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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: To investigate the effect of viral macrophage inflammatory protein Ⅱ (v MIP-Ⅱ) on CXCR4 expression in breast cancer cells and its molecular mechanism. Methods: The gene sequence of CXCR4 core promoter was designed and synthesized. The luciferase reporter vector was constructed and identified by restriction enzyme digestion. The reporter gene luciferase activity was used to detect the effect of CX21 on the CXCR4 The effect of CXCR4 interference or overexpression, HER-2 interference, and the expression of miRNAs and miRNAs after transfection of miRNAs inhibitors and analogues were detected by real-time quantitative PCR and Western blot. Cell proliferation was detected by staining, cell cycle was detected by PI staining and apoptosis was detected by Annexin V / PI staining. Results: NT21MP downregulated the expression of CXCR4 and up-regulated the expression of miR-125b, miR-135a and miR-146a (P <0.05 ~ P <0.01); NT21MP reduced the activity of CXCR4 core promoter; MiR-135a could down-regulate the expression of SP1 (P <0.01), up-regulate the expression of HER-2 (P <0.01) and up-regulate the expression of CXCR4 (P <0.01) MiR-146a and miR-146b inhibited the expression of CXCR4 (P <0.01); Compared with the blank control group, the expression of miR-146a and miR- 135a could inhibit cell proliferation, block cell cycle and induce apoptosis (P <0.01). Conclusion: NT21MP negatively regulates the expression of CXCR4 by inducing miR-125b, miR-135a and miR-146a, thereby inhibiting the proliferation of breast cancer cells and providing theoretical basis for the treatment of breast cancer patients with NT21MP.