Abnormal expression of WIF1 in hepatocellular carcinoma cells and its regulating effect on invasion and metastasis factors of TIMP-3 and caveolin-1 of hepatocellular carcinoma

Objective:To discuss the abnormal expression of Wnt inhibitory factor(WIFI) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3(TIMP-3)and caveolin-1.Methods:RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma eell lines of HepG2,Hep3 B,Huh7,PLC/PRF/5.SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3.Besides,Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.lWIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines.Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells;Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1in hepatocellular carcinoma cells,it also discussed the effect on the expression of β-catenin.Results:The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines(P<0.01);while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721.Therefore,HepG2 and SMMC-7721 were chosen as the further experimental cell lines.After transfecting the eukaryotic expression vector peDNA3.1-WEFl and blank plasmid pcDNA3.1 into hepatocellular carcinoma eell lines,compared with(he blank plasmid group,the cell viability and invasion ability in the WIF1 group were all reduced(P<0.01),the expression of TIMP-3,caveolin-1 and mRNA were all down-regulated(P<0.01),and the expression of β-catenin was decreased(P<0.01).Conclusions:Because of down-regulation or missing of expression of WIFI in hepatocellular carcinoma cell lines,the up-regulation of WIFI expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines,which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.

肝癌组织中PTEN和TIMP-3的表达及其临床意义

目的探讨抑癌基因PTEN和TIMP-3在肝细胞性肝癌(HCC)组织中的表达及其临床意义。方法收集30例HCC、癌旁组织和10例正常肝组织,用qRT-PCR、Western blot、免疫组化SP法检测PTEN、TIMP-3mRNA和蛋白表达,分析PTEN和TIMP-3表达与HCC临床病理特征的关系。结果癌组织中PTEN和TIMP-3mRNA和蛋白表达均明显低于癌旁组织、正常肝组织(P<0.01),且在癌旁组织中的表达亦明显低于正常肝组织(P<0.01)。PTEN和TIMP-3蛋白表达在肝外转移、分化差、血清AFP≥400μg/L和Ⅲ～Ⅳ期肝癌组织中的表达明显低于无肝外转移、分化好、血清AFP<400μg/L和Ⅰ～Ⅱ期肝癌(P<0.01或0.05)者;两者在是否伴有肝硬化和不同肿瘤大小肝癌组织中的表达均无统计学意义(P>0.05)。结论 HCC癌组织中PTEN和TIMP-3表达下调,表达水平有助于判断HCC侵袭转移能力、临床分期和肝外转移。

Mechanism of total glucosides from Chishao(Radix Paeoniae Rubra)on proliferation and apoptosis of hepatocellular carcinoma cells via phosphatase and tensin homolog deleted on chromosome ten/phosphatidylinositol 3-kinase/protein kinase B signaling pathway

OBJECTIVE:To investigate the possible molecular mechanism of total glycosides of Chishao(Radix Paeoniae Rubra)(TG-RPR)on proliferation and apoptosis of hepatocellular carcinoma cells.METHODS:The proliferation of TG-RPR on Hep G2 cells was detected using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.The apoptosis of Hep G2 cells was measured by annexin V-FITC/double staining.The phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt)signaling pathway was evaluated by Western Blot and reverse transcription-polymerase chain reaction(RT-PCR).RESULTS:TG-RPR can up-regulation the expression of pro-apoptotic factors such as PTEN and BCL2-Associated X(Bax),down-regulation the expression of anti-apoptotic factors including B-cell lymphoma-2(Bcl-2),PI3 K,and Akt.CONCLUSION:TG-RPR significantly inhibits the proliferation of Hep G2 cells in a dose-dependent manner and promotes apoptosis.These results demonstrated TG-RPR has significant inhibitory effect on Hep G2 cells.These results identify a critical role of TG-RPR in proliferation and apoptosis of Hep G2 cells via modulating PTEN/PI3 K/Akt signaling pathway.TG-RPR may offer a promise as a potential pharmaceutical therapy for hepatocellular carcinoma.

楮实子水提物对二乙基亚硝胺诱发小鼠肝细胞癌的抑制作用及PTEN/PI3K/Akt信号通路的影响

目的:基于同源性磷酸酶-张力蛋白(PTEN)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路探讨楮实子水提物对二乙基亚硝胺(DEN)诱发小鼠肝细胞癌(HCC)的抑制作用。方法:采用腹腔注射DEN的方法构建原发性HCC小鼠模型,将HCC小鼠随机分为模型组、索拉非尼组(0.01 g·kg^(-1)·d^(-1))、楮实子水提物低、中、高剂量组(0.9、1.8、3.6 g·kg^(-1)·d^(-1)),每组10只;另取10只C57BL/6小鼠作为空白组,腹腔注射等体积生理盐水。在小鼠肝脏出现肝癌样白色结节后灌胃给予不同浓度的楮实子水提物;索拉非尼组给予索拉非尼灌胃;空白组和模型组小鼠给予生理盐水灌胃。生化分析仪检测小鼠血清碱性磷酸酶(ALP)、天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)及γ-谷氨酰转移酶(γ-GT)活性;酶联免疫吸附测定法(ELISA)检测肿瘤标志物甲胎蛋白(AFP)、癌胚抗原(CEA)表达水平;苏木素-伊红(HE)染色及马松(Masson)染色观察肝细胞癌变程度及肝细胞损伤情况;免疫组织化学染色观察肝癌细胞增殖情况;脱氧核苷酸末端转移介导的缺口末端标记法(TUNEL)染色观察小鼠肝癌细胞凋亡情况;蛋白免疫印迹法(Western blot)检测PTEN/PI3K/Akt信号通路相关蛋白PTEN、PI3K、磷酸化-Akt(p-Akt)蛋白表达水平变化。结果:与空白组比较,模型组小鼠ALP、AST、ALT、γ-GT活性及AFP、CEA表达水平显著升高(P<0.01);小鼠肝组织癌变和炎症细胞浸润现象明显,可见大量蓝色胶原纤维增生;肿瘤增殖抗原(Ki67)阳性的增殖细胞数量显著增加(P<0.01);PTEN蛋白表达被抑制(P<0.01),并促进PI3K、p-Akt蛋白表达(P<0.01);与模型组比较,楮实子水提物中、高剂量组小鼠血清中ALP、AST、ALT、γ-GT活性及AFP、CEA表达水平明显降低(P<0.05,P<0.01);且小鼠肝组织癌变程度和炎症细胞浸润明显减轻,胶原纤维增生明显减少;楮实子水提物中、高剂量组Ki67阳性的增殖细胞数量明显减少(P<0.05,P<0.01),楮实子水提物高剂量组TUNEL阳性的凋亡细胞数量明显增加(P<0.05,P<0.01);楮实子水提物楮实子水提物中、高剂量组促进PTEN蛋白表达(P<0.05,P<0.01),抑制p-Akt蛋白表达(P<0.05,P<0.01)。结论:楮实子水提物对DEN诱发的小鼠原发性HCC具有显著的抑制作用,其作用机制可能与调控PTEN/PI3K/Akt信号通路中关键蛋白表达有关。