,to study the effect of FPN1 on malignant progression in HCC,to investigate the mechanism that FPN1 regulates the malignant progression in HCC.Methods:(1)FPN1 mRNA expression was analyzed by data-mining from Oncomine database.FPN1 protein expression in 5 human cancer tissues which include gastric cancer,breast cancer,colorectal cancer,lung cancer,liver cancer tumor tissues and para-carcinoma tissues was detected by immunohistochemistry.(2)The kaplan-meier curve was made to study the correlation between FPN1 expression and prognosis of HCC patients.(3)HepG2 and SMMC7721 cells were either or not transfectedwith lentivirus vector that carrying FPN1-shRNA or FPN1-OE,and the expression of FPN1 was detected by Western blotting and Real-time RT-PCR(RT-qPCR)after screening by puromycin.(4)EDU experiment,growth curve,plate clone formation assay,scratch assay and invasion assay were used to study the effect of FPN1 on cell proliferation,invasion and migration in HepG2 and SMMC7721 cells which were either or not transfected with FPN1-shRNA or FPN1-OE.(5)Western blotting was used to detect the expression of AKT,STAT3 proteins;Cell proliferation was detected by MTT assay after adding AKT or STAT3 inhibitors in HepG2 and SMMC7721 cells that were either or not transfected with FPN1-shRNA.Results:(1)FPN1 mRNA was high expression in neurologic tumor,esophageal cancer,lymphoma,and teratoma,on the contrary,was low expression in leukemia,lung cancer,ovarian cancer,and prostate cancer by data-mining from Oncomine database.The expression of FPN1 protein was down-regulation in all of tested tumor samples,but there was statistic difference in breast cancer and liver cancer.Kaplan-meier curve showed the total survival time of HCC patients with low expression of FPN1 was significantly shorter than that of HCC patients with high expression of FPN1.(2)Compared with vector-control cells,HepG2 and SMMC7721 cells that were transfected with FPN1-shRNA displayed higher growth which embodied in cell viability increased,the number ofclone formation and positive cells stained with EDU dye obviously increased,cell invasion and migration accelerated significantly;On the contrary,compared with vector-control,HepG2 and SMMC7721 cells that were transfected with FPN1-OE displayed growth inhibition which embodied in cell viability,clone formation,EDU staining positive rate reduced,cell invasion and migration slowed significantly.(3)Phosphorylated AKT and STAT3 pathways were significantly activated,while the total protein level of AKT and STAT3 remained unchanged in HepG2 and SMMC7721 cells that were transfected with FPN1-shRNA;Meanwhile,cell viability was detected by MTT assay after adding AKT or STAT3 inhibitors,and found that the effect of low expression of FPN1 on promoting cell proliferation in HCC was partially reversed.Conclusions:(1)Low expression of FPN1 is negatively correlated with the prognosis of HCC patients.(2)Decreased FPN1 promotes cell malignant progression in HCC.(3)Decreased FPN1 promotes cell malignant proliferation in HCC by activating p-AKT and p-STAT3 pathways.