Inhibitory Effect Of C/EBPβ Isoform LAP1 On Epithelial-mesenchymal Transition In Hepatocellular Carcinoma Cells And Its Character

Background and Objective:Epithelial-mesenchymal transformation(EMT)is a process in which epithelial cells lose their polarity and then convert to mesenchymal phenotypic cells in certain situations.The characteristic of this process is that the activity of cell motion and the ability of metastasis and invasion were increased.EMT-induced changes in epithelial plasticity are evidenced by the loss of epithelial markers,such as E-cadherin.In addition,the expression of the mesenchymal proteins such as N-cadherin and Vimentin is increased.Snail(SNA1)and Slug(SNA2)are believed to be the major nuclear transcription factors in the Snail family that regulate the occurrence of EMT in tumor cells,directly involved in the production of mesenchymal markers,such as Vimentin and N-cadherin,and inhibit the production of the epithelial cell markers,such as E-cadherin.TGF-β-Smads signaling pathway is considered to be a classical signal pathway regulating Snail production.TGF-β plays a facilitating role in the advanced tumor and is also proven to be the cause of EMT in tumor cells.With the development of the study,EMT has been found in a variety of tumor cells,including breast cancer,lung cancer and gliomas.EMT is also found in the pathological process of HCC in recent years,which is closely related to the metastasis and invasion of HCC.The study of EMT related molecular mechanism is of great significance in improving the prognosis of liver cancer patients and exploring new target therapy.CCAAT/enhancer-binding proteins(C/EBPs)comprise a class of leucine zipper transcription factors involved in the regulation of multiple tissue functions.C/EBPβ,one member of this family,produce three different protein isoforms by selective shearing.The full-length activated protein LAP1 is a transcription factor mainly expressed in the liver,which is involved in the inflammatory response,proliferation,survival,tumorigenesis and differentiation of hepatocytes.It was pointed out that the expression of LAP1 in HCC tissues was significantly lower than that in corresponding paracancerous tissues.In our previous work,we found that LAP1 can effectively inhibit the stem characteristics of hepatoma cells,including the growth rate and proliferative capacity of cancer cells,and we also found that interfering with the expression of LAP1 can affect the phenotypic changes and motiliny of hepatoma cells.It is suggested that LAP1 may be related to EMT of hepatoma cells.On the basis of human hepatoma cell lines Hep3 B and SMMC-7721,we used cell biology,biochemistry and molecular biology to investigate whether the LAP1 could inhibit the phenotype of EMT and explore the possible molecular mechanism.Methods:1.Immunohistochemical and Real-Time PCR method to detect the expression of C/EBPβ in hepatocellular carcinoma and adjacent tissues of patients with hepatocellular carcinoma was determined.At the same time,the expression of C/EBPβ and the correlation between C/EBPβ and metastasis of HCC were analyzed by comparing the expression of C/EBPβ in patients with high metastatic hepatocellular cancer with those of patients with low metastasis.2.Hep3 B and SMMC-7721 cells expressing LAP1 were established,and LAP1 overexpression was detected by Western blot assay and RT-PCR.The changes of epithelioid phenotypes of Hep3 B and SMMC-7721 cells after overexpression of LAP1 were observed under optical microscope.Immunofluorescence,Western blot and Real-Time PCR method was used to further determine whether overexpression of LAP1 could promote the expression of E-cadherin,and inhibit the expression of mesenchymal markers N-cadherin and Vimentin at the same time.3.Knockout C/EBPβ of Hep3 B and SMMC-7721 cells were established,and C/EBPβ was detected by Western blotting and RT-PCR.The changes of mesenchymal phenotype after Hep3 B and SMMC-7721 knockout C/EBPβ were observed.The expression of EMT Marker was detected by Western blotting,RT-PCR and Immunofluorescence assay.4.Transwell and Transwell containing matrigel were used to detect the metastasis and invasion ability of Hep3 B and SMMC-7721 cells which expressed LAP1 and knocked down C/EBPβ.5.The effects of C/EBPβ on the expression of EMT related transcription factors Snail and Slug were detected by Western blotting and RT-PCR experiments.The expression and phosphorylation of major factor Smad3 in TGF-β / Smads signaling pathway were detected by Western blotting assay.6.The lung metastasis model of hepatocarcinoma was established by caudal vein injection in 6-week-old male nude mice.The metastasis of SMMC-7721 cells after overexpression of lap1 and knocked down C/EBPβ was compared with that of control group.Results:1.Down-regulation of C/EBPβ expression in high metastatic hepatocellular carcinoma.2.Overexpression of C/EBPβ isoform LAP1 inhibits EMT in hepatocellular carcinoma cells.3.Knockdown of C/EBPβ promotes EMT in hepatocellular carcinoma cells.4.LAP1 inhibits invasion and migration of hepatocellular carcinoma cells.5.C/EBPβ inhibits the TGF-β / Smads pathway in hepatocellular carcinoma.6.C/EBPβ inhibits lung metastasis in hepatocellular carcinoma.Conclusions:The decrease of LAP1 expression of C/EBPβ isoform can promote the EMT process of hepatoma cells,which leads to the enhancement of metastasis and invasion ability of tumor cells.This is because LAP1 can inhibit the expression and phosphorylation of key transcription factor Smad3 in TGF-β / Smads cell signaling pathway,and then inhibit the expression of nuclear transcription factors Snail and Slug downstream.The down-regulation of LAP1 leads to abnormal activity of TGF-β-Snail axis,which leads to the enhancement of EMT in hepatoma cells.