HIFU通过下调miR-181a进而促进ICAM-1表达增强小鼠抗肿瘤免疫

Role and mechanism of miR-181a in high-intensity focused ultrasound-induced antitumor immunity in mice

目的探讨miR-181a在高强度聚焦超声(high-intensity focused ultrasound,HIFU)增强机体抗肿瘤免疫中的作用及其机制。方法 1皮下构建供HIFU治疗的黑色素瘤移植瘤小鼠模型,采用简单随机法随机分为HIFU组与荷瘤组,每组30只,采用MTT方法检测各组脾淋巴细胞对B16细胞的杀伤活性,ELISA检测小鼠血清及共培养上清中TNF-α及IFN-γ的表达水平;2qRT-PCR检测HIFU组与荷瘤组脾淋巴细胞中的差异miRNAs;3用生物信息学方法预测差异miRNAs的潜在靶基因,通过RT-PCR及Western blot验证靶基因的表达;4构建荧光素酶报告质粒进一步验证差异miRNAs的靶基因,再将差异miRNAs的mimics转染脾淋巴细胞,RT-PCR及Western blot检测其靶基因表达的变化,以进一步确认差异miRNAs对靶基因表达的影响。结果 1HIFU治疗能够抑制黑色素瘤生长,促进脾淋巴细胞分泌TNF-α和IFN-γ,增强脾淋巴细胞对肿瘤细胞的杀伤活性;2HIFU处理明显下调脾淋巴细胞中miR-181a的表达水平(P<0.01);3生物信息学方法预测发现共刺激分子细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)可能是miR-181a的潜在靶基因,且HIFU处理明显促进脾淋巴细胞中ICAM-1的mRNA和蛋白质的表达(P<0.01);4miR-181a mimics显著抑制pLUC-ICAM-1-3'UTR报告质粒荧光素酶的活性(P<0.01),并下调脾淋巴细胞中ICAM-1的mRNA(P<0.05)和蛋白质的表达(P<0.01)。结论 HIFU通过抑制miR-181a对共刺激分子ICAM-1的负调控作用从而增强荷瘤小鼠的抗肿瘤免疫。

Objective To investigate the role and mechanism of miR-181 a in anti-tumor immunity induced by high-intensity focused ultrasound （HIFU） in mice. Methods B16 melanoma models were established in C57BL/6J mice （aging 6 to 8 weeks, weighing 20 ~ 24 g） , and the 60 tumor bearing mice （ tumor mass 7 mm） were randomly divided into 2 groups ： HIFU group and tumor-bearing group. The mice in the HIFU group were treated with HIFU （3 MHz, 4.5 W, for 10 s every time, totally 120 s） and those in the tumor-bearing group were treated with a sham-HIFU procedure. In 14 d after the treatment, 20 mice of each group were sacrificed and their blood, spleen and tumor tissues were harvested. MTT assay was used to detect the cytotoxicity of splenic lymphocytes against B16 melanoma cells, and ELISA was used to measure the contents of TNF-α and IFN-γin the collected serum and in the supernatant of cocuhured splenic lymphocytes and B16 cells, qRT-PCR was used to identify the miRNAs levels of splenic lymphocytes from the HIFU group and the tumor-bearing group for the differentially expressed miRNAs. The target genes of the differentially expressed miRNAs were predicted by bioinformatics method, and then the mRNA and protein levels of the target genes were detected by RT-PCR and Western blotting, respectively. The luciferase report plasmids containing the sequences of 3＇UTR or mutant 3＇UTR of the target gene were constructed to verify the target gene of differentially expressed miRNAs. Splenic lymphocytes were transfected with the mimics or miR-NC of differentially expressed miRNA, and then the target gene was detected at mRNA and protein levels. Results HIFU treatment inhibited tumor growth and promoted the secretion of TNF-α and IFN-γ in splenic lymphocytes, which enhancing the cytotoxicity of splenic lymphocytes against B16 melanoma ceils. HIFU obviously decreased the mRNA level of miR-181a （P 〈 0. 01 ）. By bioinformatics prediction, the costimulatory molecule, intercellular cell adhesion molecule-1 （ICAM 1 ）, might be a potential target gene of miR-181a, and HIFU treatment significantly increased the expression of ICAM-1 at mRNA （P 〈0.01 ） and protein levels in the splenic lymphocytes （P 〈0. 01 ）. miR-181a mimics significantly inhibited the activity of pLUC-ICAM-1-3＇UTR luciferase （P 〈0.01 ） in the HEK293 cells and significantly decreased the mRNA （P 〈 0.05 ） and protein （P 〈 0. 01 ） level of ICAM-1 in the splenic lymphocytes. Conclusion HIFU inhibits the negatively immunity regulatory role of miR-181a for costimulatory molecule ICAM-1 and then enhances the anti-tumor in tumor bearing mice.