核小体装配蛋白1样1对鼻咽癌细胞增殖特性的影响及其机制

Effect of nucleosome assembly protein 1 like protein 1 on proliferation of nasopharyngeal carcinoma cells and its molecular mechanism

目的探讨核小体装配蛋白1样1(NAP1L1)对鼻咽癌细胞增殖能力的影响及其机制。方法选取2017年1月到2021年1月新乡市中心医院收集的60例鼻咽癌组织和癌旁组织作为研究对象, 采用免疫组织化学分析癌旁组织和鼻咽癌组织NAP1L1蛋白表达水平。采用NAP1L1短发卡RNA(shRNA)慢病毒感染低分化鼻咽癌细胞SUNE-1, 建立对照组和NAP1L1 KD组细胞系, 采用细胞计数试剂盒(CCK-8)、克隆形成实验和体外移植瘤实验分析NAP1L1对鼻咽癌细胞增殖的影响。采用流式细胞术分析两组细胞周期水平;采用荧光定量聚合酶链反应(PCR)和蛋白质印迹法(Western blot)分析肿瘤组织和癌旁组织中周期蛋白依赖性激酶4(CDK4)、周期蛋白依赖性激酶6(CDK6)和细胞周期素D1(CCND1) mRNA和蛋白表达水平。组间计量数据比较采用t检验。结果 NAP1L1蛋白主要表达于细胞核中。定量结果显示, 癌旁组织NAP1L1蛋白表达强度(31.64±7.01)明显低于鼻咽癌组织NAP1L1蛋白表达强度(91.80±15.18), 差异有统计学意义(t=27.87, P<0.05)。对照组细胞48 h和72 h吸光度(A)值(1.08±0.08、1.89±0.11)明显高于NAP1L1 KD组细胞48 h和72 hA值(0.79±0.08、1.31±0.05), 差异有统计学意义(t=6.328、11.570, P<0.05)。克隆形成实验结果显示, 对照组细胞克隆形成数量(109.50±14.45)明显高于NAP1L1 KD组细胞克隆形成数量(66.33±7.69), 差异有统计学意义(t=6.462, P<0.05)。接种30 d后对照组细胞在裸鼠成瘤体积和重量[(1 072.90±114.87) mm3、(4.96±0.52) g]显著高于NAP1L1 KD组细胞[(855.90±55.54) mm3、(3.27±0.23) g], 差异有统计学意义(t=5.387、9.425, P<0.05)对照组细胞G0/G1期比例[(55.03±3.09)%]明显低于NAP1L1 KD组细胞[(74.88±2.16)%], 差异有统计学意义(t=12.890, P<0.05)。对照组细胞G2/M期比例[(5.61±0.63)%]明显低于NAP1L1 KD组细胞[(14.01±1.03)%], 差异有统计学意义(t=17.080, P<0.05)。对照组细胞S期比例[(30.27±1.79)%]明显高于NAP1L1 KD组细胞[(11.44±2.34)%], 差异有统计学意义(t=15.650, P<0.05)。对照组细胞CDK4、CDK6和CCND1蛋白表达水平(1.23±0.06、1.03±0.09、0.88±0.05)明显高于NAP1L1 KD组细胞(0.41±0.07、0.60±0.05、0.30±0.05), 差异有统计学意义(t=21.330、10.560、19.980, P<0.05)。结论 NAP1L1在鼻咽癌组织中呈高表达, 通过调节与肿瘤细胞周期相关蛋白的表达水平, 调节细胞周期变化, 进而影响肿瘤细胞恶性增殖的能力。

Objective To investigate the effect of nucleosome assembly protein 1 like protein 1(NAP1L1)on the proliferation of nasopharyngeal carcinoma cells and its molecular mechanism.Methods A total of 60 cases of nasopharyngeal carcinoma tissues and adjacent tissues collected in our hospital from January 2017 to January 2021 were selected as the research objects.The expression levels of NAP1L1 protein in adjacent tissues and nasopharyngeal carcinoma tissues were analyzed by immunohistochemistry.The low differentiated nasopharyngeal carcinoma cell line SUNE-1 was infected with NAP1L1 short hairpin RNA(shRNA)lentivirus.The cell lines of control group and NAP1L1 KD group were established.The effects of NAP1L1 on the proliferation of nasopharyngeal carcinoma cells were analyzed by cell counting kit-8(CCK-8),clone formation test and in vitro tumor transplantation test.The cell cycle levels of the two groups were analyzed by flow cytometry.The protein expression levels of cyclin dependent kinase 4(CDK4),cyclin dependent kinase 6(CDK6)and Cyclin D1(CCND1)in tumor tissues and adjacent tissues were analyzed by Western blotting.The measurement data between groups were compared by t-test.Results NAP1L1 protein was mainly expressed in the nucleus.The quantitative results showed that the expression intensity of NAP1L1 protein in adjacent tissues(31.64±7.01)was significantly lower than that in nasopharyngeal carcinoma(91.80±15.18,t=27.87,P<0.05).The 48 h and 72 h absorbance values(A)of cells in the control group(1.08±0.08,1.89±0.11)were significantly higher than those in the NAP1L1 KD group(0.79±0.08,1.31±0.05,t=6.328,11.570,P<0.05).The number of clonogenic cells in the control group(109.50±14.45)was significantly greater than that in the NAP1L1 KD group(66.33±7.69,t=6.462,P<0.05).At 30th day after inoculation,the tumorigenic volume and weight of cells in the control group[(1072.90±114.87)mm3,(4.96±0.52)g]were significantly increased as compared with those in the NAP1L1 KD group[(855.90±55.54)mm3,(3.27±0.23)g,t=5.387,9.425,P<0.05].The G0/G1 phase ratio of cells in the control group[(55.03±3.09)%]was significantly lower than that in the NAP1L1 KD Group[(74.88±2.16)%,t=12.890,P<0.05].The G2/M phase ratio of cells in the control group[(5.61±0.63)%]was significantly lower than that in the NAP1L1 KD group[(14.01±1.03)%,t=17.080,P<0.05].The S phase ratio of cells in the control group[(30.27±1.79)%]was significantly higher than that in the NAP1L1 KD group[(11.44±2.34)%],and the difference was statistically significant(t=15.650,P<0.05).The expression levels of CDK4,CDK6 and CCND1 proteins in control group(1.23±0.06,1.03±0.09,0.88±0.05)were significantly higher than those in NAP1L1 KD group(0.41±0.07,0.60±0.05,0.30±0.05,t=21.330,10.560,19.980,P<0.05).Conclusion NAP1L1 is highly expressed in nasopharyngeal carcinoma.It can regulate the changes of cell cycle by regulating the expression level of tumor cell cycle related proteins,and then affect the ability of malignant proliferation of tumor cells.