The Role And Mechanism Of MicroRNAs In Hifu-Enduced Anti-Tumor Immunity

Backgrounds and objectivesHigh-intensity focused ultrasound (HIFU) is an emerging non-invasive technique which has been applied to treat solid tumors. The present studies have found that HIFU not only can eradicate primary tumors, inhibit tumor relapse and distant metastasis, also can trigger responsive systemic anti-tumor immunity. However, the precise mechanism of HIFU-enhanced anti-tumor immunity is sitll not fully elucidated.MicroRNAs (miRNAs) is a kind of endogenous non-coding RNA, which can regulate the expression of multiple targets by binding to the 3’UTR of target mRNAs and play an important role in the process of various life activities. Emerging evidence indicates that miRNAs have important roles in immune response and immune tolerance. In light of this, we highly postulated that miRNAs participated in the process of HIFU enhancing anti-tumor immune response. In order to validate the hypothesis, we first chose 8 miRNAs closely associated with immune response according to the related literature and bioinformatics analysis, including miR-34, miR-106a, miR-126a, miR-134,miR-155, miR-181a, miR-221, and miR-222. On the basis of verification that HIFU could enhance anti-tumor immunity, we used various of methods, including qRT-PCR, bioinformatics, luciferase report experiments and so on, to detect and validate the differential expression of miRNAs in spleen or tumor tissue after HIFU irradiation. And then the role and mechanism of these miRNAs in HIFU-enhanced anti-tumor immunity were explored, which further elucidated the mechanism of HIFU-enhanced anti-tumor immunity and provided more theoretical and experimental basis for the clinical application of HIFU treatment for tumor.Methods1. Construction of HIFU-treated murine model of B16 melanoma and validation of HIFU-enhanced anti-tumor immunity1) B16 melanoma models were constructed and then randomly divided into two groups:HIFU group and sham-HIFU group. The mice in the HIFU group were treated with HIFU at 9.3 M H z,4.5 W and in the sham-HIFU group were treated with a sham-HIFU procedure. The subsequent processing as follows:(1) mice were sacrificed after HIFU Oh and then the tumor tissues were removed and stained by HE staining to observe the ablation effect; (2) Seven days later, the single-cell suspension containing 1×106 B16 melanoma cells were injected into the tails veins and then the mice were followed up until death. During this period, the tumor volume, pulmonary metastasis nodules of tumor and survival rate were determined. (3) At 14th day after HIFU treatment, the mRNA levels of MAGE and Melan-A in the peripheral blood were detected by qRT-PCR; the levels of IFN-y and TNF-a in mice serum were detected by ELISA.2) Spleen lymphocytes from HIFU group and sham-HIFU group were isolated, dissected, and purified respectively. Purified splenic lymphocytes from each group were co-cultured with B16 cells in vitro, and then the cytotoxic activity of lymphocytes against B16 cells was measured by cytotoxicity experiment; the levels of IFN-y and TNF-a in co-culture supernatant were detected by ELISA.2. Screening of differentially expressed miRNAs in spleen caused by HIFU and identifing the target genes of these miRNAs1) miRNAs levels of spleen in HIFU group and sham-HIFU group were measured via qRT-PCR to find the differentially expressed miRNAs.2) The target genes of these miRNAs were predicted by bioinformatics method.3) The mRNA and protein levels of the target genes were detected by RT-PCR and western blot, respectively.4) 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 these miRNAs.5) Splenic lymphocytes were transfected with the mimics or miR-NC of thse miRNAs, then the target gene was detected at the levels of mRNA and protein.3. Screening of differentially expressed miRNAs in melanoma tumor tissue or B16 cells caused by HIFU and exploring the role and mechanism of these miRNAs in HIFU-enhanced anti-tumor immunity1) qRT-PCR was used to detect the differentially expressed miRNAs in tumor tissue at 14th day after HIFU treatment2) Bioinformatics softwares were utilized to predict the target genes of these miRNAs, and then the mRNA and protein levels of the target genes were detected by RT-PCR and western blot, respectively.3) 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 these miRNAs.4) B16 cells were transfected with mimics or miR-NC of these miRNAs, then the target genes were detected at the levels of mRNA and protein.5) B16 cells were irradiated by HIFU