| Primary liver cancer is a common malignant tumor,with high incidence in the worldwide.And the mortality caused by liver cancer ranks second in China.According to histological type,hepatocellular carcinoma(HCC)is the most common type,accounting for 85%-90%of the total.At present,surgical resection is still the main treatment method of hepatocellular carcinoma.Although continuous progress has been made in the diagnosis and treatment of liver cancer,5-year survival rate of the patients has not yet been greatly improved.Because of easy recurrence and metastasis as well as the effect of radiotherapy or chemotherapy is not ideal,the prognosis of patients with liver cancer is not optimistic.This is mainly due to the presence of tumor cells,especially cancer stem cells,which play an important role in the recurrence and heterogeneity of liver cancer.The cancer stem cell theory suggests that cancer stem cells are present in a small part of cell population,with stem cell characteristics as well as self-renewal and differentiation potential and can be formed into tumor cell with the heterogeneity.Bonnet D reported the existence of cancer stem cells in acute myeloid leukemia for the first time,so far,tumor stem cells have been isolated and identified in at least 30 kinds of tumors,such as melanoma,colorectal cancer,breast cancer,prostate cancer,lung cancer,glioma,pancreatic cancer and so on.Studies have confirmed that liver cancer stem cells also exist in HCC(liver cancer stem cells,LCSCs),and CD133,CD13,CD90,EpCAM,OV6and CD24 can be used as surface markers,and liver cancer stem cells are responsible for tumor metastasis,recurrence and drug resistance.So it has important scientific significance and clinical application potential to look for new and effective target of liver cancer stem cells for the development of drug or treatment strategies for liver cancer stem cells,and therefore tumor metastasis,recurrence and drug resistance which lead to high mortality of patients can be effectively treated.The transcription factor C/EBPβ(CCAAT enhancer binding protein beta)is one of 6members in the CEBPs family that were successfully cloned.In its C terminal,there is a highly conserved bZIP domain containing the DNA binding domain and a dimerization domain(leucine zipper),which can interact with the promoter and enhancer region of the target genes so as to regulate their expression.The C/EBPβgene has 3 transcription start sites,and thus can translate into3 kinds of proteins,which are LAP1(38kDa),LAP2(35kDa)and LIP(20kDa).While LAP1 and LAP2 have both transcription activation domain and DNA binding domain,LIP has only the latter.And LIP plays antagonistic effect of LAP1 and LAP2.Among them,LAP1 is the most highly expressing isoform in liver.C/EBPβ,which is a highly conserved protein existing in human and rodents,widely expressed in tissues of the whole body.C/EBPβinvolved in various life activities such as cell proliferation,differentiation,apoptosis,immune and stress response,energy metabolism,production of blood and so on.And it also involved in the occurrence and progress of tumors.In recent years,studies show that the dysregulation of C/EBPβis closely related to tumor progression,such as gliomas,Wilm,s tumor,prostate cancer,renal cell carcinoma and ovarian cancer and so on.LAP1 is an important transcription factor with a wide range of functions such as proliferation,apoptosis,invasion and EMT,and thus affects the occurrence and development of tumors.In this study,we used methods of bioinformatics and combined with clinical tissue samples,and we found that LAP1 is weakly expressed in HCC tissues,but the role of LAP1 in the occurrence and development of liver cancer and liver cancer stem cell is unclear.In this study,we studied the biological effects of LAP1 in hepatocellular carcinoma and liver cancer stem cells both in vitro and in vivo,and according to the characteristics of LAP1 related signal transduction,a small molecule drug MDV3100 was selected for preliminary clinical transformation studies.Firstly we used methods of bioinformatics and the detection of clinical samples of liver cancer tissue to compare the expression of LAP1 in hepatocellular carcinoma tissues and normal adjacent tissues,and the results showed that LAP1 was lower in hepatocellular carcinoma tissues;Furthermore,the expression of LAP1 in CD13~+liver cancer stem cells and non-stem cells was compared by flow cytometry as well as oncosphere formation experiments,and the results showed that the expression of LAP1in cancer stem cells was significantly lower than that in non-stem cells,suggesting that LAP1 may negatively regulate the malignant biological behavior of hepatocellular carcinoma.In order to further explore the biological function