The Role Of Cd133 And Aldh In Tumor Invasion And Mechanism Research

Part1. The function of CD133in human colon cell invasion and its mechanismCD133(prominin-1) is a cell surface marker of cancer stem cell which has been widely used in literature. Although CD133is widely expressed in colorectal cancer (CRC) cells, the function and mechanism of CD133in CRC invasion and metastasis remain unclear. In this study, we examined the role of CD133in CRC invasion in vitro and investigated the mechanism involved in CD133-related invasion.In order to find an appropriate cell model, the expression of CD133in six CRC cell lines was examined by FACS and Western blotting. It is shown that the expression of CD133in this six CRC cell lines were quite different, varying from1.5%to98%. HCT116with high degree of malignancy and high CD133expression level was chosen as our cell model. CD133high and CD133low HCT116cells were isolated by FACS, and then the proliferation (MTT, colony formation) and invasive ability (Transwell) of these two sub-populations were analyzed. CD133high HCT116cells exhibited greater proliferation and invasion compared with CD133low HCT116cells, suggesting that tumor cells which express high level of CSC marker have more important roles in invasion.As a highly expressed molecule on cancer cell surface, its own role on invasion and metastasis remains to be elucidated. siRNAs were employed to knockdown CD133expression in HCT116cells. Our results showed that CD133knockdown inhibited the invasive activity of HCT116cells but did not influence their proliferation. AS there is no relative references about their relationship, seven important molecules from different pathway and which were closely related to tumor invasion and metastasis were screened after CD133knockdown. Among them only TIMP-2was down regulated. In addition, knockdown of TIMP-2with siRNAs in HCT116cells significantly decreased in vitro invasion. The results demonstrated that TIMP-2is one of the downstream molecules of CD133and CD133influence the invasion ability through regulating TIMP-2. Our results further showed the MMP2and MEK/ERK signaling pathways were not involved. This is the first report about the relationship of CD133--TIMP-2and the invasion of cancer cells.In conclusion, we demonstrated that CD133plays an important role in HCT116cell invasion. And CD133affects the invasive ability of HCT116cells by regulating TIMP-2.Part2. The impact of ALDH on tumor cells ALDHs (Aldehyde dehydrogenases) belong to a family of NAD(P)+-dependent enzymes involved in detoxifying a wide variety of aldehydes to their corresponding carboxylic acids. As a recently discovered stem cell marker, the markedness of ALDH was confirmed in many normal and cancer tissues. There are19ALDH isoforms in human, and the functional research of these isoforms except ALDH1A1was very limited. The expression profiles of these isoforms in different tissues were also unclear. Reports indicated that ALDH1A3plays an important role in mammary cancers and melanoma, while its roles in colon cancers and lung cancers were still unclear.In order to know the expression of ALDH in tumor cell lines, we detected the activity of ALDH in six colon cancer cells and two lung cancer cells through ALDEFLUOR assay, and we found the percentage of positive population varying from7.4%to64.5%in different colon cancer cells. For lung cancer cells, the percentage of ALDH+cells was11.9%in A549cells and1.2%in NCI-H157cells. Colon cancer cell line HCT116and lung cancer cell line A549were chosen as our cell models. Then we separated ALDHhlgh and ALDHlow cells by FACS to study the function of ALDH, and found that ALDHhigh cells in HCT116and A549showed stronger proliferation in vitro. Besides, ALDHhigh cells in HCT116cells exhibited greater invasion in vitro, suggesting that ALDHhlgh cells have stem cell feature. After continued culture, the percentage of ALDH+cells in both ALDHhlghand ALDHlow populations tended to convert back into its original percentage in HCT116cells, suggesting that CSCs are cells that display a special functional status, but are not cells of a special entity.The main subtype of ALDH in HCT116is ALDH1A3, while ALDH1A1and ALDH1A3are both expressed in A549cells. So we further explored the influence of different subtypes to these tumor cells, and siRNAs were used to knockdown ALDH1A1and ALDH1A3. Knockdown of ALDH1A3could significantly inhibit the invasive ability of HCT116cells and A549cells, and could also significantly inhibit A549cell proliferation, which indicates that ALDH1A3have important roles in tumor proliferation and invasion ability. As tumor cells were located in hypoxia environment in vivo, we simulated this condition through adding CoCL2into the cell culture system. Hypoxia induced less apoptosis in ALDHhigh cells. Knockdown of ALDH1A3could significantly inhibit this tolerance to hypoxia caused by CoCL2in HCT116cells.In conclusion, our research revealed that ALDHs especially ALDH1A3could affect colon and lung cancers invasion and proliferation. This work increased our understanding of CSC and provided new potential therapeutic target for clinical treatment.Part3. The detection of SRS19-6MuLV in mouse dendritic cell sarcoma and its causal effects on tumorigenesisSRS19-6MuLV is a member of the MuLV family originally isolated from the Tianjin-Shanghai-Zunyi complex of murine leukemia. A notable characteristic of this virus is that it induces tumors of multiple hematopoietic lineages, including myeloid, erythroid, T-lymphoid and B-lymphoid. In previous studies, a sequence with high homology to SRS19-6MuLV was identified in a murine dendritic cell sarcoma (DCS) through cDNA expression screening with mAb983D4.To find out that if SRS19-6MuLV is the viral etiology of DCS, the existence of a specific SRS19-6MuLV DNA fragment was detected by PCR in DCS cells,15murine tumor cells,2murine tumor tissues,12normal murine cells/tissues,11human tumor cell lines and SRSV/3T3(NIH/3T3cells infected with SRS cell supernatant). The specific fragment of SRS19-6MuLV was positive in DCS, LⅡ tumor tissue from which DCS is derived and SRSV/3T3. In addition, the integration sites of SRS19-6MuLV in the positive cells were examined by inverse PCR.7integration sites for SRS19-6MuLV were detected in DCS and3in SRSV/3T3. Analysis of sequences around integration sites by BLAST revealed that some of the integration sites were associated with some Ras-regulating miRNAs and common fragile sites which always induce tumor, suggesting that SRS19-6MuLV is the viral etiology of DCS.Our results indicate that SRS19-6MuLV not only induced four types of leukemia, but also induced DCS. This virus does not infect human cells. This study enriched the understanding of the virus including its tumorigenesis spectrum and mechanism of oncogenesis.