Hong Fei, Zhang Huan-Xin, Chen Chong, Yan Zhi-Ling, Wu Qing-Yun, Zeng Ling-Yu, Li Zheng-Yu, Xu Kai-Lin
Institute of Hematology, Xuzhou Medical University, Xuzhou 221002, Jiangsu Province, China.
Institute of Hematology, Xuzhou Medical University, Xuzhou 221002, Jiangsu Province, China; Department of Hematology, Xuzhou Medical University Affiliated Hospital, Xuzhou 221002, Jiangsu Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2018 Jun;26(3):691-697. doi: 10.7534/j.issn.1009-2137.2018.03.010.
To investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism.
Specifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and β-catenin were analyzed by Western blot.
The recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total β-catenin protein, as well as the nuclear β-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of β-catenin protein.
探讨稳定下调血管内皮钙黏蛋白（VE-cadherin）表达对K562细胞化疗敏感性的影响，并探讨其可能机制。
设计针对人VE-cadherin的特异性干扰序列，构建含IRES-GFP和NEO片段的重组慢病毒载体；采用三质粒包装系统产生重组慢病毒，并转染K562细胞，然后分选稳定下调的细胞。采用CCK-8法检测K562细胞对化疗药物（伊马替尼）的敏感性。通过Annexin V/7-AAD双标记流式细胞术分析细胞凋亡情况。采用实时PCR检测CD133和ALDH1 mRNA的表达。通过Western blot分析VE-cadherin、BCR-ABL和β-连环蛋白的蛋白表达。
成功构建重组慢病毒载体pLB-shVEC-NEO-IRES-GFP，并包装成慢病毒，获得稳定下调VE-cadherin表达的K562细胞。当K562细胞中VE-cadherin表达下调时，细胞增殖率降低，凋亡率增加；CD133和ALDH1的mRNA水平也降低；BCR-ABL融合蛋白无明显变化；K562/shVEC细胞中总β-连环蛋白及核内β-连环蛋白均减少。结论：下调VE-cadherin表达时K562细胞对化疗更敏感，这可能是通过降低β-连环蛋白的稳定性及核转位实现的。
