| Invasion and metastasis is not only the malignant phenotypes of lung cancer but also the main cause of death. Elucidating the molecular mechanism of tumor invasion and metastasis will provide new molecular targets for gene-targeting treatment of lung cancer. Invasion and metastasis is complex multi-step process involving activation of tumor metastasis genes as well as inactivation of tumor metastasis suppressor genes. Nm23-Hl is originally identified as a rumor metastasis suppressor gene whose alteration is demonstrated to be closely related to many malignant tumor metastases. The reduced expression and hetero-deletion of nm23-Hl gene have been proved to be closely correlated with the high metastasis potential and poor prognosis of lung cancer. In addition, the abnormality of nm23-Hl gene is often companied with the changes of tumor metastasis-related genes. As a result, Zhou Qinghua hypothesized that there was a "Lung Cancer MetastaticSuppressive Cascade" and nm23-Hl might serve as an upstream regulatorygene to inhibit lung cancer metastasis by regulating downstream genes of thiscascade. In order to explore the function and the molecular mechanism ofnm23-Hl gene in "Lung Cancer Metastatic Suppressive Cascade", based onour previous research work, the following experimental studies were carriedout: (l)The expressive status of nm23-Hl gene was detected both in humanhigh-metastatic large cell lung cancer cell line L9981 and low-metastaticlarge cell lung cancer cell line NL9980 by RT-PCR and Western blot; (2)ThemRNA and protein expression of the metastatic-related genes, E-cadherin,integrin P 1 ^ integrin 3 3 ^ MMP-9, TIMP-1 and EGFR were determined inhuman high-metastatic large cell lung cancer cell line L9981 andlow-metastatic large cell lung cancer cell line NL9980; (3) A high-metastaticlarge cell lung cancer cell line L9981-nm23-Hl transfected with wild-typenm23-Hl gene was constructed by gene transfecting techniques; (4) Themalignant phenotype was detected in L9981-nm23-Hl, L9981-PCMVtransfected with PCMV vector and L9981 (primary high-metastatic large celllung cancer cell line) lung cancer cell line by MTT, cell colonization, celladhesion and Boyden chamber experiments; (5) The mRNA and proteinexpression of the genes related to "Lung Cancer Metastatic Suppr,essiveGenes" were detected and compared between the L9981-nm23-Hl andL9981 lung cancer cell lines by RT-PCR and Western blot. The resultsshowed as follows.1. A 462 bp mRNA expressive band of nm23-Hl gene was observed in human low-metastatic large cell lung cancer cell line, but not observed in the high metastatic large cell lung cancer cell line L9981; A 17kD protein land ofnm23-Hl existed in the human low-metastatic large cell lung cancer cell line NL9980, but not existed in the L9981 cell line.2. The mRNA expressive level of E-cadherin in the human high-metastatic large cell lung cancer cell line L9981 was remarkably lower than that in the low-metastatic large cell lung cancer cell line NL9980 (P<0.05); however, the mRNA expression of integrin P 1, integrin P 3, MMP-9 in the L9981 cell line was significantly higher than that in the NL9980 cell line (PO.05).3.The protein expression level of E-cadherin and TIMP-1 in the L9981 lung cancer cell line was remarkably lower than that in the NL9980 cell line (P<0.05), but the protein expression of integrin P 1 ^ integrin 3 3^ MMP-9 and EGFR was significantly higher than that in the NL9980 cell line (P<0.05).4. A 732 bp mRNA band of nm23-Hl gene existed in the recombinant PCMV-new-Ban-nm23-Hl plasmid, but not existed in the recombinant PCMV-neo-Bam plasmid.5. The recombinant nm23-Hl plasmid was successfully transfected in the human high-metastatic large cell lung cancer cell line L998I. The transgenic lung cancer cell line, L9981-nm23-Hl (a human high-metastatic large cell lung cancer cell line transfected with nm23-Hl gene), can stably, continuously and high-effectively express mRNA and protein of nm23-Hl gene, but neither mRNA nor protein of nm23-Hl exist both in the L9981-PCMV (L9981 cell line transfected with empty vector) and L9981 cell line (primary high metastatic large cell lung cancer cell line).6. The cell proliferation in the L9981-nm23-Hl lung cancer cell line was remarkably higher than that both in the L9981-PCMV and L9981 lung cancercell lines (PO.05); but no significant difference was observed between the L9981-PCMV and L9981 lung cancer cell lines (P>0.05).7.The colon formation in the L9981-nm23-Hl lung cancer cell line was significantly lower than that both in the L9981-PCMV and L9981 lung cancer cell lines (P<0.05), but no significant difference was existed between the L9981-PCMV and L9981 lung cancer cell lines (P>0.05).8. The adhesive ability in the L9981-nm23-Hl lung cancer cell line was remarkably lower than that both in the L9981-PCMV and L9981 lung cancer cell lines (P<0.05), however, no significant difference was existed between the L9981-PCMV and L9981 lung cancer cell lines (P>0.05).9. The in vitro invasive ability in the L9981-nm23-Hl lung cancer cell line was significantly lower than that both in the L9981-PCMV and L9981 lung cancer cell lines (P<0.05), but no significant difference was observed between the L9981-PCMV and L9981 lung cancer cell lines (P>0.05).10. The mRNA expression of E-cadherin in the L9981-nm23-Hl lung cancer cell line was remarkably higher than that in the L9981 lung cancer cell line (PO.05), but the mRNA expression of integrin 3 1 .. integrin 3 3> MMP-9 in the L9981-nm23-Hl lung cancer cell line were lower than that in L9981 lung cancer cell line (P<0.05).11. The protein expression of E-cadherin and TIMP-1 in the L9981-nm23-Hl lung cancer cell line was remarkably higher than that in the L9981 lung cancer cell line (PO.05); however, the protein expression of integrin P K integrin 3 3, MMP-9 and EGFR in the L9981-nm23-Hl lung cancer cell line were remarkably lower than that in the L9981 lung cancer cell line (PO.05).Conclusions: (1) Deletion of nm23-Hl gene exists in the human high metastatic large cell lung cancer cell line L9981, but no nm23-Hl deletion exist in the human low metastatic large cell lung cancer cell line NL9980; (2) The transgenic lung cancer cell line, L9981-nm23-Hl transtected with nm23-Hl gene, can stably, continuously and high-effectively express the mRNA and protein of nm23-Hl; (3) The molecular mechanism of nm23-Hl gene reversing the metastatic phenotype of human high-metastatic large cell lung cancer might be related to the efifects of nm23-Hl gene on regulation of mRNA and protein expression of the genes correlated with " Lung Cancer Metastatic Suppressive Cascade".
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