RNA干扰中性粒细胞相关载脂蛋白基因的表达对人脑胶质瘤细胞凋亡的影响

Effects of RNA interference on expression of neutrophil gelatinase - associated lipocalin gene in human glioma cells

目的探讨RNA干扰中性粒细胞相关载脂蛋白（NGAL）基因的表达对人脑胶质瘤细胞凋亡的影响及机制。方法提取人脑胶质瘤组织及人脑胶质瘤SHG44、U87、U251细胞中的蛋白，Western blot检测NGAL的表达；将siRNA—NC、NGAL—siRNA1、NGAL-siRNA2、NGAL—siRNA3转染U251细胞，未转染任何siRNA的作为对照组，各组细胞转染48h后，Western blot检测NGAL的蛋白表达；流式细胞仪检测细胞转染48h的细胞凋亡，Western blot检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3（Cleaved Caspase-3）、Notch1、发状分裂相关增强子1（Hes1）蛋白表达。结果脑胶质瘤组织中NGAL的mRNA和蛋白表达（0．458±0．042）显著高于瘤旁组织（0．098±0．015）（tmRNA=7．113，PmRNA=0．003；t蛋白表达=8．532，P蛋白表达=0．002），U251细胞中NGAL的蛋白表达最高，选择作为后续研究；NGAL—siRNA组细胞凋亡率[（15．62±1．94）％]及leavedCaspase-3（0．094±0．021）蛋白表达显著高于对照组[（2．18±0．67）％，0．032±0．015]，Notch1（0．204±0．028）、Hes1（0．103±0．019）蛋白表达显著低于对照组（0．453±0．041、0．215±0．029）（t调亡率=9．995，P调亡率=0．001；tCleaved Caspase-3=5．898，PCleaved Caspase-3=0．006；tNotch1=8．112，PNotch1=0．003；tHes1=8．824，PHes1=0．003）。结论NGAL基因在人脑胶质瘤组织及细胞中高表达，通过RNA干扰沉默NGAL表达可抑制人脑胶质瘤细胞增殖及促进细胞凋亡，其机制与Notch信号通路有关。

Objective To investigate the effect and mechanism of RNA interference on the expres- sion of neutrophil gelatinase associated lipocalin （NGAL） gene in human glioma cells. Methods The proteins in human glioma tissues and glioma SHG -44, U87 and U251 ceils were extracted, the expression of NGAL protein was detected ; siRNA - NC, NGAL - siRNA1, NGAL - siRNA2, NGAL - siRNA3 trans- fected U251 cells, and not transfected with any siRNA as the control group, each group of cells transfected for 48 h, expression of NGAL protein were detected by Western blotting; cell apoptosis was detected cells transfected for 48 h by flow cytometry, the expression of Cleaved Caspase - 3, Notchl, hairy and enhancer of split 1 （ Hesl ） protein was detected by Western blotting. Results The expression of NGAL mRNA and protein in gliomas was significantly higher than that in the tumor adjacent tissues （tmRNA = 7. 113, PmRNA = 0. 003; tprotein expression = 8. 532, Porotein expression = 0. 002 ） , NGAL protein expression in U251 cell was the high- est, and was selected as a follow -up study, the apoptosis rate （15.62 ± 1.94）% and expression of Cleaved Caspase- 3 （0. 094± 0. 021 ） protein in NGAL- siRNA was significantly higher than the control group （2. 18 ±0. 67）, （0. 032 ：t：0. 015）, the expression of Notchl （0. 204 ±0. 028） and Hesl （0. 103 ± 0. 019） protein was significantly lower than the control group （0. 453 ± 0. 041, 0. 215 ± 0. 029）, （ tapoptosis rate = 9. 995, Papoplosis rate = 0. 001 ; tCleaved Caspase-3= 5. 898, PCleaved Caspase--3 = 0. 006 ; tNotchl = 8. 112, PNotch1 = 0. 003 ; tHes1 = 8. 824, PHes1 = 0. 003 ）. Conclusion NGAL gene was highly expressed in human glioma tissues and cells, to inhibit the expression of NGAL by RNA interference could inhibit the prolifera- tion and promote apoptosis of human glioma cells, its mechanism was related to notch the signal pathway.