| ObjectHepatocellular carcinoma(HCC)is one of the five most common cancers,affecting about 1 million people worldwide each year.Despite tremendous advances in the diagnosis and treatment of liver cancer over the past few decades,the treatment effect remains disappointing.More and more evidence shows that cancer stem cells,also called tumor-initiating cells,can survive in chemotherapy and radiotherapy,and proliferate indefinitely and differentiate into tumor cells,which is directly related to the recurrence,metastasis and prognosis of patients.There is also a lot of evidences to suggest the existence of liver cancer stem cells,that are considered to be the cause of liver cancer initiation,metastasis,recurrence and chemotherapy resistanceJAK/STAT,Hedgehog,Wnt,Notch,and NF-?B signaling pathways are important for maintaining the growth and self-renewal of cancer stem cells,including liver cancer stem cells.Studies have reported that the abnormality of Wnt signaling pathway is associated with the development of liver cancer.40%-70%of patients with HCC can detect the nuclear accumulation of ?-catenin in early stage,which is one of the markers of Wnt/?-catenin signaling pathway activation.However,studies about Wnt signaling pathways mostly focus on the molecules in classical or non-canonical Wnt signaling pathways,such as Wnt3a,Wnt5a,etc.There are few studies on Wnt2b,and the signaling pathway type of which has not yet been identified.In the previous study,our team found that Wnt2b is highly expressed in fibrotic liver tissue and can inhibit the activation of hepatic stellate cells.The severity of liver fibrosis is related to the prevalence of liver cancer.Wnt2b plays an anti-fibrotic effect,so does it play a role in liver cancer?Therefore,in this study,we explored the effects of Wnt2b on liver cancer cells from in vitro and in vivo through molecular biology and cell biology experimental methods.Aims1.To observe the changes of Wnt2b expression levels in hepatocellular carcinoma cells and liver cancer tissues;2.To explore the eff'ect of Wnt2b on the biological characteristics of hepatocellular carcinoma cells and explore its related mechanisms.Methods1.The expressions of Wnt2b in human normal hepatocyte cell lines and hepatoma cell lines were detected by Quantitative real-time PCR(qPCR);2.In DEN/CCl4 induced mouse liver cancer,the expressions of Wnt2b and AFP were detected by qPCR;3.The effectiveness of Wnt2b-overexpression and-knockdown plasmid was verified by qPCR and Western blot;4.The expressions of inflammatory factors in Hepal-6 cells which transfected with Wnt2b-overexpression plasmid were detected by qPCR;5.The colony formation ability of Hepal-6 cells transfected with Wnt2b-overexpression or-knockdown plasmid was evaluated by clonal formation;6.The mRNA levels of liver cancer stem cell markers,such as CD44,EpCAM,CD47,ICAM-1 and CD 133,of Hepal-6 cells transfected with Wnt2b-overexpression or-knockdown plasmid were detected by qPCR,and the protein level of EpCAM was detected by Western blot;7.The sphere formation ability of Hepal-6 cells transfected with Wnt2b-overexpression or-knockdown plasmid was evaluated by sphere formation;8.The mRNA levels of stem cell pluripotency transcription factors,such as Oct4,Sox2,Klf4,c-myc and Nanog,of Hepal-6 cells transfected with Wnt2b overexpression or-knockdown plasmid were detected by qPCR,and the protein level of Sox2 was detected by Western blot;9.The mRNA levels of drug transporters MRP and MDP of Hepal-6 cells transfected with Wnt2b-knockdown plasmid were detected by qPCR;10.The chemoresistance of liver cancer cells transfected with Wnt2b-knockdown plasmid was detected by MTT;11.We constructed small hairpin or short hairpin RNA(shRNA)vector targeting Wnt2b genes,and inserted it to a lentiviral vector pLKO.1;12.The effectiveness of lentiviral vector knocking down Wnt2b was verified by qPCR;13.Male C57BL/6 WT were injected s.c.with either 1×106 or 1×107 Hepal-6 cells transfected with Wnt2b-knockdown vector and observe tumor growth.Results1.Wnt2b expresses highly in liver cancer cells and liver cancer tissuesCompared to normal liver cell HL-7702,the mRNA expressions of Wnt2b in liver cancer cells SMMC-7721,BEL-7402 and Huh7 were significantly increased.In the DEN/CCl4-induced mouse liver cancer model,the expression of Wnt2b in liver cancer tissues was consistent with the expression of the liver cancer marker alpha fetoprotein(AFP).2.Plasmids that overexpress and silence Wnt2b are effectiveWnt2b-overexpression plasmids were transfected into Hepal-6 cells in vitro,and the mRNA expression and protein expression of Wnt2b in cells were increased;Wnt2b-knockdown plasmids were transfected into Hepal-6 cells in vitro,and the mRNA expression and protein expression of Wnt2b in cells were decreased.3.Wnt2b promotes the stemness of liver cancer cellsIn liver cancer cells,by overexpressing Wnt2b,the gene levels of the stem marker molecules,such as CD44,EpCAM,and CD 133,and the protein level of EpCAM were significantly increased;colony formation and sphere formation ability were stronger than that of the control group;the gene levels of the pluripotent transcription factors,including Sox2,Klf4,c-myc,and Nanog,and the protein level of Sox2 were significantly increased.The opposite experiment results were obtained after knocking down Wnt2b.The results of these indicate that Wnt2b can promote the sternness of liver cancer cells.4.Construction and validation of lentiviral vector silencing Wnt2bWe successfully constructed a lentiviral vector that silenced Wnt2b;Meanwhile,we confirmed the silencing efficacy of Wnt2b silenced vetor in the gene level successfully.5.Wnt2b knockdown inhibits tumor initiation ability of hepatoma cellsMale C57BL/6 WT were injected s.c.with 1×106or 1×107Hepal-6 cells transfected with Wnt2b-knockdown vector.Tumor growth was measured every 2 to 3 days after tumor inoculation using digital calipersand.The group of Wnt2b-knockdown had lower tumor initiating ability and smaller tumor volume than the control group.It is indicated that silencing Wnt2b inhibits the dryness of liver cancer cells.Conclusions1.This study found that Wnt2b is highly expressed in human hepatoma cell lines,and liver cancer patients with high expression of Wnt2b have a lower survival rate;Wnt2b is also highly expressed in mouse liver cancer tissues,and its expression level is consistent with the expression of liver cancer marker AFP.2.This study demonstrates that Wnt2b can promote the expression of sternness markers in hepatocellular carcinoma cells,and also promote sternness-related biological characteristics of liver cancer cells,such as stronger colony formation and sphere formation ability and tumor initiation ability,increased expression of pluripotent transcription factors and tolerance to chemotherapeutic drugs,all of which demonstrate that Wnt2b can promote the sternness of liver cancer cells.3.This study lays the foundation for further exploring the mechanism of liver cancer development and establishing new targets and strategies for the effective treatment of liver cancer.In summary,this study demonstrates that Wnt2b can promote the sternness of liver cancer cells,and Wnt2b may be a new target for the treatment of liver cancer.Targeting Wnt2b in hepatocarcinoma cells can significantly inhibit the sternness of liver cancer stem cells,and effectively decrease the drug resistance,recurrence and metastasis.It provides a new direction and experimental basis for the treatment of liver cancer by targeting liver cancer stem cells. |
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