| Objective: Hepatocellular carcinoma(HCC)is one of the most common malignant tumors.Usually,the patients with HCC are asymptomatic and diagnosed at late stages when surgical treatment is no longer suitable.Hepatocarcinogenesis is a multi-factorial,multistep,and multicenter complex process.Its early diagnosis and effective treatments are of the most importance.Although lots of biomarkers have been reported associated with HCC diagnosis like routine alpha-fetoprotein(AFP),but most of markers can't meet the clinical need with its unsatisfactory sensitivity and specificity.Recently,a variety of signaling pathways have been reported to associated with the hepatocarcinogenesis,especially the aberrant activation of Wnt pathway during hepatocyte malignant transformation.Researches have been reported that many key molecules such as secreted frizzled related protein 1~5,Wnt1~3,Wnt3 a and ?-catenin are closely related with HCC,and Wnt3 a has been confirmed highly-expressed in breast cancer,colorectal cancer and so on.However,Wnt3 a as one of the key molecules in the Wnt pathway during HCC development,its value for targeted treatment remains to be elucidated.The aims of this study were to investigate Wnt3 a expression in sera and tissues of HCC to analyze its clinical values of diagnosis and prognosis,and explore the impact on HepG2 cell proliferation after down-regulating Wnt3 a expression.Methods: The levels of Wnt3 a expression were detected in total 186 patients including 80 HCC,53 liver cirrhosis,53 chronic hepatic B hepatitis,and 40 healthy subjects as control by the enzyme-linked immunosorbent assay,comparing with AFP to evaluate its clinical diagnosis value.Hepatic Wnt3 a expressions in 80 HCC and their matched surrounding tissues were observed by immunohistochemistry for analyzing its clinicopathological characteristics and prognostic value;Wnt3a-knockout HepG2 cell lines were established by Crispr/cas9-sg RNA system and genomic cleavage efficiency was verified at gene level by surveyor assay.The relative proteins were confirmed by Western blotting;Cell Counting Kit-8 assay was used to examine cell proliferation after knocking-out Wnt3 a successfully.Results: The average levels of serum Wnt3 a expression were significantly higher(P<0.001)in the HCC group than those in any other groups of benign liver diseases,with about 4.02,9.17 and 26.70 times higher than that in the liver cirrhosis,chronic hepatitis and normal control group,respectively.The levels of Wnt3 a expression in HCC patients were closely related to high AFP concentration(P=0.005),liver cirrhosis(P<0.001),HBV infection(P=0.024),poor differentiation(P=0.045),TNM staging(P=0.037)and extra-hepatic metastasis(P=0.023).The sensitivity,specificity,accuracy,positive predictive value and negative predictive values for HCC were 92.50%,94.34%,93.23%,96.10% and 89.29% when the cutoff value was 800 ng/L.Combining Wnt3 a and AFP test,the total sensitivity could rise up to 96.25%.According to the ROC curve,the area under curve in Wnt3a(0.994)was higher than in AFP(0.710).The positive Wnt3 a with brown staining particles was mainly distributed in cytosol and membrane of hepatocytes.The incidence of hepatic Wnt3 a expression in cancerous tissues(96.25%)was significantly higher(?2=45.266,P<0.001)than that in their surrounding tissues(48.75%).The higher strength expression(3~6 scores)was 71.25% in cancerous tissues and 13.75% in surrounding tissues.High Wnt3 a expression was associated with poorly-differentiated grade(P=0.001),liver cirrhosis(P=0.004),HBV infection(P<0.001),higher TNM stage(P<0.001)and five-year survival rate(P<0.001).According to the COX regression and Kaplan-Meier survival curves,high Wnt3 a expression was identified as independent predictive factors for poor HCC outcome and closely related with lower five-year survival rate.After knocked-out by Crispr/cas9-sgRNA system successfully,Wnt3 a expression was downregulated significantly in gene and protein level.Key molecule ?-catenin in cytoplasma was obviously inhibited.HepG2 cell lines proliferation was suppressed in time-dependent manner.Conclusions: Abnormal expression of Wnt3 a as a key signal molecule in the Wnt pathway should be a specific marker for HCC diagnosis and prognosis.Wnt3 a is expected to be a promising target for HCC gene therapy. |
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