膜泡分拣蛋白72在肝癌组织的表达及其对肝癌细胞表型的影响

目的探讨膜泡分拣蛋白72(VPS72)在肝癌组织和对应癌旁组织的表达及临床病理意义,观察其对细胞增殖、迁移能力的影响。方法选取2018年9月至2020年9月来自河南大学河南省人民医院60例肝癌组织及对应的癌旁组织标本,肝癌组织划分为中心组,取距离癌组织边缘2 cm处标本划分为癌旁组。免疫蛋白印迹法(Western blot)及免疫荧光法检测中心组和癌旁组中VPS72蛋白的表达水平及分布,分析其与年龄、性别、肿瘤大小、数目、分期、分化程度、有无脉管癌栓、包膜侵犯等临床病理参数之间的关系;小干扰RNA(siRNA)降低肝癌细胞株Huh-7中VPS72的表达,分为实验组和对照组;划痕实验、Transwell实验检测其对Huh-7迁移能力的影响;细胞计数试剂盒(CCK-8)实验和平板克隆实验检测Huh-7增殖能力。组间比较采用配对样本t检验和χ²检验。结果VPS72在癌组织中相对表达量为0.900±0.511,明显高于对应癌旁组织表达量(0.729±0.643),差异有统计学意义(t=2.027,P<0.05)。其表达量与肿瘤的分化程度、大小、脉管癌栓、细胞增殖抗原-67(Ki-67)和分期明显相关(χ²=5.621、4.855、4.275、8.418、7.202,P<0.05)。划痕实验表明24、48 h实验组迁移细胞数(38.330±3.512)、(66.670±5.508)个]低于对照组(78.670±2.517)、(165.670±5.033)个],差异有统计学意义(t=14.162、16.275,P<0.05)。Transwell实验显示穿孔细胞数实验组(32.333±3.512)个,低于对照组(52.667±2.082)个],差异有统计学意义(t=7.625,P<0.05)。CCK-8实验显示0 h实验组吸光度值(0.235±0.047)较对照组(0.237±0.068)无明显变化,差异无统计学意义(t=0.020,P>0.05)。24、48 h实验组A值(0.539±0.300、0.815±0.038)低于对照组(0.683±0.100、1.415±0.131),差异有统计学意义(t=4.654、7.373,P<0.05)。平板克隆实验显示实验组细胞团数量为(50.222±6.222)个,低于对照组(71.560±7.038)个],差异有统计学意义(t=7.875,P<0.01)。结论肝癌组织中VPS72蛋白表达量明显高表达,且与肿瘤的分化程度、大小、脉管癌栓、Ki-67和分期呈正相关,降低VPS72的表达能够明显抑制Huh-7细胞株增殖和迁移能力。

Expression of vacuolar protein sorting 72 in liver cancer tissues and its effect on the phenotype of liver cancer cells

Objective To explore the expression and clinicopathological significance of vacuolar protein sorting 72(VPS72)in liver cancer tissues and corresponding adjacent tissues,and observe its influence on cell proliferation and migration.Methods From September 2018 to September 2020,60 cases of liver cancer tissues and corresponding adjacent tissues from Henan University Henan Provincial People′s Hospital were selected.The liver cancer tissues were divided into center group,and the specimens 2 cm away from the edge of the cancer tissue served as the paracancerous group.Western blotting and immunofluorescence methods were used to detect the expression level and distribution of VPS72 protein in center group and paracancerous group,and its relationship with age,gender,tumor size,number,stage,and degree of differentiation,the presence and absence of vascular tumor thrombus,capsule invasion and other clinicopathological parameters was analyzed.human hepatoma cells-7(Huh-7)cells with VPS72 silencing by small interfering RNA were divided into experimental group and control group.The scratch assay and Transwell assay were used to verify the effect of VPS72 on the ability of migration.Cell counting kit-8(CCK-8)experiment and plate cloning experiment were done to detect the ability of proliferation in Huh-7 cells.comparisons between groups were performed by paired-sample t andχ²test.Results The relative expression of VPS72 in cancer tissues was(0.900±0.511),significantly higher than that in adjacent tissues(0.729±0.643)(t=2.027,P<0.05).The expression of VPS72 was significantly correlated with tumor differentiation,size,vascular tumor thrombus,cell proliferation antigen-67(Ki-67)and staging(χ²=5.621,4.855,4.275,8.418,7.202,P<0.05).The scratch assay showed that the number of migrating cells at24 and 48 hin the experimental group(38.330±3.512),(66.670±5.508)]was significantly less than that in the control group(78.670±2.517),(165.670±5.033)](t=14.162,16.275,P<0.05).Transwell experiment showed that the number of invasive cells in the experimental group(32.333±3.512)was significantly les than that in the control group(52.667±2.082)(t=7.625,P<0.05).The CCK-8 experiment showed that the absorbance(A)value(0.235±0.047)at 0 h in the experimental group had no significant difference from the control group(0.237±0.068)(t=0.020,P>0.05).The A values at24 and 48 h in the experimental group(0.539±0.300),(0.815±0.038)]were significantly lower than those in the control group(0.683±0.100),(1.415±0.131)](t=4.654,7.373,P<0.05).The plate cloning experiment showed that the number of cell clusters in the experimental group(50.222±6.222)was significantly reduced as compared with the control group(71.560±7.038)(t=7.875,P<0.01).Conclusion The expression of VPS72 protein in liver cancer tissue is obviously high,and it is obviously related to clinicopathological parameters.Silencing the expression of VPS72 can significantly inhibit the ability of proliferation and migration of Huh-7 cell lines.