| Canine influenza is a highly contagious disease can cause acute respiratory symptoms. As a companion animal, dogs carrying canine influenza virus causing a potential threat to human and other animal. However, the infection mechanism of canine influenza virus is still not clear, so in order to better prevention and treatment of canine influenza, it is necessary to carry out further research the mechanism of infection. Influenza virus can induce cell pathological changess, and apoptosis is one of the manifestations. Micro RNA is a kind of short chain RNA. Let-7 is a family of second high expression abundance in the lung and trachea of dogs. It plays an important role in the regulation of apoptosis. In this paper, we observed the cell apoptosis induced by H3N2 subtype CIV, and studied the effect of let-7a on CIV induced apoptosis and its pathway to apoptosis.In this study, MDCK cells were infected with H3N2 subtype CIV in vitro, and apoptosis was detected by direct microscopy, AO/EB staining and Annexin-V FITC/PI apoptosis detection method. The results showed that CIV could induce MDCK cells shrinkage, rounding, and aggregation under the microscope; The apoptosis rate of 24 h and 48 h after infection was measured by AO/EB staining. The apoptosis rate was(20.7±1.6)% and(22.3±2.2)% in infected group, and the apoptosis rate was(8.1±1.1)% and(8.9±1.7)% in the control group respectively. The apoptosis rate of 24 h and 48 h after infection were measured by Annexin-V FITC/PI double dye apoptosis assay, and the apoptosis rate was(15.53±0.49)% in 24 h and(15.87±0.58)% in 48 h in infected group, respectively, which was significantly higher than(4.13±0.24)% in 24 h and(7.2±0.38)% in 48 h in control group. The results showed that significant apoptosis could be induced in CIV infected cells.Following induction of apoptosis, the cells were collected and RNA was extracted, reverse transcripted, and finally the expression level of let-7a was measured by real time PCR. The effect of let-7a on apoptosis induced by CIV was determined by transfection of let-7a. Let-7a expression was downregulated after apoptosis occurred by the detection of real time PCR. Let-7a over-expression could inhibit the occurrence of apoptosis. And let-7a inhibition test showed that inhibition of let-7a expression could promote the occurrence of apoptosis. The results showed that let-7a could inhibit the apoptosis, and its low expression indirectly promoted apoptosis.After determining the effect of let-7a on cell apoptosis, the target gene of let-7a was predicted by the target prediction software, and the target gene was constructed on the dual luciferase reporter vector psi-check2 for cell verification. The target gene MAP4K3 of let-7a was predicted by the target prediction software. And it was constructed into dual luciferase reporter plasmid, and the experiment proved that let-7a can directly target to MAP4K3 3'UTR. Subsequently, the expression of apoptosis related proteins in the downstream pathway was detected by real time PCR to determine whether apoptosis was realized through the mitochondrial pathway. Detection of apoptosis pathway related proteins also confirmed that the effect of let-7a on cell apoptosis could be achieved by changing the expression of MAP4K3 family protein, bcl-2/bax family protein and caspase family protein.In this study, we successfully infected MDCK cells with the H3N2 subtype canine influenza virus, and induced cells apoptosis, which provided support for the basic research of canine influenza. In vitro transfection of micro RNA-let-7a, confirmed that over expression of let-7a can effectively inhibit apoptosis, while let-7a low expression promotes apoptosis. This study also provides a theoretical and data support for canine influenza virus infection and let-7a relationship, and further studies have found that the effect of let-7a on cell apoptosis can be achieved through the mitochondrial apoptotic pathway. This study provided data support for the study of the effect of let-7a on cell apoptosis. |
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