Visual Observation of Tiam1 Gene Silencing on Metastasis of ...

[Abstract]

BACKGROUND & OBJECTIVEColorectal cancer (CRC) is a common malignancy in the world. In recent years,China colorectal cancer incidence and mortality rates are risiing .year after year, themetastasis is a major cause of death. Clarification of colorectal cancermetastasis-associated factors and genes that can provides an ideal drug target toclinical treatment is the focus of colorectal cancer research.Cancer metastasis is a complex and multi-step, multi-factor process. Tumor cellsform a new metastases must be completed the following links: (1) shedded from theoriginal tumor mother; (2) invaded and entered the blood or lymphatic vessels; (3)moved to the target organ; (4) emigrated from the blood or lymphatic vessels; (5)growed and formed new tumor nodules in target organ tissues. The tumor cells arerestricted in every step of this process. The heterogeneity of tumor is the fundamentalreason in cancer metastasis. Although the majority of tumors are monoclonal origin,but because the genetic instability of tumor and the choice of target organ, tumor hasbeen evolved to a group of continuous variation. Some subpopulations with differentmetastatic potential can grow superiorly in certain specific target organ. Be compared with the original tumor cells, the subpopulations may have some special traits. Iffound these mechanisms and block the corresponding target may inhibit the metastasis.Metastasis be divided into a number of steps because of the consecutive complicatedprocess. For different targets, people use different methods to research respectively atdifferent levels. However, all experimental studies on metastasis can not do withoutanimal experiments at whatever level of research. Therefore, the establishment ofexperimental animal metastasis model occupies a very important position in thecancer metastasis researchs.Tiaml (T-lymphoma invasion and metastasis gene 1) was originally identified asthe invasion and metastasis inducing gene by proviral tagging in combination with invitro selection for invasiveness in T lymphoma cells by Habets, et al, in 1994. Then,the gene was located to the human No. 21 chromosome q22.11. Tiaml is mainexpression in the brain and testis of human, not expression or only low expression inother tissues, However, both tumor cells in a variety of sources are all express Tiaml.Tiaml was confirmed as a gene which is associated with the carcinogenesis, evolutionand metastasis in breast cancer, lung cancer, Ras-induced skin cancer and othertumors.In our previous studies, we detected the expression of Tiaml gene in colorectalcancer metastatic cell lines and tissues. Semi-quantitative RT-PCR showed thepositive rate of Tiaml in colorectal carcinoma metastatis cell line was 100%;Northern blot and in situ hybridization results also confirmed Tiaml was highexpression in metastatis carcinoma cell lines and tissue; The results that Tiaml wastransfected into human colorectal carcinoma cell line HT29 cells which almost noTiaml endogenous expression by gene transfection method, showed that theproliferation and metastasis capabilities of these cells were significantly increased;When Tiaml expression was efficiently and specifically inhibited in human colorectal carcinoma cell line SW480 which was derived from primary lesions by RNAi method,subcutaneous implantation found these cells proliferation rate in vivo was decreased.In this study, we used human colorectal carcinoma cell line SW480 with highmetastasis characteristics to establish visualizing experimental and spontaneousanimal model and observed the process of colorectal cancer metastasis. The objectiveis to clarify the possible role of Tiaml gene in human colorectal carcinoma metastasisand provide an ideal animal model for further study of human colorectal cancermetastatic mechanism.METHODS1. Identification of SW480 cell sublinesCultured colorectal cancer cell sublines SW480/EGFP~+ and SW480/EGFP~+/Tiaml which established successfully in our laboratory. Inverted fluorescencemicroscope was applied to detected green fluorescent protein expression of these twocell sublines; Immunocytochemical and western blot was applied to detect andcompare the Tiaml protein expression in the two cell sublines; And MTT assaydetection of cell growth curve compared the two cell sublines proliferation capacity invitro.2. Establishing the visible experimental metastasis nude mice model by tail veininjectionThe cultured cells of SW480/EGFP~+ and SW480/EGFP~+/Tiaml~- were collectedrespectively, then the suspension were injected into the tail vein of nude mice with2?10~5/0.2ml and 2?10~6/0.2ml for each mouse. All the euthanasia mice were observedthe tumor metastasis by whole-body fluorescence imaging after 2m inoculation.Finally, all of the mice organs and possible metastatic tissues were examined by conventional pathology.3. Establishing the visible spontaneous metastasis nude mice model by orthotopicimplantationSW480/EGFP~+ and SW480/EGFP~+/Tiaml~- cell