| Colorectal cancer (CRC) is the third most common cancer and the second cause of cancer related death both in China and worldwide. Almost 50% of patients with CRC will eventually develop liver metastases during the natural course of the disease. And liver metastases in patients is the leading reason of recurrent and death of colorectal cancer.A growing body of evidence is increasingly supporting the idea that human cancers, including CRC, can be considered as a stem cell disease. As traditional tumor cells, the cancer stem cells(CSCs) are composed by a heterogeneous population of cells different for morphology, marker expression, metastasis capacity and can give rise to a wide variety of more "differentiated" cancer cells which comprise the bulk of the tumor and provide the basis of tumor heterogeneity. According to metastasis capacity, the CSC exhits metastasis cancer stem cell (MCSC) and non-metastasis cancer stem cell (nMCSC) subpopulation, while MCSC is closely related to cancer metastasis.In previous experiments, we have successfully isolated and identified the CSCs from the colorectal cancer cell line SW480. The nMCSC and MCSC spheres were screened isolated by in vivo experiment in nude mice. Using comparative proteomics, a number of tumor metastasis-related proteins were seperated and identified, including FUBP1 and EFTU.The human far upstream element (FUSE) binding protein 1(FUBP1) is a transcriptional factor that binds to a specific single-strand DNA sequence located within the far upstream region of target promoter such as c-Myc, p21 involved in cell cycle, apoptosis, cell proliferation and matrix invasion et al. The role of its transcriptional regulation is dependent on other co-activators or repressors, such as FUBP2/3, FIR, JTV1 and GCN5. Increasing evidences suggested that FUBP1 is highly expressed in a variety of tumor tissues and cancer cell lines, including CRC. However, the role of FUBP1 in CRC stem cell, especially in CRC MCSC remains to be elucidated.The human elongation factor Tu (EFTU, Tu translation elongation factor, TUFM), one of the most abundant proteins of the mitochondrial coded by a nuclear gene, plays a key role in the elongation process of mitochondrial protein biosynthesis, delivery of aminoacyl-tRNA, maintaining the integrity of the mitochondrial membrane, the stability of the mitochondrial membrane potential and cell respiratory chain function. EFTU was found high expression in several types of tumor tissue and cell lines. The mean overall survival time for high EFTU expression patients was less than low/negative EFTU expression patients and is an independent poor prognostic factor. So far, the role of EFTU in colorectal CSC and its relationship with tumor metastasis was not reported.The nMCSC and MCSC clones of CRC were good material to investigate the function of metastasis-related genes in CRC. In this study, we will focus on the following aspects to study the role of FUBP1 and EFTU in CSCs of CRC. 1) Real-time RT-PCR and Western blot was used to confirm the data from proteomics;2) To research the role of FUBP1 and EFTU, lentivirus of coding overexpression and RNAi of them were constructed and infected into nMCSC and MCSC clone of CRC. The expression of lentivirus was tested.3) In vitro study:in order to study the role of FUBP1 and EFTU on CRC CSC biology, several experiments were performed. Cell growth curve with CCK-8 assay was used to investigate the proliferation capacity; plate colony assay was performed to test the colony formation ability; In vitro invasion assay based on transwell precoated with ECM was used to determine the change of invasion ability; wound-healing assay was used to detect the cell migrating ability change.4) In order to evaluate the affect of FUBP1 and EFTU genes on tumor-initiation ability in vivo, nude mice subcutaneous tumor forming assay was used. The effect of FUBP1 and EFTU on metastasis ability of CRC cancer stem cells were determined by intravenous injection via tain vein of nude mice.5) Clinical date verification:Immunohistochemical analysis for FUBP1 and EFTU expression was performed on paraffin-embedded specimen of 320 colorectal cancer. The correlation of FUBP1 and EFTU expression and patients’ clinicopathological parameters was statistically evaluated and the prognostic significance of FUBP1 and EFTU expression was assessed by univariate and multivariate analyses.Results:1 The real-time RT-PCR and western blot results showed that in SW480, nMCSC and MCSC cells, the quantitative expression of mRNA was responded to protein expression of FUBP1 and EFTU. The level of mRNA and protein of FUBP1 and EFTU in SW480 was higher than them in nMCSC and MCSC cells, consistant with results of proteomics. Compared with nMCSC, the expression of FUBP1 and EFTU was higher.2 Using molecular cloning, we successfully constructed the lentivirus of coding FUBP1 and EFTU overexpression and FUBP1-RNAi and EFTU-RNAi, respectively and infected them into nMCSC and MCSC. Western blot results detected a band of FUBP1/GFP or EFTU/GFP fusion protein in cells infected with overexpression lentivirus compared with control lentivirus. The cells infected with FUBPl-RNAi or EFTU-RNAi lentivirus showed significant lower level of FUBP1 or EFTU than to control RNAi lentivirus, indicating the silencing efficiency of RNAi.3 (1) CCK-8 results showed that in nMCSC and MCSC infected with overexpression lentivirus of FUBP1 or EFTU, the OD450 absorbande was higher than control lentivirus cells, the difference between target overexpression and control appeared