Effects Of ?1Integrin (ITGB1) On Proliferation And Apoptosis Of Human Colorectal Cancer

BackgroundColorectal carcinoma ?CRC? is one of the most common malignant diseases worldwide and the prognosis is still very poor. The incidence of CRC has been increasing during recent decades, and the lifetime risk for CRC in industrialized countries is about5%. Early diagnosis and better treatment of CRC requires the identification of new biomarkers as well as insights into the molecular mechanisms of CRC.Integrins are18different a and8different ? subunits heterodimeric transmembrane receptors for ligands in the extracellular matrix, and they are essential for both cell adhesion and activation of intracellular signaling pathways. At least100different splice variants have been identified for some integrin subunits. Integrins have been associated with many aspects of embryonic development, such as adhesion, cell polarity, cell proliferation and apoptosis, tumor formation and metastasis.?1integrin ?ITGB1? is the mainly expressed integrin in normal cells and in tumor-associated cells, in which they control various developmental processes including angiogenesis, tumor progression, apoptosis and metastasis. Currently, the molecular mechanisms underlying the transcriptional regulation of ITGB1expression in CRC remain poorly understood.Given the potential importance of ITGB1in tumorigenesis, we used recombinant lentiviral vectors to generate an expressing ITGB1or ITGB1-specific RNAi human colonic cancer cell lines. We aimed to determine if ITGB1may regulates apoptosis and proliferation on the development of CRC both in vitro and in vivo.Methods1. Three precursor shRNA sequences ?SRI-3? targeting ITGB1?GenBank accession number:NM₁33376? were used. GV248lentiviral vectors ?Genechem, Shanghai, China? were constructed with ITGB1-siRNA or the negative control ?NC?. Human ITGB1was inserted into GV218lentiviral vectors for up-regulating expression of ITGB1.2. Four human CRC cell lines HCT116, HT29, SW480and LoVo cells were used. They were examined by Real time RT-PCR and western blot, and the expression levels in four CRC cell lines were compared. 3. The HT29cell line which expressed lower levels was chose to be transfected the recombinant lentivirus vectors. To produce a stable transfected cell line, the cells were cultured in a selection medium containing puromycin for2weeks.4. After infecting HT29cells in vitro, expression of ITGB1was evaluated by real time RT-PCR, western blot, flow cytometry and immunofluorescence.5. The proliferation was evaluated by CCK-8and colony formation assay. The apoptosis was evaluated by Hochest33258staining and flow cytometry. Cell cycle distribution was assessed with flow cytometry. The impact of ITGB1expression on the invasiveness of HT29cells was measured using the transwell invasion assay. We assessed the effects of Bcl-2, Bax, Caspase-3, Caspase-9, cyclin D1, P21protein and the proteins related to the Hedgehog ?Hh? signaling pathway by western blotting in HT29cells.6. The influence of lentivirus infection on the tumor development capacity of HT29cells in vivo was examined by xenografting the tumor cells. At the end of the experiment, tumors were harvested and weighed. To confirm the effect of ITGB1in vivo, we dissected the tumors and conducted immunohistochemical analysis. The TUNEL technique was performed to detect and quantitate apoptotic cell death of xenograft tumor.Results1. GV248and GV218were used as recombinant lentivirus vectors. The DNA sequencing results identified that the inserts of three pre-shRNA sequences ?ITGB1-SR1, ITGB1-SR2and ITGB1-SR3? targeting to ITGB1were correct and no mutant was found in the recombinant plasmid expression vectors ITGB1-siRNA. Real time PCR and western blot analysis showed that SR1yielded the best suppression efficiency. Therefore, this construct was chosen to package the recombinant lentiviral vector for ITGB1RNAi with the GV248expression vector. The eGFP was used as a maker to detect whether the recombinant lentivirus vectors were successfully transduced in vitro and in vivo. The recombinant lentivirus could direct transferred into293T cells. Of note, the transfection efficiency is more than90%.2. We used real time RT-PCR and western blot to evaluate the expression of ITGBl in several human colorectal cancer cell lines and found that the LoVo cells expressed the lowest amount of integrin of the four cell lines tested. We chose the HT29cell line for our studies because it only moderately expressed ITGB1compared to the HCT116cell line, which had the highest levels of native ITGB1expression.3. Stably transfected cells were cultured and amplified in culture medium supplemented with2.5?g/ml puromycin. The transfected cells were called HT29-RNAi, HT29-ITGB1and HT29-NC. Real time RT-PCR, western blot, flow cytometry and immunofluorescence assay revealed that the level of ITGB1transcripts in HT29-RNAi cells were significantly decreased ?P<0.05?. The level of ITGB1transcripts in HT29-ITGB1cells was increased compared to HT29and HT29-NC cells ?P<0.05?. As expected, infection with the control lentivirus did not affect the expression of ITGB1, as similar levels of ITGB1mRNA transcripts were found in HT29-NC cells and parent HT29cells.4. The proliferative and migrate capacity of HT29-ITGB1cells increased than HT29and HT29-NC cells ?P<0.05?. The HT29-RNAi cells showed a higher apoptosis rate up to ?30.67%±5.1%??P<0.05? compared with HT29?12.1%±0.9%? and HT29-NC cells ?10.8%±1.3%?. However, the HT29-ITGB1cells only have an apoptosis rate of ?12.3%±2.5%?, and as we expected, there is no difference between HT29and HT29-NC cells ?P>0.05?. The levels of Bcl-2and cyclinD1proteins were up-regulated while Bax, Caspase-3, Caspase-9and P21levels were down-regulated in HT29-ITGB1cells compared to controls. The levels of Glil, Shh and c-Myc proteins were up-regulated while SuFu levels were down-regulated in HT29-ITGB1cells compared to controls. Taken together, these findings indicate that ITGB1activates Hh signaling in human HT29cells. However, the results were opposite in HT29-RNAi cells.5. Expression of Hugl-1in tumor tissues was analyzed by immunohistochemistry. Many cells were strongly positive for ITGB1in the HT29-ITGB1tumor sections. The solid tumors were first visible approximately5days post inoculation and grew rapidly afterwards in the HT29-ITGB1group ?1454±95.65mm3?. In contrast, the HT29-RNAi group had slower solid tumor growth rates and smaller mean tumor volumes ?889,3±61.49mm3? compared to the control groups ?1162±144.8mm3and1174±58.41mm3, P<0.05?. TUNEL staining showed markedly more positive cells in the HT29-RNAi group ?84.3%±4.0%?, which were more significant than in the HT29-ITGB1group ?48.3%±2.9%??P<0.05? and the other two control groups, HT29group ?52.0%±3.6%? and HT29-NC group ?49.7%±4.5%?. ConclusionThe overexpression of ITGB1could induce the growth and migrate of HT29cells. These results suggest that ITGB1regulate growth and apoptosis in a human colorectal cancer cell line through the mitochondria signaling pathway.