| OBJECTIVE: To study the effect of NY-SAR-35 on the malignant biological behavior of glioma cells.Methods:(1)The glioma cells overexpressing NY-SAR-35 were screened by qRT-PCR and the localization of NY-SAR-35 in cells was determined by immunofluorescence.(2)Transfection of glioma cells with a sequence with fluorescence-specific interference with NY-SAR-35 expression was performed to optimize the transfection conditions;(3)CCK-8 method to detect the effect of NY-SAR-35 on the growth of glioma cells;(4)To investigate the effect of NY-SAR-35 on glioma cell growth,(5)Western blot was used to detect the expression of cyclin A,cyclin A,CDK2,apoptosis and apoptosis in glioma cells by flow cytometry(FCM)(6)The adhesion of caspase-3 and caspase-9 was changed by matrigel adhesion test;(7)The migration ability of glioma cells was detected by Transwell migration experiment;(8)Transwell Matrigel invasion assay was used to detect the invasive ability of glioma cells.W-blot was used to detect the expression of E-cadherin and N-cadherin in epithelial cells.Results:(1)The protein expression of NY-SAR-35 was found in the cytoplasm and nucleus,but mainly in the nucleus.Two strains of glioma cells from A172 and U251 were selected as experimental subjects.(2)The two groups of interference sequences with the highest interference efficiency were selectedas the experimental group by qRT-PCR.(3)CCK-8 method showed that the proliferation of two glioma cells decreased significantly after down-regulation of NY-SAR-35,and the difference was statistically significant(P <0.05),and the difference was statistically significant(P <0.01);(4)Cell cycle was detected by flow cytometry,and the expression of NY-SAR-35 in glioma cells was decreased,S phase cells increased,G2 / M phase cells decreased(P less than0.01)(P <0.05).Western blot showed that the expression of Cyclin A and the decrease of CDK2 were down-regulated by NY-SAR-35,and the difference was statistically significant(P <0.05),and there was no significant difference between the two groups(P> 0.01).(5)Apoptosis was detected by flow cytometry.The results showed that the apoptosis of glioma cells increased with NY-SAR-35,and the difference was statistically significant(P <0.01)The results showed that NY-SAR-35 down-regulated caspase-3 table(P <0.01),and the expression of caspase-9 did not change,which was not significantly different from that of the control group(P> 0.05).(6)The results of the adhesion experiment showed that the expression of caspase-9 was not significantly different from that of the control group(P <0.01).(7)In the scoring repair experiment,when the interference with NY-SAR-35,the adhesion of the two glioma cells decreased,and the difference was statistically significant(P <0.01)(P> 0.05).When 24 hours,the repair rate of the experimental group was significantly lower than that of the control group(P <0.05),and the difference was statistically significant(P <0.05)(P <0.01).At the same time,Transwell migration experiment showed that down-regulation of NY-SAR-35 could lead to the decrease of the number of transmembrane cells,and the difference was statistically significant(P <0.01);(8)Transwell Matrigel invasion experiment showed that interfering with NY(P <0.01).The expression of E-cadherin wasincreased and the expression of N-cadherin was decreased after Western blot.The difference was statistically significant(P <0.01)P <0.01).CONCLUSION: Down-regulation of NY-SAR-35 attenuates the malignant biological behavior of glioma cells.In addition,this study lays a foundation for subsequent animal experiments and suggests that blocking the expression of NY-SAR-35 has the same effect on glioma the potential clinical value of multi-channel therapy. |
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