| Colon carcinoma is one of the most common gastrointestinal tumor.Its pathogenesis include inactivation of tumor suppressor genes and activation of oncogenes, especially the inactivation of tumor suppressor genes is very important .The model of pathogenesis of colon carcinoma which was established by Vogelstein laboratory theory ,"APC - K-ras - DCC - p53 - nm23",can't elucidate the development of colon carcinoma completely, furthermore the pathogenesis of colon carcinoma is diverse .Therefore,it has become a critical subject to reveal the pathogenesis of colon carcinoma by identifying novel tumor suppressor genes.Cancer research institute has isolated a novel gene on the region of chromosome 9p by position- candidate cloning strategy, designated human NGX6, GenBank accession number : AF188239.NGX6 can suppress the proliferation of the nasopharyngea cell linel and maybe the important candidate of tumor suppressor genes. Some study states that NGX6 is down-regulated in colon carcinoma ,especially in that with distal metastasis.Our subject is to transfect mammalian expression vector pcDNA3.1(+)/NGX6 recombinant into colon cancer line HT-29 by lipofectin ,in order to further investigate the effect of NGX6 on colon carcinoma and elucidate its molecular mechanism.NGX6 was transfected into colon carcinoma cell line (HT-29)by lipofectin and a stable cell line of pcDNA3.1(+)/NGX6/ HT-29 overexpressing NGX6 gene was established .The overexpression of NGX6 was identified by PCR, RT-PCR and Dot blot analysis. The effect of NGX6 on the malignant behavior of HT-29was assessed by growth curves of cells , MTT, Clone formation in soft agar, FCM, tumor formation into nude mice.Its Molecular mechanism in vivo and in vitro was analyzed by immunohistochemical, Western Blot and immunofluorescence.pcDNA3.1(+)/NGX6/HT-29 ceils had a significant inhibition in cell proliferation by the growth curves of cells and the ability of clonogenesis in soft agar compared with HT-29 and pcDNA3.1(+) /HT-29 cells. Its double proliferation time of HT-29 cells prolonged from 23.0 hours to 34.5 hours after transfected with NGX6. The result of MTT showed that NGX6 had a negative effect on the cell viability. There was a delay in tumorigenesis and a reduction in tumor size when pcDNA3.1(+)/NGX6/ HT-29 cells were transplanted into nude mice. HE stain showed that xenografts were poorly differentiated adenocarcinoma. Taken together, these results indicated that the overexpression of NGX6 can reverse the malignant phenotype of the HT-29 cell line.The disorder of the cell cycle regulation can lead to the proliferation arid division of the cells out of control,Then the cells show its malignant characterization. So tumor is a kind of cell cycle disease .To investigate the possible effect of NGX6 gene on the distribution of cell cycle and apoptosis, FCM analysis were used to evaluate the cell cycle distribution and cyclins expression of cells. Flow cytometry analyses indicated that the overexpression of NGX6 could delay the progression of G1-S in cell cycle, while it had no effect on the apoptosis of cells. Flow cytometry analysis demonstrated that the expression of cyclin E, cyclin B, cyclin D1 decreased in NGX6-expressed HT-29 cells , especially the decrease of cyclin E and cyclin D1. All these studies provided evidence that NGX6 gene can repress the colon cancer cell's uncontrolled proliferation,which mechanism was that NGX6 mainly exerted at the G1/S boundary of the cell cycle by down-regulating the expression of cyclin Eand cyclin D1.It was predicted that NGX6 gene possessed several structure including a EGF-like domain and two transmembrane regions. Study stated the phosphorylation degree of EGFR and MAPK , an important protein kinase on the EGFR relevant signal pathway, decreased in pcDNA3.1(+)/NGX6/HT-29 cells. Our study was designed to further investigate the alteration of critical molecular which included on the tyrsine kinase signal pathway , related cyclins and CKIs after transfected with NGX6 by of immunohistoc...
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