Analysis Of Hugl-1 Gene Expression And The Biological Effects In Colorectal Cancer

Loss of lethal giant larvae (lgl) in Drosophila results in the neoplastic transformation of the brain hemispheres and the imaginal discs which lose their epithelial monolayer stucture and grow in large amorphous masses resembling mammalian carcinomas. Upon transplantation into wild-type recepients, these tumorous tissues grow and migrate to distant sites, thus behaving like mammalian metastatic tumours. Lgl-induced metastases overexpress typeⅣcollagenase and NDP kinase, thus showing some of the biochemical features of metastatic tumours in humans. Homologues of lgl have been identified in many species including man, mouse, insect, worm, slime mold and yeast. Indeed, the function of Lgl is conserved as evidenced by rescue of the Drosophila lgl mutation with its human homologue, Hugl-1, and the complementation of the yeast mutations by the human and Drosophila proteins. The human gene Hugl-1 has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). Hugl-1 gene maps to the short arm of chromosome 17 in a pericentromeric region, 17p11.2-12.. The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. but to date, a role of Hugl-1 in human cancer remains largely unknown. Whether loss of Hugl expression plays a role in human tumorigenesis has not been extensively investigated. According to the mentioned above, we performed the following experiments. Firstly, we investigated the expression of Hugl-1 gene in colorectal cancer samples and explored its correlation with clinical and pathological characters. Then we established and identified colorectal adencarcinoma cell lines(LS174) transfected by exogenous gene Hugl—1. Based on above work, we investigated whether the exogenous gene Hugl-1 transfer could inhibit the growth of colorectal adencarcinoma cell lines in vitro and explored the possible mechanism of this effect. Purpose a new theoretical basis of genic diagnosis and therapy for colorectal cancer.Part1. Expression of Hugl-1 in colorectal cancer(CRC) and its possible implications Objective: To determine the expression of Hugl-1 gene in colorectal cancer(CRC), and to investigate the association of Hugl-1 with CRC. Methods: RT-PCR and Western-blot mothods were used to detect the expression level of mRNA and protein of Hugl-1 gene in 63 pairs of fresh colorectal cancer tissues and according para-cancer normal tissues. Analysis implications between the expression of Hugl-1 and clinical and pathological characters. Results: The expression levels of mRNA and protein in para-cancer normal tissues were3.59-fold and 3.18-fold (P<0.05) higher than those in cancer tissues respectively.The decreased expression of Hugl-1 gene in colorectal cancer tissues were associated with advanced stage and particularly with lymph node metastases (P<0.05).Conclusions: Decreased expression of Hugl-1 gene in colorectal cancer is tumor specific, and may be a result of alterations of the gene translation and (or) protein metabolism. Hugl-1 might contribute to restrain malignant transformation and aggressiveness of CRC.Part2. Recombinant pcDNA3.1-Hugl-1 transfect colorectal carcinoma cell line LS174 Objective: To study biological effects of exogenous gene Hugl-1, the custom-crafted pcDNA3.1-Hugl-1 was transfected intocolorectal carcinoma cell line LS174.Methods: Two different plasmids including a recombinate pcDNA3.1-Hugl-1 and an empty pcDNA3.1 were prepared by transformation of bacterium, amplification and purification of plasmids.Then two kinds of plasmids were reseparately transfered into colorectal carcinoma cell line LS174 cultured in vitro by using lipfectamine. After transfection, positive clones were screened with G418 and expanded by culture. The expression of the Neor tag-gene was detected by PCR to confirm whether the recombinant vector DNA integrated with the genomic DNA of LS174 cells. RT-PCR and Western-blot methods were used to analysis expression of Hugl-1 mRNA and protein in LS174 cells before and after transfection. Results: All of the two cell lines transfected by corresponding plasmid acquired resistance to neomycin. The Neor-tag gene was detected by PCR in cell lines transfected by recombinate pcDNA3.1-Hugl-l and empty pcDNA3.1, wherease, it did not show in cell lines un-transfected due to lack of Neor-tag. RT-PCR and western-blot showed respectively that the Hugl-1 gene were expressed higher in cell lines transfected by pcDNA3.1-Hugl-l than in cell lines un-transfected and transfected by an empty vector both on mRNA and protein lever.Conclusions:The eukaryotic vector was transfected successfully to LS174 cells with liposome, which integrated with LS174 cell genome and markedly enhanced the expression of Hugl-1 mRNA and protein in LS174 cells.Part3.The biological effects of recombinate vector in stably transfected colorectal cancer cell lines LS174.Objective: To observed the biological behaviors of LS174 before and after transfection in vitro. Methods: MTT growth test, soft agar colony formation , wound-healing experiment,adhesion assay and Matrigel movement assays were used in vitro to study the effects of Hugl-1 expression on transfected cells proliferation, movement and invasion. Results: Transfected with Hugl-1 eukaryotic expression vector could significantly increase cell adhesion and decrease cell movement and invasion capabilities compared with the untransfected cells. Conclusions: Hugl-1 gene transfer could inhibit movement and invasion capabilities of colorectal cancer cell line LS174 in vitro.