| MicroRNA (miRNA) is a class of small non-coding RNA with approximately 22nt. It’s involved in the regulation of gene expression. miRNA plays important roles in most biological process, including cell proliferation, immune and tumorigenesis and tumor development. Phagocytosis is a conserved immunized way in animals. Some miRNA is highly conserved from invertebrates to higher animals. Therefore, there may be a same regulatory mechanism in cell phagocytosis of invertebrates and mammalian. In this paper, we investigated a conserved mechanism by focusing a conservative miRNA. Tumorigenesis and development is a complex process. In this process, miRNA is not only involved in the regulation of cell activity of immune cells including macrophages, but also involved in regulating the activity of cancer cells, such as growth and migration. Therefore, we investigated the roles and mechanisms of the deregulated miRNA in cancer.Phagocytosis plays an important role in immune responses. However, the regulation of phagocytosis mediated by microRNAs has not been extensively investigated. To address this issue, based on our previous research, the regulation of phagocytosis by miR-1 was characterized in this study. The results showed that miR-1 played an important role in the phagocytosis regulation in shrimp in vivo. The sequence analysis indicated that miR-1 was highly conserved from invertebrates to mammals, so we further investigated the role of miR-1 in phagocytosis regulation of mammal macrophages. The data presented that miR-1 was significantly downregulated in cancerous macrophage RAW264.7 cells compared with those in the isolated murine macrophage and in the immortalized macrophage ANA-1. The findings showed that miR-1 had a great effect on the regulation of phagocytosis in cancerous macrophage by the inhibition of clathrin heavy chain 1 (CLTC1) gene. Therefore, our study presented a novel miR-1-mediated regulation of phagocytosis both in invertebrate and in vertebrate.To further investigate the function of miR-1 in cancer.we analyzed the expression level in gastric cancer cell lines (MGC-803, HGC-27, MKN45) and breast cancer cell lines (MDA-MB-231, MDA-MB-435). Real-time PCR result showed that miR-1 expression was reduced in all cancer cells compared with non-cancerous gastric cells (GES-1) or non-cancerous breast cells (MCF-10A). And then,42 pairs of cancerous tissue and corresponding normal tissue from the same patients with gastric cancer were examined. The results indicated that the expression level of miR-1 in cancer tissues was decreased compared with normal tissues. Above data demonstrated that it is presumably served as a suppressor in tumor progression. Overexpression of miR-1 led to significant inhibitions of the proliferation rates of cancerous cells compared with the negative control and initiated the cell cycle arrest at G1 phase of cancer cells but not non-cancerous cells. Further research including expriments in cells and animals found that miR-1 overexpression resulted in decrease of CDK4, cell cycle arreast and inhibition of tumor growth in vivo. Additionally, miR-1 overexpression significantly inhibited the ability to migration, invasion and adhesion of cancer cells. The confocal microscopy data indicated that the structure of fiber actin was changed in the miR-1 overexpressed cells compared with the non-treated cells. And the phase contrast microscope data displayed the obvious morphological changes of cells, indicating the motility ability of cancer cells was inhibited. We analyzed the miR-1 target gene and found that miR-1 can directly target 3’UTR of twinfilin actin binding protein 1 (TWF1), calponin 3, acidic (CNN3), coronin, actin binding protein (CORO1C), WAS protein family, member 2 (WASF2), thymosin beta 4, X-linked (TMSB4X), leading to the inhibition of migration, invasion and adhesion of cancer cells. Expriments in cells and animals showed that miR-1 overexpression resulted in decrease of TWF1、CNN3、CORO1C、WASF2 and TMSB4X and inhibition of cancer cells metastasis in vivo. Cleavage with Ago2 in vitro and RNA Fluorescence in situ hybridization in cancer cells demonstrated that miR-1 can simultaneously target these six gene (CDK4, CNN3, CORO1C, WASF2, TMSB4X, TWF1) related to proliferation and migration of cancer cells. In vivo assay, miR-1 can efficiently inhibit tumorigenesis and metastasis of cancer cells in nude mice, further revealing that miR-1, as a tumor suppressor, can simultaneously inhibit cancer cells growth and metastasis. |
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