| Backgroud and objectivesMelanoma has the highest mortality in skin diseases, which prone to metastasis at the early stage. Metastasis is an important factor affecting survival and prognosis in patients with the disease, but also the biggest problem of its treatment. Therefore, an effective treatment for melanoma metastasis is urgent to improve the survival of melanoma patients.High Intensity Focused Ultrasound (HIFU) is a non-invasive means of thermal ablation for tumors. This technology began to rise from 1990 in our country. It takes advantages of ultrasound, let the low-energy ultrasound focus on tumor tissue and kill tumor cells accurately through heat effect and the others, without damage to surrounding normal tissue. Currently HIFU technology is widely used in uterine-fibroids, breast cancer and other benign and malignant solid tumors, and its treatment achieved remarkable results. But for HIFU applications in melanoma treatment and its mechanisms had few study so far.microRNAs(miRNAs) is a class of about 18 to 25 nucleotide sequence of the endogenous small non-coding small RNA, by regulating the expression of the target genes at post-transcriptional level, and they participate in the incidence and development of malignant tumors. In recent years, some evidence proved that the biological effects of miRNAs in physical therapy, such as radiation tolerance and tumor radiation therapy-induced apoptosis. miRNAs may become a potential new target to improve radiation therapy for tumor. Many studies focused on microRNA-21(miRNA-21). The study about the mechanisms of HIFU exposure focused on the physical mechanisms such as thermal coagulation effect and cavitation effect, but had few studies about other small moleculars like miRNAs.The effects of HIFU ablation on the primary tumor had no doubt. But in clinical ablation process, due to equipment, imaging guild, operator techniques and tumor location, tumor size and other factors, the complete ablation of tumor is relatively difficult, so the residual cells are hard to avoid, and then the biological behavior of the residual tumor cells is one of the key factors affecting treatment and prognosis. Some studies have reported that radio frequency ablation of liver cancer promotes the proliferation of residual liver cells. So after HIFU ablation, what changes will happen to proliferation, apoptosis of the residual tumor cells, and their metastatic ability, which is the key impact to the prognosis of patients. These answers are essential to HIFU ablation for melanoma.Therefore, this study was to investigate the changes on proliferation, apoptosis and migration of the residual melanoma cells (RMCs) after HIFU exposure in vivo and vitro, especially the change and potential mechanisms of metastasis, which play a key role in the prognosis of patients with melanoma. Our study is to provide an experimental basis for the clinical application of HIFU ablation in melanoma.Methods1.To investigate the effects of HIFU ablation on growth and metastasis of the RMCs in mouse model:1)We built subcutaneous melanoma models, did HIFU or sham-HIFU exposure, recorded the volume of subcutaneous melanoma, did TUNEL test to assess the number of melanoma cells undergoing apoptosis.2)We recorded metastasis rate and the number of metastases to represent the metastatic ability of RMCs. The number of melanoma cells in mice blood circulation was detected by qPCR, in order to reflect the ability of leaving from primary site into the blood circulation of residual cells. Meanwhile, to test the lung colonization capacity of RMCs after HIFU ablation, we injected the same cell line of melanoma cells intravenously on the 14th day after HIFU ablation. The expression of molecules associated with tumor metastasis, including E-cadherein, Vimentin, VEGF and MMP9, was detected by western blot, immunohistochemistry.2.We exposed murine melanoma B16-F10 cells in vitro on HIFU for 1s,2s, 3 s and then kept culturing them for 12h. The residual cells were adherent and used to investigate the effects of HIFU exposure on cell survival, proliferation, apoptosis and migration of RMCs by trypan blue dye test, MTT assay, flow cytometry, wound healing assay and transwell cell migration assay respectively.3. To explore the mechanisms of migration/metastasis inhibition of the RMCs after HIFU treatment:1)The changes of miRNA-21, PTEN expression and the activation of AKT signaling pathway in residual tissues and cells after HIFU exposure were tested by qPCR, western blot, and immunonistochemistry assay.2)miRNA-21 targets PTEN in B16-F10 cells was verified by luciferase activity reports and western blot.3) The roles of miRNA-21 and PTEN in migration inhibition were tested by wound healing assay and transwell cell migration assay.Results1.HIFU ablation prolonged survival time, suppressed growth and reduced lung