Objective      To investigate the role and mechanism of miR-181a 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 2

0~24 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-γ in the collected serum and in the supernatant of cocultured 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 cells. HIFU obviously decreased the mRNA level of miR-181a (P0.01). By bioinformatics prediction

the co-stimulatory 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 (P0.01) and protein levels in the splenic lymphocytes (P0.01). miR-181a mimics significantly inhibited the activity of p

LUC-ICAM-1-3′UTR

luciferase (P0.01) in the HEK293 cells and significantly decreased the mRNA (P0.05) and protein（P0.01)  level of ICAM-1 in the splenic lymphocytes.       Conclusion      HIFU inhibits the negatively regulatory role of miR-181a for costimulatory molecule ICAM-1 and then enhances the anti-tumor immunity in tumor bearing mice.