MiR-221调控CXCL12/CXCR4生物轴诱导NKT细胞浸润致HBV相关HCC的发病机制

The HBV infection is related to Hepatocellular Carcinoma (HCC ),but the pathogenic mechanism is not clear yet. We have found HBX protein could upregulate the miR-221 to induce HCC, which has been published. HBX protein could upregulate CXCL12/CXCR4 axis. Bioinformatics analysis demonstrated that the 3’-UTR of human CXCL12 contains a conserved binding site for miR-221 in different species. Our experiment showed that CXCL12 in HepG2 cells transected miR-221 was up-regulated.The receptor of CXCL12 was CXCR4 which expressed on the NKT cell.NKT cell could induce liver cirrhosis on the HBV transgenic mice model. Our experiment showed the NKT ratio in liver tissue of HBV-related HCC was higher than the HCC without HBV infection. The programme may employ HBV-related HCC mouse as research model, and use the method and technology such as luciferase assay, EMSA and CHIP and so on to elucidate the molecular mechanism that HBX protein regulate the miRNA-221/ CXCL12/CXCR4 to induce HBV-related HCC .The research can provide the new molecular target in clinical HCC therapy.

HBV感染与肝细胞肝癌（HCC）的发生有关，但致病机制尚不清楚。我们前期发现HBX蛋白可以上调miR-221导致HCC的发生，相关成果已发表SCI论文。HBX能上调CXCL12/CXCR4生物轴，生物信息学分析显示miRNA-221存在与趋化因子CXCL12转录调控序列相结合的结构基础。预实验表明转染miR-221的HepG2细胞中的CXCL12表达上调。CXCL12的特异性受体CXCR4表达在NKT细胞。在HBV感染的小鼠模型中，NKT细胞参与介导肝硬化过程。预实验发现HBV感染的肝癌组织中的NKT细胞比例明显高于未感染HBV的肝癌组织。本项目以罹患HBV相关HCC的小鼠为模型，拟采用免疫印迹法，荧光素酶活性分析、流式细胞术，免疫组织化学染色和CHIP等方法探讨miR-221通过调控CXCL12/CXCR4致NKT细胞浸润导致HBV相关HCC发生的分子机制，为HCC的防治开辟新的途径。

在我国，慢性HBV感染是HCC的首要原因。我们之前发现HBx上调肝癌细胞中的miR-221,但HBx调控miR-221后引起HCC的机制仍未清楚。Qpcr和Western Blot表明将HBX转染HepG2 细胞，miR-221，CXCL12，CXCR4上调表达，促进细胞增殖，S期细胞增多。但用抗CXCR4抗体后，细胞增殖减弱，S期细胞减少，说明HBX能上调CXCL12/CXCR4轴。虫荧光素酶实验，Qpcr和Western Blot表明CXCL12是miR-221的顺式作用元件，miR-221上调CXCL12表达。进行裸鼠成瘤实验， qPCR, Western Blot和免疫组化表明HBX转染HepG2细胞后，促进肿瘤的生长，HBX， miR-221，CXCR4，CXC12表达上调，但加入抗CXCR4抗体治疗后，肿瘤生长减慢，HBX， miR-221，CXCR4，CXC12表达下调，表明HBX上调miR-221后，继而上调CXCR4/CXC12轴促进HepG2-HBX肿瘤生长。免疫荧光显示肿瘤组织中，HBX上调CXCR4和NKT细胞的表达，但抗CXCR4抗体治疗后，CXCR4和NKT细胞的表达下调，表明HBX通过CXCR/CXCL12轴上调肿瘤微环境中的NKT细胞。ELISA检测发现HBX上调小鼠CXCL12，IL-2，TNF-α的表达。收集HBV相关的HCC病人和对照组的血清， ELISA显示HBV相关的HCC患者的CXCL12，IL-2，IL-6，TNF-α的水平升高。ROC曲线表明将CXCL12, IL-2 或IL-6联合使用，诊断价值更高。qPCR结果显示HBV相关的HCC患者血清中的CXCL12与miRNA-221表达上调。流式细胞术发现HBV相关的HCC患者外周血中NKT细胞高于正常对照组。综上研究表明HBX上调miR-221，激活CXCL12-CXCR4轴，招募NKT细胞，分泌炎性因子，促进HCC发生。