The Role Of MiR-1000 In Shrimp Antiviral Immunity And The Mechanism Of MiR-71 Promoting Autophagy Of Gastric Cancer Cells

MicroRNA (miRNA), a class of endogenous and about 22nt small non-coding RNA, is evolutionarily conserved and widely exists in animals, plants, viruses and others. Most miRNAs target mRNAs by recognition and binding with the target mRNA 3’-untranslated region (3’UTR) by the seed sequences (2-8 bases). In this study, the role of miRNA in virus infection and the mechanism of miRNA regulating autophagy are explored.Studies have shown that miRNA plays a key role in the virus-host interaction. White spot syndrome virus (WSSV), a virus with a double-stranded DNA genome, is a fatal pathogen of shrimp. It can infect most shrimp and crab, causing white spots. WSSV also leads to high mortality and makes serious economic loss. Based on previous studies in our laboratory, the expression profiles of shrimp miRNAs were found to be reshaped in response to WSSV infection, and shrimp miR-1000 might participate in the antiviral immune response. Therefore, the mechanism of shrimp miR-1000 in antiviral immunity was further investigated. This study revealed that miR-1000 was upregulated in response to WSSV infection, indicating that it was related with the antiviral immunity. In insect High Five cells, the synthesized miR-1000 could directly interact with WSSV early genes wsv191 and wsv407. On the one hand, it was revealed that the host miR-1000 overexpression led to a significant decrease of the wsv191 and wsv407 transcript levels compared with the control in vivo. On the other hand, the wsv191 and wsv407 gene expressions were significantly upregulated when the miR-1000 expression was knocked down. These results demonstrated that miR-1000 could regulate the expression of these two target genes. When the synthesized miR-1000 was injected into shrimp, the WSSV copies and shrimp mortality were lower than those of WSSV only. Conversely, the blocking of endogenous miR-1000 by AMO-miR-1000 led to increases of WSSV copies and shrimp mortality in WSSV-infected shrimp compared with the controls. The results showed that the knock down of viral early genes wsv191 and wsv407 by sequence-specific siRNA could significantly decrease the WSSV copies and shrimp mortality, indicating that wsv191 and wsv407 played important roles in WSSV infection. Therefore, miR-1000 could inhibit the virus infection in shrimp by targeting the viral wsv191 and wsv407 genes. To investigate whether the host miRNA could simultaneously regulate the expressions of different target genes in vivo, the interaction between miR-1000 and wsv191/wsv407 was studied. Electrophoretic mobility shift assay (EMSA) showed that miR-1000 could target the wsv191 and wsv407 in Ago complex simultaneously. It was further revealed miR-1000 and wsv191/wsv407 were colocalized in shrimp hemocytes by fluorescence in situ hybridization (FISH). Our study presented that animal miRNAs could regulate the expression of two target genes simultaneously.Autophagy is a highly conserved catabolic process that degrades misfolded or damaged proteins or organelles to provide energy for cells. The activity of autophagy is generally low in normal cells. However, in different stages and types of tumor, autophagy plays different roles. On the one hand, autophagy plays a crucial role in preventing the accumulation of damaged proteins and organelles which are toxic and carcinogenic. On the other hand, autophagy can promote tumor growth by supplying nutrition and energy. As reported, miRNAs are involved in the regulation of expressions of autophagy-related genes (ATGs). The alternation of these miRNAs expression profiles is able to change the level of autophagy and then influences cancer growth and development. Based on previous studies in our laboratory, miR-71 could regulate the immune-related genes of shrimp, and then induce the shrimp autophagy to facilitate the virus infection. Because of the conservation of miR-71 in animals, it is hypothesized that miR-71 may take effects on autophagy of gastric cancer cells. In this study, the results indicated that the expression levels of miR-71 in different gastric cancer cells were higher than stomach immortalized cells (GES). Overexpressing miR-71 could increase autophagy of cells, whereas inhibiting the miR-71 expression could decrease the autophagy level, indicating that miR-71 had influence on autophagy of gastric cancer cells. In insect High Five cells, miR-71 mimic could directly interact with G3BP1, MTMR4 and PAK2 which are related with autophagy. It was revealed that the miR-71 overexpression resulted in significant decreases of G3BP1, MTMR4 and PAK2 transcription levels compared with the control in gastric cancer cells. At the same time, the G3BP1, MTMR4 and PAK2 gene expressions were significantly upregulated when miR-71 was knocked down. And it was found that the three target genes could inhibit the level of autophagy in gastric cancer cells. These results indicated that miR-71 could influence autophagy by targeting G3BP1, MTMR4 and PAK2 genes. To investigate whether miR-71 could simultaneously regulate the expressions of various target genes in cells, the interaction between miR-71 and its targets was explored in this study. The data of electrophoretic mobility shift assay (EMSA) showed that miR-71 could target G3BP1, MTMR4 and PAK2 in the Ago complex simultaneously. Fluorescent in situ hybridization (FISH) assays revealed that miR-71 was colocalized with G3BP1, MTMR4 and PAK2 in gastric cancer cells. Our study presented that miR-71 facilitated autophagy of gastric cancer cells by simultaneously regulating three target genes.