Roles Of MiR-100in Apoptosis Of Shrimp Hemocytes And Breast Cancer Cells

Apoptosis is a programmed cell death which is critical for the harmony of the internal environment. It is not only an important component of immune system, but also associated with many diseases especially the development of cancer. Shrimp is one kind of invertebrates, which merely possesses innate immunity. Apoptosis belongs to the hemocyte defence system of the shrimp innate immunity, which plays an essential role in the process against bad environment. It is demonstrated that apoptosis plays an important role in the process against virus infection. Therefore, it would be meaningful to investigate the mechanism of apoptosis in shrimp, which could provide new horizon and new method to deal with virus infection. Recently, numerous reports reveal that non-encoding small RNA---miRNA could be involved in apoptosis through regulating the post-transcription level of genes. However, miRNAs involved in the regulation of apoptosis are not intensively investigated.Based on previous studies in our laboratory, the shrimp miRNAs were obtained through miRNA sequencing of shrimp. In this study,10miRNAs were found to be related with apoptosis through shrimp miRNA microarray using three different treatments (normal shrimp hemocytes, apoptosis-induced hemocytes, apoptosis-inhibited hemocytes). The northern blot data revealed that6miRNAs were consistent with the date of microarrays. In order to further study the function of miRNA in apoptosis of shrimp, an evolutionarily conserved miR-100was chosen to for further investigations. The anti-miR100-oligonucleotide (AMO-miR100) was injected into shrimp. The expression of miR-100was knocked down in shrimp. Then the caspase3/7enzyme activity in the miR-100-silenced hemocytes was determined. The results showed that the caspase3/7enzyme activity was significantly increased in AMO-miR100-treated shrimp hemocytes. To further investigate the function of miR-100in the process of shrimp antiviral immunity, the WSSV genome copies and shrimp mortality were evaluated when shrimp were treated with WSSV only or WSSV+AMO-miR100, respectively. The results presented that AMO-miR100could reduce both the WSSV copies and the shrimp mortality, indicating its antiviral feature.Till now, more and more researches have reported that dysregulation of miRNA in cells is highly associated with the development of cancer. In our studies, it was found that miR-100was upregulated in breast cancer cell line SK-BR-3compared with those in breast cell lines (MCF-7and MDA-MB-453), suggesting that miR-100might serve as onco-miRNA in SK-BR-3.In order to study the function of miR-100in apoptosis of breast cancer cell, AMO-miR-100or precursor of miR-100was used to knock down or over-express miR-100in breast cancer cell line. Then apoptosis of breast cancer cells was evaluated by caspase3/7enzyme activity assay, MTS assay, western blot and Annexin V assays. The results revealed that AMO-miR-100could induce apoptosis in breast cancer cell, whereas precursor-miR-100had no any impact on apoptosis of breast cancer cell. These results demonstrated that miR-100could inhibit apoptosis in breast cancer cell.To further investigate the mechanism of apoptosis mediated by miR-100, the target genes of miR-100were assayed through mRNA microarray and bioinformatics analysis. It was found that FGFR3and MTMR3might be target genes of miR-100. The duel-luciferase reporter system assays revealed that MTMR3was directly targeted by miR-100. The findings showed that the expression level of MTMR3was higher in apoptosis-induced breast cancer cells than that in normal breast cells, which could promote the expression of p27, leading to G2/M cell cycle arrest. However, when the mRNA level of MTMR3was knocked down through RNAi and AMO-miR-100was simultaneously transfected into breast cancer cells, the apoptosis of cells happened. This phenomenon suggested that miR-100was involved in apoptosis by regulating the expressions of several target genes.In summary, miR-100, a highly conserved miRNA in animals, shared the same roles in shrimp and breast cancer cells, inhibiting the process of apoptosis. Therefore our study would provide novel insights for antiviral immunity of shrimp and provide new approaches for the diagnosis and treatments of breast cancer.