Function And Mechanism Of CD133in Human Colon Cancer Cells Invasion In Vitro Exogenous GFP Labeling Affects Tumor Cells Biological Behavior

Cancer is still a major public health problem in most parts of the world, although tremendous progress on cancer molecular biology and treatment has achieved. Colorectal cancer (CRC) remains the third most common cause of cancer-related death, although the incidence and mortality of colorectal cancers has continued to decrease for the sake of early detection and treatment. Although radical colorectal resection by standardized guidelines and adjuvant chemo-radiotherapy (CRT) administered to improve survival have achieved some advancement, recurrence and metastasis of CRC remains one of the most common cause of cancer-related death.CD133(prominin-1) is a five-transmembrane glycoprotein, with a molecular weight of120kDa, and CD133was used as a marker for enrichment of CSCs in various solid tumors, including brain, prostate, liver and also colon. However, some authors have denied the role of CD133as a CSC marker for colon cancer, since it has been found expressed by the full spectrum of undifferentiated and differentiated colonic epithelial cells.Recent investigations suggest tha CD133may be useful indicator for clinical assessment of cancer (CRC) and bean effective indicator for prognosis. Still, the role and mechanism of CD133in CRC metastasis remains to be elucidated. Metastatic subpopulation were highly enriched in CD133+CD44+HCT116cells, and the in vitro invasion and in vivo tumor growth of CD133+HCT116cells were enhanced in the presence of CD10+fibroblasts. These results suggested that CD133may be involved in the CRC invasion.In the current thesis, function and mechanism of the CD133+sub-population were studied in CRC cell proliferation and invasion in vitro.First, the CD133expression of6CRC cell lines was examined by FACS and Western blotting assay. Data show that all of6cell lines are positive for CD133. CD133+/high and CD133-HCT116(or COLO320DM) cells were separated by FACS, and the biological behavior of these two sorted cell sub-populations was tested by MTT and Transwell assay. CD133+/higt HCT116cells maintained higher in vitro proliferation and higher invasion compared with that of CD133-HCT116cells, and CD133+COLO320DM cells maintained higher in vitro proliferation. Secondly, the CD133+-GFP-COLO320DM (labeled with EGFP) and CD133--RFP-COLO320DM (labeled with dsRed) sub-populations seprated by FACS were mixed in different ratio and cultured for several passages. It proved that CD133+cells played a major role for the in vitro expansion of tomor cell population, and the percentage of CD133+population gradually restored to the original level. In the following study, CD133siRNA was employed for CD133down-regulation in HCT116cells. After that Cells showed lower invasive ability, while no significant difference was found in their proliferation. These data suggested that CD133have important role in CRC cells invasion, and the "marker role" and "function role" of CD133is different.Various pathways were proved to be involved in the process of invasion. In order to find the downstream signaling molecules regulated by CD133, RNA expression of various proteins involved in proliferation, mobility and invasion were tested by Real-time PCR assay, including Hif-1α,Hif-1β,cdc42, RhoA, Smad7, TIMP-2and TIMP-2. Data showed that TIMP-2is the only one protein that was significantly decreased after CD133down-regulation in HCT116cells, while no difference was found for other molecules. Several studies have established the role of MMP-2and TIMP-2and MMP-2/TIMP-2complex in the growth and progression of CRC.Consequently, the following study focused on the function and mechanism of TIMP-2in CD133-related invasion in vitro.Protein expression tested by Western blotting confirmed the down-regulation of TIMP-2after the knock down of CD133, while the mRNA and protein expression of TIMP-2in CD133+/high HCT116cells was similar with that of CD133-HCT116cells. These data further suggested that the difference between the marker role and function role of CD133, CD133may play different role with different status (distribution, glycosylation and truncated form).The effect of TIMP-2on CD133expression and the biological characteristics of CT116cells were tested after TIMP-2siRNA transfection. It is found that the mRNA and protein expression of CD133was not changed following the knockdown of TIMP-2; the invasive ability of TIMP-2si-HCT116cells decreased sharply.These data suggested that in vitro invasion of CD133si-HCT116cells was decreased by down-regulation of TIMP-2and there existed a functional relationship between CD133and TIMP-2involved in tumor invasion. No relevant reports were found about these data until now.In conclusion, we demonstrated that CD133have important role in CRC cells invasion, and for the first time we demonstrated that CD133knockdown significantly down-regulated TIMP-2expression, which suggests that CD133affects the invasion of HCT116cells via regulation of TIMP-2. The current study provides a theoretical basis for further understanding of the role and mechanism of CD133in CRC, and thus provides target basis for the diagnosis and treatment of relevant diseases. Osamu Shimomura, Martin Chalfie, Roger Y. Tsien were awarded the Nobel Prize ir Chemistry in2008for their outstanding contribution to the the discovery anc development of the green fluorescent protein (GFP). As a non-cytotoxic protein, GFP has become one of the most important tools used in contemporary bioscience.Used as a tagging tool in bio-science, GFP makes it convient to trace at the level of living cells, tissues and the whole organisms. The deriviation of GFP, enhanced GFP (EGFP), was developed to meet the application of various studies. GFP and derivations have been widely used in the field of genetics, neurobiology, regeneration medicine and tomor,especially in the research about tumorigenesis in vivo, model of metastasis, drug screening and therapy of tumor. No paper was found to report the influence of exogenousGFP/EGFP on the biological properties of cells themselves.In the current study, the lentiviral or retroviral carried stably-expressing exogenous GFP/EGFP human colon cancer cell lines (HCT116-GPF, HCT116-EGFP, COLO320DM-EGFP, and DG6-EGFP) were successfully established by infection or tranfection Their growth was measured by MTT assay. The growth of HCT116-EGFP. COLO320DM-EGFP and DG6-EGFP was significantly decreased. The average cell size tested by Cell Analyzer (CasyTT) changed after expression of exogenous GFP, the cell volume of HCT116-EGFP cells was bigger than that that of HCT116cells. The invasion, measured by Trans well assay, of HCT116-EGFP was significantly lower than that of HCT116. Thus, our study suggested that exogenous GFP/EGFP labeling of tumor cells may affect the biological characteristics of the tumor cells in vitro, indicating that before the use of these models, researchers should evaluate its effects on the relevant parameters.