探讨了PinX1 基因在乳腺癌MCF-7 细胞生长和细胞周期中的作用, 初步探讨了该基因用于乳腺癌临床治疗的可行性. 采用RT-PCR 技术从293-T 细胞中扩增PinX1 基因, 将其克隆入真核表达载体pEGFP-C1 中, 再将重组质粒转染MCF-7 细胞. 通过real-time PCR 检测PinX1 基因的mRNA 表达, 用MTT 法检测转染前后细胞生长曲线的变化, 用流式细胞仪检测转染目的基因后细胞生长周期的改变. 检测结果表明, PinX1 基因已经在转染后MCF-7 细胞的细胞核内稳定表达, 乳腺癌细胞生长明显减缓(P <0.05), 增殖变慢(P <0.05), 细胞生长阻滞于G0/G1 期, 说明PinX1 基因可抑制乳腺癌MCF-7 细胞的生长和增殖.
This paper explores the effects of PinX1 gene on the growth and the cell cycles of breast carcinoma cell line MCF-7 and the potential in treatment of breast cancer. The PinX1 mRNA was amplified from 293-T cell with RT-PCR and inserted into pEGFP-C1 vector. The recombined eukaryotic expression vector of PinX1 was confirmed by enzyme digestion, and transfected into the breast carcinoma cell line MCF-7 using a liposome method. The expressed mRNA was detected by real-time PCR, and the cell growth and cell cycles by MTT and flow cytometry, respectively. Results showed that PinX1 transfection into the breast cancer MCF-7 cell line was stable and the transfected gene was integrated into nucleolus DNA of transfected cells. Transfection of PinX1 gene could significantly inhibit the growth and proliferation of breast cancer cells (P < 0.05) and the cell growth was arrested at G0/G1 stage. The conclusion is that PinX1 gene could inhibit growth and proliferation of breast cancer cells.
[1] Blasco M A. Telomeres and human disease: ageing, cancer and beyond [J]. Nat Rev Genet, 2005, 6(8): 611-622.
[2] Zhou X Z, Lu K P. The Pin2 /TRF1 interacting protein PinX1 is a potent telomerase inhibitor [J]. Cell, 2001, 107 (3): 347-359.
[3] Liao C, Zhao M J, Song H, et al. Identification of the gene for a novel liver-related putative tumor suppressor at a high frequency loss of heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma [J]. Hepatology, 2000, 10: 721-722.
[4] 孙文旦. PinX1 基因真核表达载体的构建及其在Hek293 细胞中的表达[J]. 临床检验, 2009, 27: 445-447.
[5] Won S P, Jong H L, Jik Y P, et al. Genetic analysis of the liver putative tumor suppressor gene in hepatocelluar carcinomas [J]. Cancer Let, 2002, 178(2): 199-207.
[6] de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres [J]. Genes Dev, 2005, 19(18): 2100-2110.
[7] van Steensel B, De Lange T. Control of telomere length by the human telomeric protein TRF1 [J]. Nature, 1997, 385: 740-743.
[8] Chong L, Van S B, Broccoli D, et al. A human telomeric protein [J]. Science, 1995, 270(5242): 1663-1667.
[9] Ancelin K, Brunori M, Bauwens S, et al. Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2 [J]. Mol Cell Biol, 2002, 22(10): 3474-3487.
[10] Soohoo C Y, Shi R, Lee T H, et al. Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation [J]. J Biol Chem, 2011, 286: 3894-3906.
[11] Zhou X Z, Huang P, Shi R, et al. The telomerase inhibitor PinX1 is a major haploinsufficient tumor suppressor essential for chromosome stability [J]. J Clin Invest, 2011, 121: 1266-1282.
[12] Chen G, Da L, Wang H, et al. HIV-Tat-mediated delivery of an LPTS functional fragment inhibits telomerase activity and tumorigenicity of hepatoma cells [J]. Gastroenterology, 2011, 140(1): 332-343.
[13] Zhang L, Jiang Y, Zheng Y, et al. Selective killing of Burkitt’s lymphoma cells by mBAFF-targeted delivery of PinX1 [J]. Leukemia, 2011, 25(2): 331-340.
[14] Wang H B, Wang W Q, Wang X W, et al. PinX1 gene transfection enhances the sensitivity of gastric carcinoma cell line to 5-fluorouracil [J]. Hepatogastroenterology, 2011, 58(106): 682-686.
[15] Harley C B. Telomerase and cancer therapeutics [J]. Nat Rev Cancer, 2008, 8(3): 167-179.
[16] Joseph I, Tressler R, Bassett E, et al. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines [J]. Cancer Res, 2010, 70(22): 9494-9504.
