Wang Tian-Bao, Hu Bao-Guang, Liu Da-Wei, Shi Han-Ping, Dong Wen-Guang
Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, P.R. China.
Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong, P.R. China.
Int J Oncol. 2014 Jun;44(6):1861-9. doi: 10.3892/ijo.2014.2348. Epub 2014 Mar 19.
The aim of this study was to construct a lentiviral vector of CXCR4-siRNA (Lenti-CXCR4-siRNA) and investigate whether the vector can inhibit the growth, migration, invasion and hepatic metastasis of colorectal cancer (CRC). RT-PCR and western blotting were employed to identify the ideal RNA interference sequence. Lenti-CXCR4-siRNA was constructed and transfected into the SW480 cell line. We used RT-PCR and western blotting to measure the expression of CXCR4 RNA and protein, respectively; the MTS assay to assess the proliferation of SW480 cells; transwell chambers to estimate the inhibitory effect on migration and invasion; and the Balb/c nude mouse model of CRC to examine the inhibition of hepatic metastasis. The relative expression of the CXCR4 gene and protein was 5.4 and 18.95%, respectively, in the siCXCR4 group. The genes in the expression plasmid pLenti-CXCR4-siRNA were in the correct order. In the SW480, nonsense control (NC) and the Lenti-CXCR4-siRNA groups CXCR4 RNA levels were, respectively, 0.54±0.06, 1.00±0.03 and 0.11±0.04 (P=0.0001); CXCR4 protein levels were 0.60±0.03, 0.72±0.03 and 0.18±0.02 (P=0.0001); the OD value was 1.38±0.04 (P=0.0050), 1.28±0.05 (P=0.0256) and 0.92±0.06; SW480 cell number in migration test was 32±6.85, 32.63±1.69 and 0.75±0.71 (P=0.0000); SW480 cell number in the invasion test was 29.13±10.3, 30.38±6.09 and 0.63±0.74 (P=0.0000); hepatic metastasis number was 7.10±3.98 (P=0.034), 7.50±4.09 (P=0.019) and (3.50±2.51); hepatic metastasis mean weight (in g) was 2.25±2.51 (P=0.000), 2.11±2.38 (P=0.000) and 1.45±2.07. Lenti-CXCR4-siRNA constructs were correctly constructed and effectively inhibit the expression of CXCR4 RNA and protein, reducing the proliferation, migration, invasion capacity of SW480 cells and hepatic metastasis of CRC.
本研究旨在构建CXCR4-siRNA慢病毒载体（Lenti-CXCR4-siRNA），并研究该载体是否能抑制结直肠癌（CRC）的生长、迁移、侵袭及肝转移。采用RT-PCR和蛋白质印迹法鉴定理想的RNA干扰序列。构建Lenti-CXCR4-siRNA并转染至SW480细胞系。我们分别用RT-PCR和蛋白质印迹法检测CXCR4 RNA和蛋白质的表达；用MTS法评估SW480细胞的增殖；用Transwell小室评估对迁移和侵袭的抑制作用；用CRC的Balb/c裸鼠模型检测对肝转移的抑制作用。在siCXCR4组中，CXCR4基因和蛋白质的相对表达分别为5.4%和18.95%。表达质粒pLenti-CXCR4-siRNA中的基因顺序正确。在SW480、无义对照（NC）和Lenti-CXCR4-siRNA组中，CXCR4 RNA水平分别为0.54±0.06、1.00±0.03和0.11±0.04（P = 0.0001）；CXCR4蛋白质水平分别为0.60±0.03、0.72±0.03和0.18±0.02（P = 0.0001）；OD值分别为1.38±0.04（P = 0.0050）、1.28±0.05（P = 0.0256）和0.92±0.06；迁移试验中SW480细胞数分别为32±6.85、32.63±1.69和0.75±0.71（P = 0.0000）；侵袭试验中SW480细胞数分别为29.13±10.3、30.38±6.09和0.63±0.74（P = 0.0000）；肝转移数分别为7.10±3.98（P = 0.034）、7.50±4.09（P = 0.019）和（3.50±2.51）；肝转移平均重量（g）分别为2.25±2.51（P = 0.000）、2.11±2.38（P = 0.000）和1.45±2.07。Lenti-CXCR4-siRNA构建体构建正确，能有效抑制CXCR4 RNA和蛋白质的表达，降低SW480细胞的增殖、迁移、侵袭能力及CRC的肝转移。