treatment systems:(1) B16 cells were exposed to HIFU at 4.5 W for 0,5,10 s, respectively. After evaluating the survival rate by trypan blue staining, B16 cells treated with HIFU were cultured in T25 flask for detecting the levels of miRNAs and target genes. (2) In addition, a fraction of B16 cells after HIFU treatment were co-cultured with B16-activated splenic lymphocyte, then the survival rate was assessed by cytotoxicity experiment and the levels of IFN-y and TNF-a in co-cultured supernatant were detected by ELISA.6) CD86 was stably knocked down by siRNA in B16 cells, and then the treated cells were exposed to HIFU and co-cultured with activated splenic lymphocytes. Finally, the survival rate was assessed by cytotoxicity experimentResults:1. HIFU can treat melanoma and enhance anti-tumor immunity1) HIFU treatment reduced the tumor size instantly and caused coagulation necrosis in the focused zone; the tumor volumes in the HIFU group were significantly smaller than the ones in the sham-HIFU group from 11 days after HIFU to the end of our study; the average number of tumor nodules in HIFU group was lower than that of sham-HIFU group (P<0.01) and the cumulative survival rate of HIFU-treated mice was statistically higher than that of the sham-HIFU group (P<0.01)2) HIFU treatment significantly reduced the mRNA levels of MAGE and Melan-A in the peripheral blood of mice(P<0.01), increased the levels of IFN-γ(P<0.05) in serum. No significant differences of TNF-a levels in each group were found (P>0.05), but there still was a trend showing a steady increase after HIFU treatment.3) HIFU treatment increased the cytotoxic activity of lymphocytes against B16 (P<0.05) and the levels of IFN-y (P<0.05) and TNF-α(P>0.05) in co-culture supernatant.2. HIFU can promote the expression of ICAM-1 by down-regulating miR-181 a in spleen1) Compared with sham-HIFU group, miR-181a level was significantly decreased in HIFU group (P<0.01)2) By bioinformatics prediction, the co-stimulatory molecule ICAM-1 might be a potential target gene of miR-181a.3) HIFU treatment significantly increased the ICAM-1 mRNA (P<0.01) and protein level of the splenic lymphocytes (P<0.01).4) miR-181a mimics significantly inhibited pLUC-ICAM-1-3’UTR luciferase activity (P<0.01) and significantly decreased the mRNA (P<0.05) and protein (P<0.01) levels of ICAM-1 in the splenic lymphocytes.3. HIFU enhances anti-tumor immunity by inhibiting the negatively regulatory role of miR-134 on CD86 in a murine model of B16 melanoma1)miR-134 (P<0.01) and miR-222 (P<0.01) were significantly decreased in tumor tissues after HIFU treatment.2) Bioinformatics indicated that costimulatory molecule CD86 and ICAM-1 was one of the potential target genes of miR-134 and miR-222, respectively.3) In tumor tissues of HIFU group:(1) CD86 levels were highly increased both in mRNA level (P<0.05) and protein level (P<0.05);(2) ICAM-1 levels were highly elevated both in transcriptional level (P<0.01) and post-transcriptional level (P<0.01)4) miR-134 mimics effectively inhibited luciferase activity of pLUC-CD86-3’UTR reporter plasmid,downregulating its target gene CD86 both in mRNA level (P<0.05) and protein level (P<0.05); However, miR-222 did not suppress luciferase activity of pLUC-ICAM-3’UTR reporter plasmid.5) HIFU iradiation efficiently decreased miR-134 and increased CD86 in B16 cells and there is a negative correlation between them.6) when B16 cells treated with HIFU were co-cultured with B16-activated splenic lymphocytes for 24 h,48 h:(1)HIFU efficiently decreased the survival rate of B16 cells (P<0.05); (2) HIFU increased the levles of IFN-γ (P<0.05) and TNF-α (P<0.05) in co-cultured supernatant.7) CD86 knockdown using RNA interference markedly rescued viability of HIFU-treated B16 cells (P<0.01) when co-cultured with lymphocytes.Conclusions1) HIFU irradiation not only eradicates primary tumors, inhibits tumor growth and distant metastasis, and improves host survival rate, also can trigger responsive systemic anti-tumor immunity.2) HIFU irradiation can inhibit the negatively regulatory role of miR-134 on costimulatory molecule CD86 and then enhance the anti-tumor immunity.3) HIFU irradiation can inhibit the negatively regulatory role of miR-181a on costimulatory molecule ICAM-1 in spleen, which is possibly one of the mechanism of HIFU-enhanced anti-tumor immunity.This study provides a new train of thought and experimental basis for elucidating the mechanism of HIFU-enhanced anti-tumor immunity,and has a promoting effect for the clinical application of HIFU treatment for tumor.