Hepatocyte-specific MANF Deficiency Accelerates The Progression Of Hepatocellular Carcinoma By Promoting Epithelial Mesenchymal Transformation In Mice

Objective: As an endoplasmic reticulum stress protein,Mesencephalic Astrocyte-derived Neurotrophic Factor(MANF),is expressed in almost all tissues of the body,especially in the brain and liver.MANF has been shown to protect and repair neurons in neurological diseases.And our group found that the m RNA expression of MANF in HCC tissues was lower than that in adjacent para-cancerous,and subsequently protein level detection is also like this.In the cases followed up of HCC patients,it was found that HCC patients with high MANF expression had a longer postoperative survival time and a lower probability of HCC recurrence than those with low MANF expression.This suggests that MANF plays a role in inhibiting HCC.In this study,we tested the effect of MANF on HCC in hepatocyte-specific MANF deficiency mice.Methods: Hepatocellular carcinoma(HCC)model was established by intraperitoneal injection of DEN(30 mg/kg)on the 15 th day of birth in wild-type(WT)mice and hepatocyte-specific MANF deficiency(KO)mice.WT mice were sacrificed at 8 months after DEN treatment,and the expression of MANF in HCC tissue and adjacent noncancer tissues were detected by immunohistochemical(IHC).The tumor formation was compared between WT and hepatocyte-specific MANF deficiency mice in DEN-induced HCC by HE staining.IHC detected the pulmonary metastasis in DEN-induced HCC mice.IHC,western blot(WB)and real-time quantitative PCR detecting system(QPCR)experiments were used to detected the m RNA and protein level of EMT(epithelial-mesenchymal transition)indexes,E-cadherin,Vimentin and β-catenin in vivo.In order to determine the interaction between MANF and p65,we conducted Co-IP assay in human liver cancer samples and hepatoma cell line Hep G2 cells to determine whether there is an interaction between MANF and p65 in HCC.Then we tested the expression and phosphorylation level of p65 in mice HCC model.Additionally,IHC,WB or QPCR experiments were used to detected expression of NF-κB target genes and Snail1+2 in mice HCC model.Results: 1.The expression of MANF was detected in the liver tissue of DEN-induced mice HCC model Immunohistochemical data indicated that MANF expression was lower in liver cancer tissues than that in adjacent noncancer tissues.2.The effect of hepatocyte-derived MANF deficiency on HCC Anatomical and histological data showed that hepatocyte-specific MANF deficiency promoted the growth of the liver tumor nodule and the cell atypia in adjacent noncancer tissues.3.The effect of hepatocyte-derived MANF deficiency on EMT in DEN-induced HCC mice model Hepatocyte-specific MANF deficiency decreased E-cadherin level and increased β-catenin,Vimentin levels,suggesting that MANF suppressed EMT in DEN-induced HCC mice model.Meanwhile,more serious pulmonary metastasis was found in the hepatocyte specifical MANF deficiency mice 20 months after DEN treatment,compared with the WT controls.4.The interaction between MANF and p65 in HCC We performed Co-IP assay to verify the interaction pf MANF and p65 in the cancer tissues of HCC patient.And the major interaction was localized in the nuclei of hep G2 cells.5.The effect of hepatocyte-specific MANF deficiency on NF-κB pathway The expressions of IL-1α and TNF-α were up-regulated in m RNA and protein levels in the liver tissues of hepatocyte specifical MANF deficiency mice,compared with the WT mice.6.The effect of hepatocyte-specific MANF deficiency on the expresion of Snail We found Snail1 m RNA level in the liver tissues of hepatocyte specifical MANF deficiency mice was increased,compared with the WT mice.However,there is no statistical difference in Snail2 m RNA levels.Additionally,both Snail1 and Snail2 protein levels in the liver tissues of hepatocyte specifical MANF deficiency mice were much higher than those in WT mice.Conclusion: Hepatocyte-specific MANF deficiency accelerates the progression of hepatocellular carcinoma by promoting epithelial mesenchymal transformation in mice.