【作者】 付军科；

【导师】 周清华；

【作者基本信息】 四川大学 ， 外科学， 2004， 博士

【摘要】 肺癌的侵袭和转移是肺癌的恶性标志和特征,也是导致肺癌患者治疗失败和死亡的主要原因。肺癌的侵袭和转移是一个多因素作用、多基因参与、涉及细胞多个信号通路改变,并经过多个阶段才最终形成的复杂生物现象。因此,探讨肺癌侵袭转移相关细胞信号传导通路的变化,不仅有助于揭示肺癌侵袭转移的分子机制,而且将为阻断肺癌侵袭转移的信号传导和逆转肺癌侵袭转移表型提供新的靶点和途径。已经证明肿瘤转移抑制基因nm23-H1的低表达和杂合性缺失与肺癌的高转移性和预后不良有密切关系,并在调控“肺癌转移抑制级联”中发挥上游关键基因的作用。在nm23-H1基因缺失的人高转移大细胞肺癌细胞株L9981中转染野生型nm23-H1基因后,nm23-H1可通过调控“肺癌转移抑制级联”中多个转移相关基因的表达,逆转肺癌细胞的转移表型。Nm23-H1基因对“肺癌转移抑制级联”的调控作用可能是通过细胞中一些关键的信号传导通路来实现的。近年来的研究发现,Wnt信号通路的异常与肿瘤发生和侵袭转移有密切关系。为了探讨nm23-H1基因在“肺癌转移抑制级联”中对Wnt信号传导通路的调控作用及其分子机制,本研究在已经筛更多还原