metastasis of subcutaneous melanoma mouse model.1) Compared with the control group, HIFU ablation prolonged survival time of these mice(P<0.0001). The tumor volume had no difference immediately after HIFU ablation between two groups, but the difference began to appear significantly at 20th day(P<0.05, n= 15). The tumor volume of HIFU group was significantly smaller than that of the control group. HIFU exposure increased the number of apoptotic cells in residual tissues (P<0.01).2) In the control group, the lung metastasis rate was 20%(3/15), and the number of black lung metastases in each mouse was 1.67±0.58. But in HIFU group, only one lung metastasis in tissue sections was found under the microscope after H-E staining, which was not visible with naked eyes, so the HIFU group metastasis was 6.7%(1/15).3) qPCR showed that after HIFU ablation for 7 days, the expressions of MAGE-A3, MART-1 and PAX3 were 3.183±0.323,1.416±1.374, and 1.654±1.290 in control group, and 0.578±0.482,0.822±0.309, and 0.794±0.356 in HIFU group,respectively. The three markers in HIFU group was lower than that in control group. At the 14th day after HIFU ablation, the MAGE-A3 level was still much lower in HIFU group(P<0.05),4.852±2.583 in control group,0.882±0.514 in HIFU group. It means that HIFU ablation decreased the number of circulating melanoma cells.4) Intravenous injection of B16-F10 cells at 14th days after the HIFU treatment, the number of lung metastases was 26.4±6.6 in each mouse, in the control group was 45.8±7.6(P<0.05, n=10). It means that HIFU ablation could lead the inhibition of circulating tumor cells colonized in lung.5) E-cadherin, a epithelial cell marker, was increased after HIFU ablation (P<0.05), and the mesenchymal cell marker Vimentin was lower (P> 0.05). Meanwhile, the expressions of VEGF and MMP9 were significantly lower than the control group (P<0.05). It means that HIFU ablation could reverse EMT partially and reduce the expression of VEGF, MMP9 at RMCs.2.Compared with the control group, the alterations of survival rate, proliferative capacity and the number of apoptotic cells of RMCs after HIFU exposure for Is,2s and 3s had no significant difference(P> 0.05). The wound healing rate was decreased (P<0.001) and the number of penetrating cells was decreased (P1s<0.05, P2s<0.001, P3s<0.001) at 48h after HIFU exposure. It means that HIFU exposure inhibited the migration of RMCs.3.HIFU exposure inhibited migration of residual murine melanoma cells by the down-regulating miRNA-21 and up-regulating PTEN.1) In the residual tumor tissues and B16-F10 cells, after HIFU exposure:(1)the relative expression of miRNA-21 was decreased (P<0.001,P<0.05).(2)The expression of PTEN was increased (P< 0.05,P< 0.05).(3)The levels of pAkt(T308)/t-Akt and p-Akt(S473)/t-Akt were reduced (P <0.05, P<0.05). It means that HIFU treatment down-regulated the expression of miRNA-21, up-regulated the expression of PTEN and thus inhibited the activation of AKT pathway.2) The luciferase activity report showed that the luciferase activity was decreased after transfecting miRNA-21 mimic and PTEN-3’UTR region predicted binding fragment, especially the complementary fragment number 1; while the luciferase activity after transfecting miRNA-21 negative control or PTEN-3’UTR mutant was not significantly decreased. It means that miRNA-21 directly targets PTEN in B16-F10 cells.3) Wound healing assay showed that scratch healing rate was significantly lower after transfecting anti-miRNA-21 and HIFU exposure (P<0.01), and transwell cell migration assay showed that the number of penetrating cells was reduced (P<0.01). These changes of wound healing rate and the number of penetrating cells partially restored while transfecting anti-miRNA-21 and psiPTEN. It means that HIFU exposure inhibited migration of residual murine melanoma cells through the down-regulating miRNA-21 and up-regulating PTEN.Conclusion1. HIFU ablation could prolong survival time, inhibit melanoma growth, promote apoptosis in residual melanoma cells.2.HIFU treatment could inhibit the migration and metastasis of residual melanoma cells.3.HIFU ablation could inhibit the tumor cells of the primary site into the blood, which may be related to the reversal of EMT partially in residual cells; at the same time, HIFU ablation could inhibit colonization of circulating tumor cells in lung.4.The one of the mechanisms of migration inhibition after HIFU exposure is through miRNA-21 down-regulation, increased PTEN expression and decreased AKT activity. Our study identifies a miRNA-21/PTEN/AKT pathway involved by HIFU for the inhibition of melanoma cell metastasis.This study lays a preliminary foundation for the study of metastasis inhibition mechanisms after HIFU ablation and the clinical application of HIFU treatment for melanoma. |
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