RNA干扰 z-bo 2020-11-10 281

　　OBJECTIVE: To use RNA interference technology to inhibit the expression of autophagy regulatory gene Beclin 1, and to detect the effect of Beclin 1 expression on the proliferation and apoptosis of naked mole rat skin fibroblasts and the effect of p53, BAX, Bcl2 and other genes.

　　Method: To detect the expression of Beclin 1 in naked mole rat fibroblasts after starvation and H2O2 stimulation, and then use the designed interfering RNA of Beclin 1 gene and negative control to transiently transfect naked mole rat fibroblasts. Real-time PCR and Western blot were used to detect the silencing effect, CCK-1 experiment was used to detect the cell proliferation activity after silence, flow cytometry was used to detect cell apoptosis, and Western blot was used to detect the protein expression level of related genes.

　　Result: Both starvation and H2O2 stimulation can cause changes in the expression level of Beclin 1. The transfection efficiency of naked mole rat skin fibroblasts with gene expresso transfection reagent can reach more than 90%. The results of real-time PCR and Western blot show that the designed Beclin 1 siRNA can effectively reduce the expression of Beclin 1. After Beclin 1 gene silencing, the proliferation inhibition rate of naked mole mouse skin fibroblasts was significantly higher than that of the control group, and the early and late apoptosis rates of cells were significantly increased. At the same time, p53, BAX, Bcl2, LC3B, p-AKT, The expression level of mTOR etc. decreased.

　　Conclusion: The expression of Beclin 1 changes significantly during the process of naked mole rat fibroblasts resisting starvation and H2O2 stimulation. At the same time, inhibiting the expression of Beclin 1 can inhibit the proliferation of naked mole rat cells and promote their apoptosis. This suggests that Beclin 1 gene pairs Naked mole rats play a regulatory role in autophagy, proliferation and apoptosis.