suspension (1?10~7/0.2ml) themice were executed euthanasia. The tumor tissues were taken down and stripped into1mm~3 particles. The tissue particles were inoculated into cecum subserosa of nudemice respectively (8/group). Then these tumor growth, evolution and metastasis in thenude mice were observed by whole-body fluorescence imaging at intervals. Twomonths later, all the cachexia mice were killed, the primary tumor and metastasis fromthe mice were observed by conventional pathology.4. Histopathologic examinationAll tissues from the experimental mice were fixed by 10% neutral formalin,embedded by paraffin and stained by HE. Finally, the morphological changes andmetastasis of the tumor cells were observed and photographed with microscopy.5. Statistical analysisThe Application of SPSS13.0 statistical package to analysis the experimentaldatas: Factorial analysis was used for MTT assay and independent-samples T test wasused for comparation of the tumor size of colonic grafts in situ.RESULTSThe main results and findings are as follows:1. Identification of SW480 cell sublinesBoth of the two human colorectal carcinoma cell sublines, SW480/EGFP~+ andSW480/EGFP~+/Tiaml~-, stably expressed EGFP and the expression level reached 100%; SW480/EGFP~+ cells were strong positive for Tiaml, while SW480/EGFP~+/Tiaml~- cells were negative by immunocytochemical detection; Tiaml gene productionreduced to a 30% level in SW480/EGFP~+/Tiaml~- as much as that in SW480/EGFP~+ bywestern blot detection; The growth rate in vitro by MTT of the two cell lines had nosignificant difference (P?0.05).2. Tail-vein-injection experimental metastasis modelThe 2?10~5/0.2ml tail-vein-injection group: SW480/EGFP~+ and SW480/EGFP~+/Tiaml~- both developed no metastasis in nude mice; The 2?10~6/0.2ml tail-vein-injection group: SW480/EGFP~+ cells induced 4 mice metastasis of 8, the metastasisrate was 50%. The metastasis were distributed over the heart, lung and bone, butwithout in two nude mice, the metastasis rate was 25%; SW480/EGFP~+/Tiaml~- cellscould not induce any metastasis in all the mice.3. Colon orthotopic implantation spontaneous metastasis modelSW480/EGFP~+ and SW480/EGFP~+/Tiaml~- tumor particles which wereinoculated into the colon of nude mice both can survive. But the tumor size ofSW480?EGFP~+/Tiaml~- group were all less than SW480/EGFP~+ (P?0.05). Only onemouse from SW480/EGFP~+/Tiaml~- group was detected abdominal wall invasion andmetastasis, and the metastasis rate was 16.6%; SW480/EGFP~+ group had 6 miceperitoneal metastases of 8, of which 2 mice got hepatopulmonary and pleuralmetastases, the metastasis rate was 75%.4. Histopathologic examinationTumor metastasis by histopathologic examination were basically consistent withwhat observed by whole-body fluorescence imaging. Heart, lung, liver, bone and allthe other sites which were visually detected metastasis in the mice were comfirmed cancer tissues in the lesions under the optical microscopy; Furthermore, one of thetwo mice which be found heart metastasis by tail-vein-injection was detected a lot ofmicro-lung metastatic lesions which could not be inspected by fluorescence imaging.Another finding was that there were micro-metastatic tumor cells in lymphatic andvascular tissues in primary grafts and metastases of SW480/EGFP~+ mice. But all ofSW480/EGFP~+/Tiam1~- mice had not represented any of these features.CONCLUSIONS1. It was successfully established a tail-vein-injection experimental model forcirculation propagation of human colorectal cancer, and an orthotopicimplantation animal model for spontaneous metastasis to assess the effect ofTiam1 gene silencing upon the tumorigenesis and metastasis of SW480 cell line;2. Silencing of Tiam1 gene could suppress the metastasis of blood vesselpropagation and colon orthotopic implantation of human colorectal carcinomacellline SW480;3. Tiaml gene promote tumorigeness and metastasis in vivo that could play animportant role in the signal pathways which controlling cells morphology,adhesion, movement and interaction with vascular endothelium;4. Human colorectal carcinoma cell line SW480 had transplanted to heart, lung andbone but not to liver in tail-vein-injection experimental metastasis model. Itsuggested that the higher level of hepatic metastasis of colorectal cancer may beinvolved with colorectal carcinoma cells adapting to the environment of liver, andthe anatomical position may be a pivotal factor for metastasis of colorectalcarcinoma cells;5. SW480/EGFP~+ and SW480/EGFP~+/Tiam1~- are two stable and reliable human colorectal carcinoma cell sublines which can be widely used in the research of themetastasis mechanisms and the anti-metastatic drugs evaluation for colorectalcancer.

Title: Visual Observation of Tiam1 Gene Silencing on Metastasis of Human Colorectal Carcinoma Cell Line SW480

Category: Stomach Cancer

Filename: Visual Observation of Tiam1 Gene Silencing on Metastasis of Human Colorectal Carcinoma Cell Line SW480.pdf

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