摘要
目的研究长链非编码RNA核旁斑长点组装转录本1(Neat1)在紫外线B诱导人晶状体上皮细胞(LECs)焦亡中的作用及其机制。方法体外培养人晶状体上皮细胞系HLE-B3,将处于对数生长期的HLE-B3细胞分别采用紫外线B照射0、2、4和8 h;采用Western blot法检测照射不同时长后焦亡相关蛋白胱天蛋白酶1(caspase-1)的表达,采用实时荧光定量PCR法检测照射不同时长后细胞中Neat1 mRNA相对表达量,采用细胞计数试剂盒8(CCK-8)法检测细胞活力并以此筛选紫外线诱导LECs焦亡的最佳照射时长,最终确定为4 h。另将HLE-B3细胞分为阴性siRNA转染组、siRNA Neat1转染组、阴性siRNA转染+照射组和siRNA Neat1转染+照射组,采用相应试剂转染24 h,其中阴性siRNA转染+照射组和siRNA Neat1转染+照射组转染相应试剂后采用紫外线B照射4 h。采用CCK-8法检测各组细胞活力,流式细胞术检测细胞焦亡,Western blot法检测caspase-1、gasdermin D蛋白(GSDMD)、NOD样受体蛋白3(NLRP3)表达,ELISA法检测白细胞介素(IL)-1β质量浓度,透射电子显微镜下观察各组HLE-B3细胞超微结构变化。结果随照射时间的延长,caspase-1蛋白表达条带灰度呈递增趋势。照射0、2、4、8 h caspase-1蛋白相对表达量分别为0.05±0.01、0.25±0.07、0.51±0.04和0.74±0.02,总体比较差异有统计学意义(F=168.223,P<0.001),其中照射不同时长两两比较,差异均有统计学意义(均P<0.05)。Neat1 mRNA相对表达量随照射时间延长呈递增趋势,细胞活力值呈递减趋势,照射不同时长两两比较,差异均有统计学意义(均P<0.05)。与阴性siRNA转染组比较,siRNA Neat1转染组细胞活力值升高,阴性siRNA转染+照射组细胞活力值降低,差异均有统计学意义(均P<0.01);与阴性siRNA转染+照射组比较,siRNA Neat1转染+照射组细胞活力值升高,差异有统计学意义(P<0.05)。阴性siRNA转染组和siRNA Neat1转染+照射组细胞焦亡率明显低于阴性siRNA转染+照射�
Objective To investigate the role of long non-coding RNA nuclear paraspeckle assembly transcript 1(Neat1)in pyroptosis of ultraviolet B(UVB)-induced human lens epithelial cells(LECs)and to explore the possible mechanism.Methods The human lens epithelial cell line HLE-B3 was cultured in vitro,and cells at log phase were exposed to ultraviolet B for 0,2,4 and 8 hours,respectively.The expression of cysteine aspartic acid-specific protease-1(caspase-1),a protein related to pyroptosis,was detected by Western blot.The relative expression level of Neat1 in cells after different irradiation durations was determined by real-time quantitative PCR.Cell viability was determined by the cell counting kit-8(CCK-8)method to screen the optimal irradiation duration for UVB-induced LECs pyroptosis,which was finally determined to be 4 hours.HLE-B3 cells were divided into negative siRNA transfection group,siRNA Neat1 transfection group,negative siRNA transfection+irradiation group and siRNA Neat1 transfection+irradiation group,and were transfected with corresponding reagents for 24 hours.The negative siRNA transfection+irradiation group and siRNA Neat1 transfection+irradiation group were irradiated with UVB for 4 hours after transfection.The cell viability was detected by the CCK-8 method.The pyroptosis rate was detected by flow cytometry.The expression levels of caspase-1,gasdermin D(GSDMD)and nod-like receptor protein 3(NLRP3)proteins were detected by Western blot.The concentration of interleukin(IL)-1βwas detected by enzyme-linked immunosorbent assay(ELISA).Ultrastructural changes in HLE-B3 cells were observed under a transmission electron microscope.Results The grayscale of caspase-1 protein bands increased with the extension of irradiation duration.The relative expression levels of caspase-1 protein at 0,2,4 and 8 hours of irradiation were 0.05±0.01,0.25±0.07,0.51±0.04 and 0.74±0.02,respectively,with a statistically significant overall difference(F=168.223,P<0.001),and significant differences were found in paired comparison
作者
王敏
王妍茜
陈颖
赵越越
杨涛
康刚劲
Wang Min;Wang Yanxi;Chen Ying;Zhao Yueyue;Yang Tao;Kang Gangjin(Department of Ophthalmology,Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2023年第6期536-544,共9页
Chinese Journal Of Experimental Ophthalmology
基金
西南医科大学-泸州市中医医院基地项目(2019-LH007)。