【Abstract】 Tumor metastasis is not only the malignant marker and characteristics of lung cancer, but also the main cause of failure to cure and lose their life of the patients with lung cancer. It is a complex biological behavior that dealed with many factors, genes, signal pathways and processes. Therefor, exploration of the changes of cell signal transduction related to invasion and metastasis in lung cancer will not only illuminate the molecular mechanism of tumor invasion and metastasis, but also provide a new targeting molecule and route for blocking signal transduction and reversing metastatic phenotype of lung cancer. Now, it has been proved that low expression and hetero-deletion of tumor metastasis suppressor gene nm23-Hl was closely correlated with the high metastasis ability and poor prognosis of patient with lung cancer. Nm23-Hl gene is a key and upstream regulative gene in the "lung cancer metastatic suppressive cascade". Transfection of wild type nm23-Hl cDNA into human high-metastatic large cell lung cancer cell, which has been proved that it had LOH of nm23-H1 gene, can regulate theexpression of metastatic relative genes and reverse the metastatic phenotypeof lung cancer cell lines. The molecular mechanism that nm23-Hl genesuppress lung cancer metastasis may be involved some important signalpathways. At present, it has been proved that the abnormality of Wnt signalpathway was closely correlated with the oncogenesis and development ofmalignant tumor. Up to now, however, we had not seen any reports on that ifnm23-Hl gene regulate "lung cancer metastatic suppressive cascade"simultaneously with Wnt signal pathway or not? How do the nm23-Hl generegulate the expression and activity of the key proteins and kinases in Wntsignal pathway? That if block of the activities of the key kinase in Wnt signalpathway can influnce the reversion effects of nm23-Hl gene for malignantbiological behavior and metastatic phenotype of lung cancer or not ? In orderto explore the regulation effects and mechanism of nm23-Hl gene in "lungcancer metastatic suppressive cascade" for Wnt signal transduction in lungcancer, based on our previous research results that human high-metastaticlarge cell lung cancer cell line L9981 had been screened and identified, thatthe L9981, L9981-pLXSN ( transfected with vector ) and L9981-nm23-Hl(transfected with nm23-Hl gene) lung cancer cell lines have beensuccessfully constructed, we carried out the following experimentalstudies:(l)The expression and kinase activity of GSK-30, P -catenin andphospho- ^ -catenin were detected in human high-metastatic large cell lungcancer cell lines L9981, L9981-pLXSN and L9981-nm23-Hl before and aftertransfection of nm23-Hl gene by Western blot, immunoprecipitation andisotope scintillative count; (2)The proliferative and invasive abilities of theL9981 lung cancer cell line were detected and compared by MTT andBoyden chamber, before and after transfection of nm23-Hl gene; (3)The changes of expressive level and kinase activities of GSK-3P, P-catenin and phospho-P-catenin were determined in the L9981, L9981-pLXSN and L9981-nm23-Hl lung cancer cell lines before and after treating with GSK-3P kinase inhibitor, LiCL, by Western blot and immunoprecipitation and isotope scintillative count; (4)The effects of GSK-3P kinase inhibitor, LiCL , on the proliferative and invasive activities were detected in L9981, L9981-pLXSN and L9981-nm23-Hl lung cancer cell lines by MTT and Boyden chamber. The result in this study first showed in the world as follows:1.Western blot analysis showed that:(l) The expression of GSK-3P of cytoplasm and nucleus in L9981-nm23-Hl lung cancer cell line was remarkably higher than those in L9981 and L9981-pLXSN lung cancer cell lines (PO.001); (2)The expression of P -catenin of cytoplasm in L9981-nm23-Hl lung cancer cell line was significantly higher than those in L9981 and L9981-pLXSN lung cancer cell lines(P<0.01), but no significant difference of P -catenin expression of nucleus was found among the three lung cancer cell lines; (3)The expression of phospho- P -catenin of necleus in L9981-nm23-Hl cell line was remarkably higher than those in L9981 and L9981-pLXSN cell HnesCPO.OOl), but the expression of phospho- P -catenin of cytoplasm in L9981-nm23-Hl cell line was remarkably lower than those in L9981 and L9981-pLXSN cell lines(/><0.001).2.LiCL is a selective inhibitor of GSK-3P and activitor of Wnt signal pathway. The comparisons of time- and dose-dependent suppression of GSK-3P expression by LiCL showed that the most significant effection of LiCL on GSK-3P expression of cytoplasm and nucleus in the three lungcancer cell lines was that the cell lines were treated with 20 mmol/L concentration for 60 min.3.After treatment of the three lung cancer cell lines with 20 mmol/L LiCL for 60 min, Western blot analysis showed that:(l) The GSK-3(3 expression of cytoplasm and nucleus in L9981-nm23-Hl lung cancer cell line was remarkably higher than those in L9981 and L9981-pLXSN cell lines (/><0.001);(2) The expression of P -catenin of cytoplasm in L9981-nm23-Hl lung cancer cell line was remarkably lower than those in L9981 and L9981-pLXSN cell lines(P<0.001), but the expression of 3 -catenin of nucleus in L9981-nm23-Hl cell line was significantly higher than those in L9981 and L9981-pLXSN cell lines(P<0.001);(3) The expression of phospho-3 -catenin of necleus in L9981-nm23-Hl cell line was remarkably higher than those in L9981 and L9981-pLXSN cell lines(P<0.001), however, the expression of phospho- P -catenin of cytoplasm in L9981-nm23-Hl cell line was remarkably lower than those in L9981 and L9981-pLXSN cell Iines(P<0.001).4. After treatment of the three cell lines with 20 mmol/L LiCL for 60 min, Western blot analysis showed that:(l)No significant difference of GSK-3p expression of cytoplasm and nucleus in L9981-nm23-Hl cell line was observed betwwen before and after treatment with 20mmol/L LiCL for 60min(P>0.05), but significant down-regulation of GSK-3P expression both in cytoplasm and nucleus was existed in L9981-pLXSN and L9981 cell lines after treatment with 20 mmol/L LiCL for 60 min(P<0.001); (2) P -catenin expression was remarkably increased in L9981-nm23-Hl nucleus and decreased in cytoplasm after treatment with 20mmol/L LiCL for60min(/><0.001), but the ^ -catenin expression both of cytoplasm and nucleus was significantly upregulated in L9981 and L9981-pLXSN cell lines after treatment with 20 mmol/L for 60 min(.P<0.001); (3)The expression of phospho- P -catenin of cytoplasm and nucleus was downregulated in L9981-nm23-Hl , L9981-pLXSN and L9981 cell lines after treatment with 20mmol/L LiCL for 60min(P<0.001).5.GSK-3P activity assay were detected by immunoprecipitation and isotope scintillative count. GSK-3P activity of cytoplasm and nucleus in L9981-nm23-Hl cell line was remarkably higher than those in L9981 and L9981-pLXSN cell lines (PO.001), but no significant difference was observed between L9981-pLXSN and L9981 cell lines (P>0.05).6.The comparison of time- and dose-dependent suppression showed that: (l)The most significant effection of LiCL on GSK-3P activity in the three cell lines was observed when the cell lines were treated with 20 mmol/L LiCL for 60 min;(2) GSK-3P activity of cytoplasm and nucleus in L9981-nm23-Hl cell line was significantly higher than those in L9981-pLXSN and L9981 cell lines after treatment with 20 mmol/L LiCL for 60 min (P<0.00l), but no significant diffirence of GSK-3P activity of cytoplasm and nucleus was observed between L9981 and L9981-pLXSN cell lines (P>0.05); (3)The activities of GSK-3P both of cytoplasm and nucleus in L9981, L9981-pLXSN and L9981-nm23-Hl lung cancer cell lines after treatment with LiCL were remarkably lower than those before treatment with LiCL (PO.001).7.The cell proliferative and invasive abilities of L9981-nm23-Hl cell line were significantly lower than those in L9981 and L9981-pLXSN celllines (PO.001); The cell proliferative and invasive abilities of L9981-nm23-Hl, L9981, and L9981-pLXSN cell lines after treatment with 20 mmol/L LiCL were significantly higher than those before treatment with LiCL (PO.001); The cell proliferative and invasive abilities of L9981-nm23-Hl cell line was still significantly lower than those in L9981 and L9981-pLXSN cell lines after treatment with 20mmol/L LiCL(P<0.001).8.N0 significant difference of GSK-3P expression and activity, P -catenin and phospho- P -catenin expression of cytoplasm and nucleus was existed between L9981 and L9981-pLXSN lung cancer cell lines after treatment with LiCL (P>0.05). There was no significant difference of the proliferation and invasion in L9981 cell line after treatment with LiCL compared with L9981-pLXSN cell line (P>0.05).Conclusion:(l)Transfection of nm23-Hl gene can significantly upregulate the expression and activity of GSK-3p of cytoplasm and nucleus in the human high-metastasis large cell lung cancer cell line L9981; (2)nm23-Hl can significantly upregulate the expression of P -catenin of cytoplasm in the human high-metastasis large cell lung cancer cell line L9981, but not for P -catenin of nucleus; nm23-Hl can also significantly upregulate the expression of phospho-P -catenin of nucleus in L9981 lung cancer cell line; (3) LiCL can not only antagonize the effection of nm23-Hl gene on the upregulation effect for GSK-3p-. P -catenin and phospho- P -catenin in human high metastasis lung cancer cell line L9981, but also antagonize the reversion effects for cell proliferation and invasion of human high metastasis lung cancer cell line L9981. (4)The transduction of Wnt signal might be directly or indirectly suppressed through the upregulation of expression andactivity of GSK-3P in human high-metastasis large cell lung cancer cell lines by nm23-Hl gene. It may be one of the molecular mechanism that nm23-Hl gene regulate "lung cancer metastatic suppressive cascade" and reverse metastatic phenotype of human high-metastasis large cell lung cancer cell lines.更多还原