The Functional Analysis Of Colorectal Cancer Metastasis-related Genes DR-nm23

Object:DR-nm23 gene was choosed as the target of studying in the research project. From molecular biology to study the expression of DR-nm23 in colorectal cancer cells and its biological behavior Impact to colon cancer cell line, Discussion the Relationship of DR-nm23 and colorectal cancer development, invasion and metastasis and clarify its clinical significance, to laid the theoretical basis for guide clinical diagnosis, treatment and prognosis.Methods:We apply the RT-PCR to screen and identify the colon cancer cell lines with high expression of DR-nm23 gene and with low or no endogenous DR-nm23 gene expression in colon cancer cell line. We stably transfected the DR-nm23 gene into the non-endogenous expressed colorectal cancer cell, to observe the impact of the colon cancer cells in vivo and in vitro invasive ability before and after transfection, and concluded by statistical analysis.Results:1. DR-nm23 in three colorectal cancer cell lines:SW620, SW480 and HT-29 expression and clinical significance.There is no endogenous expression of DR-nm23 gene in the highly metastatic cell line SW620, in the low metastatic cell line SW480 and HT-29 with endogenous DR-nm23 gene expression. Conclude that DR-nm23 gene may be negatively prompted with the metastatic of colorectal cancer cell lines.2. Lentivirus constructing, packaging and stability transfected to the SW620 cell lineUsing Age I digested pGC-FU vector to make it linear, the target gene fragment which is amplified by PCR exchanged into the linearized plasmid pGC-FU Vector. After exchange, converse into competent cells of bacteria, identify the PCR for positive clones and DNA sequencing, transfect the Purpose plasmid into 293T cells. After identified by Western--blot, the lentiviral packaging, identification, and the Titer Determination. To obtain the lentiviral with the objective gene transfected SW620 cell line. 3. The impact of the biological characteristics after DR-nm23 gene over-expression of cancer cells1) Observe cell proliferation in vitro, compared with SW620 and SW620 cells transfected with empty vector, SW620 transfected with target gene slow down the cell proliferation, Differences have Significant Significance (P<0.01). Colony formation assay also showed that SW620 cells transfected of the target gene of the proliferation in a single cell was significantly lower than other groups, Differences have Significant Significance (P<0.01). These results both indicated that after DR-nm23 gene over-expression, tumor cells which was in vitro was significantly inhibited.2) Transwell chamber detectied the changes of exercise capacity in vitro, the results showed that after DR-nm23 gene over-expression, cells exercise capacity significantly decreased (P<0.01), illustrate that DR-nm23 overexpression inhibited the ability of colon cancer cells movement.3) Observed the proliferation ability of the colon cancer cells after the DR-nm23 gene overexpression through the subcutaneous tumor experiments. Using the SW620, the SW620 transfected with empty vector and the SW620 transfected with the target gene inoculated subcutaneously in nude mice, through 30 days of continuous observation, found that after DR-nm23 gene over-expression, significantly inhibited the proliferation of colon cancer in vivo.4) Through the colon transfer experiments in vivo, Observed the invasion, metastasis of the cells after DR-nm23 gene over-expression in vivo, The results showed that in the original SW620 cells and the SW620 cell transfected with empty vector have (3/8), about 37.5% of the formation of liver metastases in nude mice. the SW620 cells after Transfected the target gene formed (1/8), about 12.5% of liver metastases in nude mice.Conclusions:1. Successfully screened out the SW620 without endogenous expression of DR-nm23 gene.2. Successful obtained the colon cancer cell line which DR-nm23 gene stability expression, For the subsequently experimental study in vitro lay the basis about the DR-nm23 gene to the proliferation, adhesion and invasion on human colorectal cancer cell.3. Proliferation experiments, cloning experiments and exercise test plate illustrated that DR-nm23 gene expression in colon cancer cells reduced proliferation and athletic ability.4. Subcutaneous tumor experiments and colon Transfer experiments in vivo illustrated that DR-nm23 gene expression in colon cancer cells can significantly inhibit the proliferation and metastasis in vivo.