significant (P<0.001). In constrast, in nMCSC and MCSC infected with RNAi lentivirus of FUBP1 or EFTU, the absorbance of OD450 was attenuated compared with control RNAi lentivirus. The difference was also significant (P<0.001); (2) Plate colony assay results showed that the nMCSC and MCSC overexpressed with FUBPl or EFTU had more colony-forming potential than control lentivirus. The difference was statistically significant (P<0.01). In nMCSC and MCSC infected with RNAi lentivirus of FUBP1 and EFTU, the colony formation nubmers were less than RNAi control lentivirus, the difference of colony formation capacities appeared statistically significant (P<0.05); These results indicate that FUBP1 and EFTU overexpression enhances the colony-forming capacity of CRC cancer stem cells; (3) in vitro invasion assay results showed that in nMCSC and MCSC infected with overexpressed lentivirus of FUBP1 and EFTU, the number of cells invading to the under surface were more than control lentivirus. The difference of invasion activities was significant (P<0.01); In constrast, in nMCSC and MCSC infected with RNAi lentivirus of FUBP1 or EFTU, the invading cells were less than RNAi control cells. The difference also apeared significant (P<0.05); (4) wound-healing results showed that in nMCSC and MCSC infected with overexpression lentivirus of FUBP1 or EFTU had more significantly higher capacity of migration compared with control lentivirus, while the nMCSC and MCSC infected with RNAi lentivirus of FUBP1 or EFTU showed an inhibitory migration capacities compared with control RNAi lentivirus.4 Nude mice subcutaneous tumor forming assay results showed that in MCSC infected with FUBP1 overexpression lentivirus, the gross xenografts volume were larger than control lentivirus at 28-day, the difference of gross volume was statistically significant (P=0.047), however, the difference of gross volume at 35-day was not significant (P=0.063). In EFTU overexpressed MCSC, the gross xenografts volume was larger than in control lentivirus cells, the difference had statistical significance (P<0.001). Similar to the results of MCSC, in nMCSC and MCSC infected with FUBP1 and EFTU overexpression lentivirus, the gross xenografts volume was larger than control lentivirus, too. The difference was statistically significant (P<0.001). In constrast, in nMCSC and MCSC infected with FUBP1 or EFTU RNAi lentivirus, the gross xenografts volume was smaller than those in control lentivirus cells. The difference was significant (P<0.001). The results suggest that FUBP1 and EFTU have an increased ability to generate tumor xenografts in CRC stem cells. Metastasis tumor model of tail vein injection results showed that all control cells of nMCSC and MCSC including overexpression control and RNAi control generated tumor xenografts. Among them, the lung was the main organ that metastasis xenograft occurred. In MCSC infected with overexpression control and RNAi control, nMCSC infected overexpression control and RNAi control, the rate of lung metastasis was 2/6,3/6, 2/6 and 2/6, respectively. The nMCSC infected with RNAi control had one case of ear metastasis. The FUBP1 overexpressed nMCSC and MCSC generated an increasing metastasis of different organs and rate, including lung (4/6), thyroid (2/6), spine (1/6). MCSC infected with EFTU overexpression lentivirus showed lung (4/6) and subcutaneous/intestinal metastasis (1/6). nMCSC infected with EFTU overexpression lentivirus was similar to MCSC, the organ and rate of metastasis xenografts was lung (3/5), thyroid (2/5), Subcutaneous and breastbone (1/5). In contrast, the nMCSC and MCSC infected with FUBP1 or EFTU RNAi lentivirus generated metastasis xenografts only one case limited at lung, except for 2/6 of MCSC/FUBP1-RNAi group. These data suggested that FUBP1 and EFTU overexpression has the potential to promote metastasis of CRC stem cells.5 The immunohistochemical analysis of clinical colorectal cancer data showed that high expression of FUBP1 and EFTU was 53.73%(172/320),38.75%(124/320), respectively. EFTU high expression was associated with AJCC stage (x2=8.875, P=0.031) and metastasis (x2=5.334, P=0.021). FUBP1 high expression was associated with metastasis (x2=4.137, P=0.041). The The mean overall survival time for high FUBPl and EFTU expression group was shorter than low/negative FUBP1 and EFTU expression group, and the difference in survival was significant (P<0.001). Multivariate Cox ProPortional analyses of overall survival rate showed that the risk parameters of colorectal cancer prognosis had AJCC stage (OR=2.103, P=0.001), size (OR=0.671, P=0.039), FUBP1 expression (OR=1.911, P=0.002) and EFTU expression (OR=2.411, P=0.001). The expression of FUBP1 and EFTU served as an independent poor prognostic factor.Conclusion:1 The mRNA and protein level of FUBP1 and EFTU in MCSC of CRC is higher than that in nMCSC;2 We successfully constructed the overexpression and RNAi lentivirus of FUBP1 and EFTU and built the stable strains of nMCSC and MCSC beared the various lentivirus;3 In vitro experiments data suggeste that in nMCSC and MCSC, FUBP1 and EFTU enhances the proliferation capacity, colony forming capacity, invasion ability and migrating capacity;4 In vivo experiments data indicate that FUBP1 and EFTU may increase the tumor-initiating capacity and promote metastasis in nMCSC and MCSC of CRC.5 Clinicopathological data support that FUBP1 and EFTU high expression correlates with metastasis of CRC and can serve as an independent poor prognostic factor. |
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