| ObjectHepatocellular carcinoma(HCC)is one of the most malignant tumors with the highest fatality rate,and is also a serious disease which threaten human health,and its incidence rate has been high.In our country,the mortality rate of HCC is second,and the incidence rate is fourth.For the diagnosis of Hepatocellular carcinoma,the detection of tumor markers and imaging examination.There are many ways to treat HCC,such as surgical resection.However,despite the effective treatment,HCC still has the possibility of recurrence and metastasis.Therefore,it is necessary to clarify the molecular mechanism of the occurrence of liver cancer,and to reveal the different molecular and abnormal signal pathway in the occurrence of liver cancer providing a new direction for clinical treatment.In recent years,some scholars have found that there is an abnormal activation of the signal pathway in the development and progression of HCC.Here,we pay a attention to Wnt classical signaling pathway dring the process of hepatocarcinogenesis.Wnt signaling pathway is highly conserved and it could regulate the development in the embryo of the organism,tissue repair and regeneration,regulation of organism and cell growth,apoptosis,self-renewal and survival of gene expression.Wnt is generally divided into canonical and noncanonical signal transduction pathway.At present,the relationship between Wnt signaling pathway and liver fibrosis has been described.Studies have shown that abnormal activation of Wnt signaling pathway could activate hepatic stellate cells and lead to hepatic fibrosis.However,there are few studies on Wnt signaling pathway and liver cancer.In the previous work,we found that the Wnt2b expression in liver was higher than that in the normal liver tissue,and there was a certain correlation with the severity of the disease.As we know,TLR4 promoted HCC and TLR2 inhibited the occurrence of HCC under the premise,we found that TLR4 agonist induced the Wnt2b in human hepatocellular carcinomacell lines(HepG2,H7702)and murine hepatocellular carcinoma cell line(Hepal-6),while TLR2 agonist inhibited Wnt2b expression.These results suggested that Wnt2b may play an important role in the development of HCC.Based on the above phenomena,this study investigated the effects of Wnt2b on the biological characteristics of hepatocellular carcinoma cells in vitro.Also,it was found that Wnt2b could promote the growth of tumor in mice,and the number and proportion of NK cells in tumor tissue were descreased.In this study,we discussed the feasibility of the interference of Wnt2b expression in the treatment of liver cancer,providing a new direction and reference for the clinical treatment of liver cancer.Methods1.In several human and mouse hepatoma cell line as a model,using MTT method,cell clonogenic assay for detection of Pam3CSK4 and LPS on the proliferation of hepatoma cells.2.Immunofluorescence,immunohistochemistry and Western blot were used to detect?-catenin in HCC cell lines and tissues.3.Wnts mRNA was detected in TLR2-/-,TLR4-/-,TLR9-/-mice by semi quantitative RT-PCR method.4.Semi-quantitative RT-PCR method was used to detect Wnts mRNA level in t human hepatocellular carcinoma cell line HepG2.5.MTT method to performed to study the effect of Wnt2b on proliferation of HCC.The effects of Wnt2b on cell viability of HCC were detected by cell plate cloning assay and quantitative PCR assay.The effects of Wnt2b on cell cycle and apoptosis of HCC were detected by PI staining and Annexin V/PI double staining,respectively.6.Western blot,quantitative PCR,MTT method were used to detect the effect of Wnt2b on hepatoma cells by activating Wnt signaling pathway.7.Animal experiments:Using subcutaneous tumor tissue tumor,plasmid transfection and intratumoral injection method to detect the impact of Wnt2b on the tumor growth,and the flow cytometry was used to detect liver lymphocytes and tumor tissue.Results1.TLR2 inhibited the proliferation of HCC,while TLR4 promoted the proliferation of HCC cell lines.TLR2 inhibited the sternness of liver cancer cells,TLR4 promoted the sternness of liver cancer cells.2.TLR4 promoted the expression of(3-catenin in HCC and promoted the entry into the nucleus.The expression level of ?-catenin in liver in TLR4-/-was higher than that in WT mice,which was incorporated into the nucleus.3.The expression profiles of Wnts in liver tissue of mice were in TLR2-/?,TLR4-/-,TLR9-/-mice.TLR2 agonists stimulate human hepatocellular carcinoma cell line HepG2,the expression levels of Wnt2 and Wnt2b were down-regulated.Under the stimulation of TLR4 agonist LPS,the expression of Wnt5a was down-regulated,and the expression of Wnt2b was up-regulated.4.TLR2 inhibited the expression of Wnt2b in hepatocellular carcinoma cell line,while TLR4 promoted the expression of Wnt2b in hepatocellular carcinoma cell line.5.Wnt2b promoted proliferation and inhibited the apoptosis of hepatocellular carcinoma cell lines,while cell cycle was not affected by Wnt2b.The expression levels of anti-apoptotic gene Bcl-2 and Mcl-1 were promoted by Wnt2b.6.The promotion effect of Wnt2b on liver cancer is achieved by activating Wnt signaling pathway.Wnt2b could not promote liver cancer when ?-catenin is silencing.7.Wnt2b promoted tumor growth by inhibiting the number of NK cells in tumor tissue.Conclusion1.TLR4 signaling pathway promoted the activation of ?-catenin.2.Wnt2b could promote the proliferation of HCC.3.Wnt2b promoted tumor growth by inhibiting NK cells in vivo,providing a new direction for future HCC therapy. |
PDF Full Text Request