The Regulation And Mechanism Of Hepatocyte Nuclear Factor-1? On Stemness Of Liver Cancer Cells

Background and Aims: The primary liver cancer was divided into hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC)and combined hepatocellular-cholangiocarinoma(CHC)by World Health Organization(WHO).The prognosis of hepatocellular carcinoma with different pathological types is very different.The prognosis of ICC is significantly worse than that of HCC,which is due to tumor heterogeneity.Many researches indicated that the tumor heterogeneity was associated with cancer stem cells(CSCs).CSCs are a group of cells that exist in tumor tissue,with similar characteristics of stem cells,which have unlimited self-proliferation and differentiate potential to maintain tumor cell vitality.Previously researches show that liver CSCs may contribute to recurrence of HCC.Several studies have shown that a portion of HCC expresses progenitor cell phenotype such as CK7 and CK19.Actually,this part of liver cancer has a significantly worse prognosis and a higher recurrence rate after Surgery or other treatment.hepatocyte nuclear factor-1?(HNF-1?)is a member of the hepatocyte nuclear factors family,which regulates the complex gene networks involved in lipid,carbohydrate,and protein metabolism13 and plays an important role in regulating liver development and hepatocyte differentiation.In recent years,it has been reported that the expression of HNF-1? is associated with a variety of cancer risk.However,there are few studies on the relationship between HNF-1? and liver cancer.Our previously study showed that HNF-1? expression was associated with pathological subtype of primary liver cancer.The expression of HNF-1? in ICC tumor tissue was higher than that in HCC.Therefore,we hypothesized that high expression of HNF-1? may promote HCC cells de-differentiate into liver cancer stem cell,thus increasing the malignancy of HCC and affecting the prognosis of HCC.Firstly,we retrospectively investigate the relatiation between the expression of HNF-1? in patients with hepatocellular carcinom and the prognosis.Then,we observe the change of stemness of liver cancer cells with HNF-1? overexpression.Finally,we discuss the mechanism of HNF-1? effect the stemness of liver cancer cells.Methods:(1)Patients and histopathologic analysis: We retrospectively reviewed the liver tumor tissue specimens collected from 90 liver cancer patients and analyzed the correlation between the prognosis and the HNF-1? expression level.The 90 patients underwent surgical treatment.The patients were followed up for recurrence and survival.Immunohistochemistry was used to test the expression of HNF-1?in tumor.The level of HNF-1? expression was independently evaluated by two pathologists.The 90 patients were divided into HNF-1? high expression group and HNF-1? low expression group.The correlation between HNF-1? expression level and prognosis of patients was statistical analyzed.(2)HNF-1? was stably transfected into the HCC cell lines to establish the HNF-1? stably overexpressing cell lines.Real-time PCR ? Western-Blotting and Immunofluorescence were used to detect the expression of HNF-1?and HPC markers(CK7,CK19,SOX9 and CD133).Colony formation assay was used to observe the clonogenic ability.(3)Cell invasion ability of HNF-1?overexpression HCC was determined using the transwell assay.The effect of HNF-1? on epithelial-mesenchymal transition(EMT)of hepatocellular carcinoma cells wae detected by Real-time PCR?Western Blotting and Immunofluorescence.(4)To further elucidate the ability of HNF-1? in tumor formation in vivo,normal HCC cells or HNF-1? overexpression HCC cells were subcutaneously transplanted into nude mice.After four weeks,tumor size was measured in all nude mice.Immunohistochemistry were used to detect the expression of HPC markers(CK7,CK19,SOX9 and CD133).(5)Further to explore the mechanism of HNF-1? in enhancing the stemness of HCC.HNF-1? and Notch1 signaling pathway were detected by Real-time PCR and Western Blotting.Result:(1)High expression of HNF-1? promotes HCC cells de-differentiate into liver cancer stem cell,and then maintenances the stemness of liver cancer cells and regulates EMT,enhancing the invasion and metastasis of liver cancer.(2)HCC cells with HNF-1? overexpression strongly expressed liver progenitor cell markers.These results demonstrated that HNF-1? positive malignant cell may retain some progenitor-like characteristics,and the correlation between the malignant degree of liver cancer and HNF-1? is probably due to upregulation of liver progenitor cell markers and the stemness of tumor cells.(3)In transwell invasion assay,the invasion ability of HCCs is higher in the HNF1 b overexpression group than in the control group.(4)Normal HCC cells or HNF-1? overexpression HCC cells were subcutaneously transplanted into nude mice.After four weeks,tumors were detected in all nude mice.The tumor size of nude mice injected with HNF-1? overexpression HCC cells was significantly larger than that of the control group.In addition,IHC showed that the expression of HPC markers(CK7,CK19,SOX9 and CD133)was higher in the tumor formed by HNF-1? overexpression HCC cells than the control group.This results illustrate that HNF-1? overexpression enhanced the ability of HCC cells to form tumor in vivo.(5)HNF-1? upregulated expression of Notch signaling related genes(NOTCH1and HES1)in HCC.These results strongly supported that HNF-1? could enhance the stemness of hepatocellular carcinoma cells through Notch pathway.Conclusion: Our results indicate that HNF-1? contributes to the stemness maintenance of HCC through Notch pathway,and HNF-1? can promote the epithelial-mesenchymal transition,and thus enhance the ability of self-renewal and invasion and metastasis of hepatocarcinoma cells,worse the prognosis of HCC patients.