Role Of Bmi1 On Stemness Features And Biliary Phenotype Transition Of Hepatocellular Carcinoma Cells

Backgroud and aims:Hepatocellular carcinoma(HCC) has a very poor prognosis over the world cause of the much complex heterogeneity of liver cancer. Recent studies have shown that, heterogeneity of liver cancer is mainly caused by the stemness characteristics of tumor, the different extents of stemness expression in tumor determine different biological features, including tumor cell proliferation, invasion and metastasis ability, chemoresistance and individual response to clinical treatment. Therefore, clarify the different stemness characteristics of tumor can provide a theoretical basis for tumor heterogeneity, and provide guidance and strategies for individual treatment and prevention of recurrence.Our recent study showed that HPC activation was associated with recurrence and prognosis in combined hepatocellular-cholangiocarcinoma in the chronic background of liver. Proliferative index of ductular reaction(PIDR) was significantly associated with HPC activation. Moreover, HPC activation could also influence the expression of some stemness markers in tumor. Besides, our results showed that stemness marker Bmi1 was positively associated with PIDR, suggesting the role of Bmi1 on HPC activation and carcinogenesis and development of liver cancer in some extent.Bmi1 is widely involved in cell proliferation and differentiation, regulating cell senescence, changing biological features and is closely related to the occurrence and development of many kinds of tumors. Recently, some experts found that Bmi1 could induce EMT and promote cancer metastasis, then affecting the patient’s prognosis. Thus it has been a hot point in high metastatic and poor clinical prognostic liver cancer in recent years.Our recent results also showed that the activation status of HPC was positively associated with CK7 and CK19 expression in tumor. It suggested that HPC could activate the expression of progenitor/biliary epithelium markers in tumor and result to biliary phenotype of liver cancer cells. Meanwhile, researchers confirmed that overexpression of HNF-1β led to biliary phenotype and upregulated expression of CK7 and CK19 in HCC. These results may be used to explain the clinical characteristic of postoperative tumor subtype transition.Methods and results:Part 1: Effect and mechanism of Bmi1 on the biological features and metastasis of hepatocellular carcinoma. Bmi1 overexpression cells of Huh7 and L02 were constructed respectively using lentivirus. Bmi1 knock down SMMC-7721 cell and its negative control were also constructed. Cell proliferation rate was determined by CCK-8 assay, foci formation assay was used to check the colony forming ability, 5- Fluorouracil was used to test the drug resistance ability. Then we carried out would healing and transwell assays to evaluate the metastatic capacities of HCC cells. We also examined the EMT markers expression in mRNA and protein levels.Results: 1. Bmi1 could enhance the proliferation, chemoresistance and colony formation abilities of HCC cells, then changing the biological characteristics of HCC cells. 2. Overexpression of Bmi1 could enhance the metastatic ability of HCC cells and induce EMT program. Loss of function assays showed the reverse results.Part 2: Effect of expression of stemness markers on the stemness and biliary phenotype in hepatocellular carcinoma cell line Huh7. Bmi1 or EpCAM overexpression cells and Bmi1, EpCAM co-overexpression cell were constructed using lentivirus. CCK-8 assay, foci formation assay, transwell assay and xenograft tumor growth assay were used to test the stemness characteristics. Western-Blot experiment was performed to analyze the expression of EMT markers, E-Cadherin and Vimentin. We also examined the expression of hepatic marker HNF4 a and progenitor/ biliary epithelium marker CK19 in protein level.Results: 1. Stemness markers Bmi1 and EpCAM could enhance the proliferation and colony formation abilities of Huh7 cells, while co-expression of Bmi1 and EpCAM enhanced the most extent. 2. Bmi1 overexpression and Bmi1-EpCAM co-expression Huh7 cells underwent EMT process and had higher invasion ability than EpCAM overexpression cell. 3. Overexpression of stemness markers Bmi1 and EpCAM significantly led to the downregulation of hepatic marker HNF4 a and upregulation of progenitor/biliary epithelium marker CK19.Conclusion:Bmi1 could influence the biological characteristics and enhance the metastatic capacity via inducing EMT in HCC cells. Expression of different stemness molecules led to different stemness features of HCC cells. The more expression of stemness markers comes the stronger stemness characteristics. Expression of stemness markers could result to biliary phonetype of HCC cells, and this may be associated with the clinical characteristic of postoperative tumor subtype transition.