| Laryngeal caner(LC) is one of the commonest cancers in human. Laryngeal squamous cell carcinoma(LSCC) was the major pathological type. The current therapies include surgical removal of the tumor, chemotherapy and radiotherapy, or combination of these remedies. To date, the therapeutic effect is very poor, which largely relies on the early diagnosis and early treatment. Therefore, to develop novel therapeutics for the treatment is of significance. The tumor tolerance indicates a condition that the immune system does not respond to the tumor antigen stimulation, which endues tumor cells to escape from the immune surveillance and grow out. The immune suppressive cells producing immune regulatory molecules, play major roles in the tumor tolerance. Micro RNAs(mi RNA) are small non-coding RNA molecules. Published data indicate that mi RNAs are involved in the pathogenesis of cancer. Some of them are associated with the development of cancer. Thus, we hypothesize that mi RNAs may be involved in the tumor tolerance in LC. In this study, we observed that the expression of hsa-mi R-193 b in LSCC and the effect on the tumor immune regulation.Objective:We aim to study the expression of mi R-193 b in LSCC and the impact on the biological functions of laryngeal cancer cells. Further, we study the expression of several immune markers, such as IL-10, CD14, CD68 in LSCC with the clinicopathologic factors and investigate the their value in LSCC. Lastly, we verify the effect of mi R-193 b on tumor immune regulation.Methods:We used the qRT-PCR to identify the expression of mi R-193 b in LSCC. Cell proliferation was monitored using the MTT assay kit according to the manufacturer’s protocol. Cell cycle and cell apoptosis were analyzed immediately by flow cytometry. Cell invasion was monitored using the Transwell assay. Secondly, we study the expression of several immune markers, such as IL-10, CD14, CD68 in 46 primary LSCC tumor tissues with the clinicopathologic factors. Expressions of the markers were detected with immunohistochemistry staining. Thirdly, we analyzed the expression of mi R-193 b in the culture supernatant of LC cells isolated from the LSCC tissue. The immune cells mixing in the isolated single cells from the LSCC tissue were isolated out by the magnetic cell sorting with commercial reagent kits. IL-10-expressing monocytes were detedted with the Flow cytometry. We analyzed the effect of mi R-193 b on CD14+CD16-monocytes and generated IL-10+monocytes. CD8+CD25-T cells collected from healthy subjects were co-cultured with IL-10+ monocytes. Finally, the proliferation of CD8+T cells was assessed by flow cytometry and the cytotoxic cytokines in the culture supernatant by ELISA. All statistic analyses were performed with SPSS20.0.Results:We collected the surgically removed tumor tissues from 20 patients with LSCC, compared with corresponding pericarcinous tissues, the expression level of mi R-193 b was significantly enhanced in LSCC tissue. After 48 h of mi R-193 b inhibitor transfection, we observed a significant inhibition of Hep-2 cells growth. Flow cytometry revealed that mi R-193 b inhibitor significantly affected cell apoptosis and cell cycle of Hep-2 cells. The mi R-193 b inhibitor also inhibited the invasive property of Hep-2 cells. Secondly, we found that in T stage grouping, the overexpressions of IL-10, CD14 and CD68 were significantly higher in T3+T4 compared with T1+T2(all P<0.01). In clinical stage grouping, those were significantly higher in III+IV compared with I+II(all P<0.01). In histological differentiation grouping, the overecpression of CD68 increased significantly in poorly and moderately differentiated LSCCs compared with the well differentiated LSCCs(P<0.05). Meanwhile the overecpression of IL-10 and CD14 were significantly higher in LSCCs with lymph node metastasis compared with those without lymph node metastasis(both P<0.01). There is a positive correlation trend between the expression of IL-10 and CD14(P<0.01). Finally, we found that IL-10-expressing monocytes were detected in the LSCC tissue-derived single cells. Treating na?ve CD14+CD16-monocytes with mi R-193 b induced expression of IL-10 in the monocytes. Our data revealed that IL-10+ monocytes suppress effector CD8+T cells activities.Conclusion:In summary, the present data indicate that the mi R-193 b expression level is significantly enhanced in LSCC tissue. Acting as an oncogene, the mi R-193 b can promote cell growth and the invasive property of Hep-2 cells and inhibit cell apoptosis significantly. We confirm that the high level expression of IL-10, monocytes and tumor-associated macrophages contribute to the tumor development of LSCC and there is a positive correlation trend between the expression of IL-10 and monocytes. We also reveal that there is a fraction of IL-10+monocytes in LSCC tissue. The mi R-193 b can up regulate the expression of IL-10 in monocytes. This IL-10-expressing monocytes have an immune suppressive function on CD8+T cell activities. |
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