Effects Of Silencing APN Gene Expression On Proliferation?Invasion And Apoptosis Of Human Glioma U251 Cells And Mechanism

OBJECTIVE:To explore the effects of Aminopeptidase N?APN?on glioma cell U251 proliferation,migration and invasion and apoptosis,and its possible mechanism.Methods:After silencing the expression of APN in glioma cell U251by RNA interference technology,the biological function of glioma U251after silencing was detected by appropriate functional experimental methods.RT-qPCR was used to detect changes in mRNA expression levels before and after APN in silent glioma cells U251.Western blot was used to detect changes in APN protein expression levels.Cell Counting Kit 8?CCK-8?reagent was used to detect cell proliferation activity;flow cytometry was used detection of apoptosis rate and cell cycle distribution;Transwell migration test and cell scratch test were used to detect the migration ability of glioma cell U251 in each group;Transwell experiment was used to detect different groups Invasion ability of glioma cells U251;Western blot was used to detect the protein expression levels of t-STAT3,p-STAT3,Bcl-2,MMP-2,PCNA and Cyclin D1.Results:Compared with the blank control group and the negative control group,the expression of APN in gene and protein decreased after silencing the expression of APN in U251?P<0.05?,indicating that RNA interference has achieved the desired effect;the proliferation activity of U251 after silencing APN significantly decreased?P<0.05?;cell cycle detection by flow cytometry methods revealed that the S-phase proportion of cells in the blank control group,NC-siRNA group,and APN-siRNA group was?33.72±4.79?%,?29.28±4.31?%,and?8.3±2.88%?,respectively;G₂/M phase ratios are?10.35±4.85?%,?9.2±1.42?%and?7±1.61?%,respectively;G₀/G₁ phase ratios are?55.93±9.57?%??61.52?±3.29)%and?84.7±2.66?%,respectively,compared with the blank control group,the proportion of S phase in the APN-siRNA group was significantly reduced?p<0.01?.Cell cycle arrest was in the G₀/G₁ phase,and the apoptosis rate was significantly increased?P<0.01?;Apoptosis rate detected by elapsed cell technology showed that the overall apoptotic rates of the blank control group,NC-siRNA group and APN-siRNA group were?4.89±3.38?%,?2.72±2.55?%,and?30.58±9.51?%,respectively,the apoptosis rate of the APN-siRNA group was significantly increased compared with the blank group,and the difference was statistically significant?p<0.05?;In the wound healing assay,we found that the mobility of the blank control group,NC-siRNA group and APN-siRNA group was?60±1.73?%,?55±4.58?%,and?27±4.58?%,respectively.The mobility was significantly weakened in the APN-siRNA group compared with the blank group,and the difference was statistically significant?p<0.01?.Transwell's method was used to detect changes in the migration capacity of U251 in different groups.We found that each of the blank control group,NC-siRNA group,and APN-siRNA group The visual field cells were 35±4,34±5,and 22±2,respectively.Compared with the blank control group,the cells in the APN-siRNA group were significantly reduced,and the difference was statistically significant?p<0.01?.Transwell invasion method detected U251 invasion In the capacity test,the cells in each field of the blank control group,NC-siRNA group,and APN-siRNA group were 33±3,31±3,and 15±2,respectively.Compared with the blank control group,the cells in the APN-siRNA group Significantly reduced,and the difference was statistically significant?p<0.05?;Western Blot explored the changes in STAT3 signaling pathway protein expression after silenced APN in glioma cell U251 and found that p-STAT3 and bcl-2,MMP-2,PCNA and Cyclin D1 protein expression levels in the APN-siRNA group were compared with the Blank group were significantly reduced.There was statistically significant?p<0.05?.Conclusion:Through a series of cell experiments,we found that silencing APN can inhibit the proliferation,migration and invasion of glioma cells U251 cells,and can induce apoptosis.APN could affect the biological activity of glioma cells U251 through the STAT3 signaling pathway.