RAR? Promotes Hepatocellular Carcinoma Invasion And Metastasis Through Regulating NF-?B/E-cadherin Signaling Axis

Background and Objective Hepatocellular carcinoma(HCC)is one of the most common cancers in the world,particularly in China.Many studies have showed that lots of risk factors such as hepatitis B(HBV)and hepatitis C(HCV),dysregulation of signaling pathway and molecules,are correlated with HCC development.Although current advances have been made in the pathogenesis of HCC,the overall survival rate of the HCC patients is very low because of recurrence and metastasis.For example,HCCs that undergo early vascular metastasis have poor efficacy of existing chemotherapy.In general,little is known about how HCC undergoes metastasis.Therefore,how to identify the molecular mechanisms underlying HCC metastasis is particularly important.Tumor metastasis is a complex biological process,which involves tumor cells detaching from the original site,migrating or vascular invasion,locating in distant organs and continuing to grow.In recent years,accumulating evidence proves that epithelial-mesenchymal transition(EMT)plays a significant role for cancer cell invasion and metastasis initiation.Downregulation of E-cadherin is a essential feature of EMT.Loss of E-cadherin correlates with poorer survival for patients with many cancers such as gastric cancer,breast cancer and HCC.Absent or reduced expression of E-cadherin in human tumors can be caused by somatic mutations,chromosomal deletions,DNA methylation and also can be regulated by EMT related transcription factors such as Twist,Snail,Slug,Zeb1,Zeb2 and others combining with E-cadherin promoter.We recently reported that Nur77,a member of the orphan nuclear receptor superfamily,induces E-cadherin reduction in a Matrix metalloproteinase 9(MMP-9)-dependent manner,and subsequently promotes invasion and metastasis of colorectal cancer.Although understanding the molecular mechanism of E-cadherin loss or reduction in cancer is continuously increasing,regulating machanism of E-cadherin expression in cancer still remains to be studies.Retinoic acid receptor ?(RAR?)is a member of the nuclear receptor subfamily.There are conflicting views regarding RAR? role in cancer.On one hand,RAR? play a role as an oncogene to drive tumor cell growth and metastasis.For example,RAR? upregulation in cholangiocarcinoma promotes cancer cell growth and metastasis.RAR? is overexpressed in HCC and leads to the occurrence of HCC.In contrast,RAR? has also been reported as a tumor suppressor that inhibits cancer cells proliferation and invasion.For example,RAR?,by regulating the expression of carbohydrate sulfotransferase 10(CHST10),can inhibit melanoma invasion.RAR? is downregulated in clinical colorectal cancer tissues,inhibits colorectal occurrence and metastasis by adjusting the Hippo-Yap pathway-mediated tumorigenic signaling.Taken together,these results strongly indicated that RAR? plays an important role in tumor development.RAR?,however,whether and how to play a role in HCC invasion and metastasis remains to be further discussed,and whether there is a certain relationship between RAR? and E cadherin still needs further research.In this study,we aimed to study the role of RAR? in the invasion and metastasis of HCC,clarify that RAR?,acting through NF-?B-mediated E-cadherin reduction,drives the molecular mechanism of HCC cell invasion and metastasis.In order to clear the role of RAR?/NF-?B/E-cadherin signal transduction mechanisms in hepatocellular carcinoma and its clinical significance.Methods 1.Using lentiviral sh RNA and plasmid carrier to construct hepatocellular carcinoma cell lines that knockdown RAR? and overexpress RAR? respectively.2.Western blot was performed to examine the expression of RAR? in the tissues and cells of hepatocellular carcinoma.3.The correlation between the expression of RAR? and clinical pathological parameters of hepatocellular carcinoma,RAR? and patient's prognosis were analyzed by immunohistochemistry.4.Hepatocellular carcinoma cell migration and invasion test in vitro were performed,we analyse its influence on cell migration and invasion ability by changing RAR? expression in HCC lines.5.We examined the effect of RAR? on hepatocellular carcinoma metastasis ability by establishing nude mouse model through injecting hepatocellular carcinoma cells into mouse tail vein to induce pulmonary metastasis of tumor cells.6.The correlation between the expression of RAR? and E-cadherin was researched by western blot,immunofluorescent staining and q PCR.7.Analyse the role of NF-?B signal in the process of RAR? regulating E-cadherin through the method of drug intervention.8.The expression of RAR? and E-cadherin in hepatocellular carcinoma cell lines and clinical hepatocellular carcinoma samples was studied by western blot,RT-PCR and immunohistochemistry,then the correlation between RAR? and E-cadherin was analysed.Result Section 1 To detect the expression of RAR in clinical HCC tissues We examed the expression of RAR? in human HCC tissues and their matched surrounding tissues by western blot and found that the expression of RAR? protein was higher in primary HCC tissues than matched surrounding tissues.Furthermore,we analysed gene expression data from Oncomine,and found that RAR? m RNA levels are obviously increased in hepatocellular carcinoma tissues compared with liver cancer precursor tissues.We further proved RAR? expression is overexpressed in 56 cases of human hepatocellular carcinoma tissues by immunohistochemical methods,and its expression significantly associated with distant metastasis.Kaplan-Meier analysis revealed that high RAR? expression closely related to the prognosis of patients with hepatocellular carcinoma.Section 2 Knockdown of RAR? expression affects the biological characteristics of HCC cells We used a lentiviral-mediated sh RNA technique to stably knock down RAR? expression in MHCC-97 H cell lines,Migration and invasion assays showed that knockdown of RAR? largely inhibited the ability of HCC cell migration and invasion.We further found that deletion of RAR? greatly restrained HCC metastasis ability in nude mouse model through injecting hepatocellular carcinoma cells into mouse tail vein to induce pulmonary metastasis of tumor cells.Section 3 RAR? regulates E-cadherin expression Western blot and immunofluorescent staining confirmed knockdown of RAR? in MHCC-97 H cells markedly induced the expression of E-cadherin.Conversely,overexpression of RAR? in Huh-7 cells significantly decreased E-cadherin expression.Real-time PCR assays confirmed that changing RAR? expression in HCC cells could significantly affect E-cadherin m RNA expression,and this regulation preseted a dose-dependent manner.At the same time,we found that ATRA,an agonist for RARs,could significantly enhance RAR?-induced E-cadherin reduction.Section 4 RAR?-induced E-cadherin reduction is dependent on NF-?B Activation Further experiments found that BMS-345541,an inhibitor of NF-?B signaling pathway,completely blocked RAR?-induced E-cadherin reduction both at m RNA levels and at protein levels.However,tumor necrosis factor ?(TNF?),an agonist against the NF-?B signaling pathway,significantly enhanced RAR?-induced E-cadherin reduction.Section 5 RAR? expression is negatively correlated with E-cadherin expression in HCC cell lines and clinical HCC samples The expression of RAR? and E-cadherin in HCC cell lines and clinical HCC samples was analyzed.we noticed a significant correlation in the expression of RAR? and E-cadherin both at the m RNA and protein levels in six HCC cell lines.The correlation of RAR? and E-cadherin was further approved by examining the expression of these two molecules in 56 cases of HCC tissues using immunohistochemical staining.Our results showed that low RAR? expression was associated with high E-cadherin expression.Inversely,the high levels of RAR? correlated with the low levels of E-cadherin.Spearman's Rank Correlation analysis confirmed that there was a significant negative correlation between RAR? and E-cadherin expression.