MicroRNA-7 In Exosomes Regulates Autophagy In Lung Cells By Target Gene Bcl-2 After Mouse Brain Received Radiation

ObjectiveTo investigate the mechanism of mi R-7 in regulating lung cells autophagy by target gene Bcl-2 after the brain of mouse received radiation, and finally provided the basis for further study on injury in which normal tissue may produce after cancer patients receiving radiotherapy and injury prevention.Method1. Immunohistochemical stain and Western Blot were used to detect the expression levels of related proteins in lung tissues including Cleaved caspase-3, Beclin-1, LC3 B, Bcl-2 and so on.2. Real-time fluorescence quantitative PCR was used to detect the expression levels of mi R-7 and m RNA Bcl-2 in the samples of mouse brain and lung tissues, blood, astrocyte cells and so on.3. The Bcl-2-3’UTR wild-type and mutant reporter gene plasmids were constructed, and the dual luciferase activity experiment was used to determine whether Bcl-2 is a direct target gene of mi R-7.4. The extra high speed gradient centrifugation method was used to extract the Exosomes of the cell culture medium.5. Exosomes were stained by Di I dye, and added to the normal primary astrocyte cell cultures, and confocal technique was used to observe Exosomes into cytoplasm.Results1. The expression levels of Beclin-1 and LC 3B II / I were increased significantly but the expression level of Cleaved caspase-3 was not significantly changed after mouse brain received irradiation.2. The expression level of mi R-7 in lung tissue was higher in irradiated mouse than that in normal one while the expression level of Bcl-2 was opposite to that of mi R-7, and the expression level of Bcl-2 in lung tissue was lower compared to the control.3. The dual luciferase activity experiment found that mi R-7 could significantly inhibit the reporter gene plasmid luciferase activity of Bcl-2-3’UTR wild-type, while had no significant effect on mutant type, which confirmed that Bcl-2 was a direct target gene of mi R-7.4. Treatment of astrocyte cells with Exosomes isolated from radiated astrocyte cells(rad-exo) showed that the expression level of mi R-7 was significantly higher than that with Exosomes isolated from unradiated astrocyte cells(nc-exo), while the expression level of m RNA Bcl-2 was decreased significantly. The espresstion levels of LC 3B II / I and Beclin-1 were higher in rad-exo group than that in nc-exo group, while the expression level of Bcl-2 was decreased. Exosomes were injected into mice from tail vein and the results showed that the expression level of mi R-7 was significantly higher in rad-exo group than that in nc-exo group, while the expression level of Bcl-2 was decreased significantly, and the expression levels of LC 3B II / I and Beclin-1 significantly increased.Conclusion1. Bcl-2 is a direct target gene of mi R-7.2. Exosome/mi R-7 mediates the bystander effect in the lungs after the brain exposed to radiation via regulation of Bcl2.