vMIP-Ⅱ N端肽对趋化因子受体CXCR4表达调控的机制研究

The study of chemokine receptors CXCR4 expression regulation by N-peptide of v MIP-Ⅱ

目的：探讨病毒巨噬细胞炎性蛋白Ⅱ（v MIP-Ⅱ）对乳腺癌细胞CXCR4表达的影响及分子机制。方法：设计并合成扩增CXCR4核心启动子基因序列,构建荧光素酶报告载体,酶切鉴定;通过检测报告基因荧光素酶活性反映病毒巨噬细胞炎性蛋白N端21肽（NT21MP）对CXCR4启动子的作用;通过荧光定量PCR技术和Western blot检测CXCR4干扰或过表达、HER-2干扰,以及miRNAs抑制剂和类似物转染后相关基因及miRNAs表达;采用磺酰罗丹明B（SRB）染色法检测细胞增殖活性,PI染色法检测细胞周期,Annexin V/PI染色法检测细胞凋亡。结果：NT21MP下调CXCR4的表达,并上调miR-125b、miR-135a和miR-146a的表达（P〈0.05~P〈0.01）;NT21MP降低CXCR4核心启动子的活性;SP1为CXCR4核心启动子高频结合转录因子,miR-135a可下调SP1的表达（P〈0.01）;过表达CXCR4上调HER-2表达（P〈0.01）,而干扰CXCR4表达则下调HER-2表达（P〈0.01）;miR-125b可下调HER-2的表达（P〈0.01）;HER-2表达下调诱导miR-146a的表达;miR-146a抑制CXCR4表达（P〈0.01）;相对于空白对照组,miR-146a和miR-135a均可抑制细胞增殖,阻断细胞周期,并诱导凋亡（P〈0.01）。结论：NT21MP通过诱导miR-125b、miR-135a和miR-146a负调控CXCR4的表达,从而抑制乳腺癌细胞的增殖能力,为NT21MP应用于乳腺癌患者的治疗提供了理论基础。

Objective： The aim of this study is to investigate the effect of viral macrophage inflammatory protein II（ VMIP-II） on the expression of CXCR4 in the human breast cancer and the molecular mechanism. Methods： To designed and synthesized the sequence of CXCR4 core promoter were designed and synthesized,and established fireflyluciferase reported carrier; By detecting the fireflyluciferase activity of reported gene to reflect the effect of NT21 MP on CXCR4 promotor; Western blotting and qRT-PCR were used to assess the expression of the related gene and miRNAs after depletion or overexpression of CXCR4,depletion of HER-2 and transfected with miRNAs inhibitor and mimic; SRB assay was used to measure the cell viability,while PI staining and Annexin V / PI stainning was performed to analyze the cell cycle and apoptosis. Results： NT21 MP down-regulated CXCR4 and up-regulated miR-125 b,miR-135 a and miR-146a（ P〈0. 05- P〈0. 01）; NT21 MP inhibited the activity of CXCR4 core promoter; SP1 is a transcription factors,which connected with CXCR4 core promoter frequently,miR-135 a mimic down-regulated SP1（ P〈0. 01）; overexpression of CXCR4up-regulated HER-2（ P〈0. 01）,while depletion of CXCR4 down-regulated HER-2（ P〈0. 01）; miR-125 b down-regelated HER-2（ P〈0. 01）; the downexpression of HER-2 can induce the expression of miR-146a; miR-146 a inhibited CXCR4（ P〈0. 01）; compared to the blank group,miR-146 a and miR-135 a all can inhibit cell proliferation,arrest cell cycle and induce apoptosis. Conclusions： NT21 MP down-regulated the expression of CXCR4 by inducing miR-125 b,miR-135 a and miR-146 a,inhibited the proliferation ability of breast cancer cell,and promoted theoretical basis to NT21 MP applied to breast cancer treatment.