of LAP1,we further overexpressed LAP1 in hepatoma cells,and with in vitro and in vivo experimental models,we systematic analysed the effects of LAP1 on the malignant biological behavior of liver cancer stem cells and hepatocellular carcinoma population cells.Firstly,the hepatoma cell lines Huh7 and CLC13,which stably overexpressed the LAP1 and GFP control plasmids,were established using a lentivirus infection method.Then sphere forming assay was performed to analyze the effect of LAP1 overexpressing on sphere forming rate,and the effect of LAP1 overexpressing on the expression of stem markers was analyzed by flow cytometry and qPCR.The results suggest that overexpression of LAP1 can inhibit sphere forming ability,down regulate the proportion of hepatoma stem cells as well as the expression level of liver cancer stem cell markers.At the same time,CCK8 proliferation assay,colony formation assay,scratch test,Transwell migration and invasion assay were used to analyze the effect of LAP1 overexpressing on hepatoma cells,cell cycle and apoptosis assays were also performed.And the results suggest that overexpression of LAP1 can significantly inhibit the proliferation,migration and invasion ability of hepatoma cells,and induce cell cycle arrest.Finally,tumorigenesis in nude mice was performed to observe and analyze the effects of overexpression of LAP1on tumorigenicity and tumor growth.The results suggest that LAP1 overexpression reduces the tumorigenicity of hepatoma cells in nude mice:the number,volume and weight of subcutaneous tumors are lower than those in the control group.At the same time,LAP1 overexpression reduced the expression of liver cancer stem markers CD13,CD133 and EpCAM,and significantly reduced the expression of proliferation marker Ki67.Cellular function studies shown that LAP1 exhibited a significant inhibitory effect on tumor biological function and participated in the stem-ness regulation of liver cancer stem cells.Many results suggest that LAP1 may serve as an effective target for the treatment of liver cancer.But there is great risk in gene therapy,so there are still many challenges in clinical application.Therefore,if small molecule drugs that can upregulate LAP1 are screened,they may have greater potential for clinical transformation and application.Studies have shown that androgen receptors have a negative regulatory effect on LAP1,and the expression of androgen receptors is correlated with the prognosis of liver cancer.This suggests that upregulation of LAP1 expression by inhibiting androgen receptor has potential application value in the treatment of liver cancer.The androgen receptor antagonist MDV3100 is a highly selective antagonist of the androgen receptor and is widely used in endocrine therapy of prostate cancer.In this study,the androgen receptor antagonist MDV3100 has been further used for the research of liver cancer treatment,which can promote the transformation and application of LAP1 as a therapeutic target of liver cancer.In Huh7 cells,different concentrations of MDV3100 were added to examine the effect on cell viability.The results showed that MDV3100 can reduce the cell viability of Huh7 cells.qPCR and WB assays were performed on cells treated with different concentrations of MDV3100,and it was found that MDV3100 increased the expression of LAP1 and the expression of P53,P21 and P16 were also increased.After treatment with MDV3100,as the volume of cell and the ratio of positive staining of SA-β-Gal were became larger,it can be concluded that MDV3100 may cause liver cancer cells to be aged.In addition,cell cycle assay was performed on Huh7 cells treated with different concentrations of MDV3100,and MDV3100 was found to inhibit the cell cycle of them.In summary,MDV3100 can upregulate the expression of LAP1,then induce liver cancer cell to be senescent,and as a result,inhibit the growth of tumors.Therefore,LAP1 may serve as a new target for liver cancer treatment.And we hoped that this study will provide new ideas and feasible methods for the diagnosis and treatment of liver cancer.Conclusion:In this study,we found that C/EBPβwas weekly expressed in liver and liver cancer stem cells.The most important and most abundant subtype of C/EBPβin liver,LAP1,inhibits the stemness of liver cancer stem cells and inhibits the progression of liver cancer.The mechanism remains to be further studied.Through upregulation of LAP1,the androgen receptor antagonist MDV3100 can induce senescence of hepatocellular carcinoma cells,and as a result,inhibit the development of liver tumors.These findings have expanded the study of LAP1 function and also provided strong evidence for LAP1 as a new therapeutic target of liver cancer. |
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