跨膜emp24结构域蛋白4在肝癌患者肝组织中的表达及其对肝癌细胞生物学行为的影响

Expression of the transmembrane emp24 domain-containing protein 4 in liver tissues of patients with hepatocellular carcinoma and its effects on biological behavior of hepatocellular carcinoma cells

目的检测跨膜emp24结构域蛋白4(TMED4)在肝癌患者肝组织中的表达情况,并初步探究TMED4基因对肝癌细胞增殖和迁移能力的影响及其相关分子机制。方法采用蛋白质印迹法和免疫组织化学染色检测肝癌患者癌组织、癌旁组织中TMED4的蛋白质表达水平,并分析其表达与患者的临床病理参数之间的相关性;分别通过细胞增殖实验、Transwell实验、划痕愈合实验和裸鼠皮下成瘤实验探究过表达或敲减TMED4基因对肝癌细胞体内外增殖、迁移和愈合能力的影响;通过通路分析初步探究TMED4基因调控肝癌细胞生物学行为的可能分子机制。统计学方法采用独立样本t检验、Mann-Whitney U检验和卡方检验。结果蛋白质印迹法结果显示,TMED4在肝癌组织中的蛋白质表达水平低于其对应的癌旁组织(0.52±0.29比0.83±0.22),差异有统计学意义(t=2.54,P=0.022)。免疫组织化学染色结果显示,TMED4在肝癌组织中的蛋白质表达水平低于其对应的癌旁组织(5.46±3.37比7.58±3.08),差异有统计学意义(t=3.49,P<0.001)。TMED4的蛋白质表达水平与患者是否发生肿瘤血管侵犯和巴塞罗那临床肝癌(BCLC)分期显著相关(χ^(2)=6.83、4.20,P=0.009、0.040)。细胞增殖实验结果显示,SMMC-7721细胞中TMED4过表达组细胞的光密度值低于对照组(1.38±0.05比2.37±0.08),HepG2细胞中TMED4敲减组细胞的光密度值高于对照组(0.76±0.04比0.54±0.01),差异均有统计学意义(t=18.23、8.85,均P<0.001)。Transwell实验结果显示,SMMC-7721细胞中TMED4过表达组的迁移细胞数少于对照组(286.30±13.01比439.70±12.34),HepG2细胞中TMED4敲减组的迁移细胞数多于对照组(249.00±6.00比160.00±6.56),差异均有统计学意义(t=14.81、17.34,均P<0.001)。划痕愈合实验结果显示,SMMC-7721细胞中TMED4过表达组的细胞愈合率低于对照组[(0.21±0.01)%比(0.45±0.01)%],HepG2细胞中TMED4敲减组的细胞愈合率高于对照组[(0.46±0.01)%比(0.20±0.01)%],差异均有统计学意义(t=200.10、30.46,均P<0.001)。裸鼠皮下成瘤实验结果显示,TMED4过表达组细胞的生长速度较对照组缓慢,细胞接种6周后,TMED4过表达组小鼠的皮下肿瘤体积小于对照组[27.36 mm^(3)(138.70 mm^(3))比1741.62 mm^(3)(1783.39 mm^(3))],肿瘤质量低于对照组[0.06 g(0.14 g)比1.46 g(1.09 g)],差异均有统计学意义(均Z=-2.31,均P<0.001)。蛋白质印迹法结果显示,SMMC-7721细胞中TMED4过表达组锌指转录因子(Snail)的蛋白质水平低于对照组(0.32±0.01比0.90±0.03),HepG2细胞中TMED4敲减组Snail的蛋白质水平高于对照组(1.03±0.01比0.97±0.01),差异均有统计学意义(t=28.49、12.31,均P<0.001)。实时荧光定量聚合酶链反应结果显示,SMMC-7721细胞中TMED4过表达组Snail的mRNA水平低于对照组(0.13±0.05比1.00±0.15),HepG2细胞中TMED4敲减组Snail的mRNA水平高于对照组(1.25±0.32比0.21±0.14),差异均有统计学意义(t=9.62、5.10,P<0.001、=0.007)。结论TMED4可能通过调控Snail的表达进而影响肝癌细胞的增殖和迁移能力,其有望成为肝癌治疗的潜在靶点。

Objective To examine the expression of transmembrane emp24 domain-containing protein 4(TMED4)in liver tissue of patients with hepatocellular carcinoma,and to investigate the effects of TMED4 gene on the proliferation and migration of hepatocellular carcinoma cells and related molecular mechanisms.Methods The expression of TMED4 at protein level in liver cancer tissue and paracancerous tissue of patients with hepatocellular carcinoma were detected by Western blotting and immunohistochemical stainning,and the correlation between its expression and clinicopathological features was analyzed.The effects of TMED4 overexpression or knockdown on proliferation,migration and healing ability of hepatocellular carcinoma cells in vitro and in vivo were determined by cell proliferation test,Transwell test,wound healing test and subcutaneous tumor formation in nude mice.The molecular mechanism of TMED4 in regulating the biological behavior of hepatocellular carcinoma cells was preliminarily explored by pathway analysis.Independent sample t test,Mann-Whitney U test and chi-square test were used for statistical analysis.Results The results of Western blotting showed that the expression of TMED4 at protein level in hepatocellular carcinoma tissue was lower than that in paracancerous tissue(0.52±0.29 vs.0.83±0.22),and the difference was statistically significant(t=2.54,P=0.022).The results of immunohistochemical examination indicated that the expression of TMED4 at protein level in liver cancer tissue was lower than that in paracancerous tissue(5.46±3.37 vs.7.58±3.08),and the difference was statistically significant(t=3.49,P<0.001).The expression of TMED4 at protein level was significantly correlated with vascular invasion and Barcelona clinic liver cancer stage(χ^(2)=6.83 and 4.20,P=0.009 and 0.040).The results of cell proliferation assay showed that the absorbance value of SMMC-7721 cells in TMED4 overexpression group was lower than that in control group(1.38±0.05 vs.2.37±0.08),while the optical density value of HepG2 in TMED4 knockdown group was higher than that in control group(0.76±0.04 vs.0.54±0.01),and the differences were statistically significant(t=18.23 and 8.85,both P<0.001).The results of Transwell test showed that the number of migrated SMMC-7721 cells in TMED4 overexpression group was less than that in control group(286.30±13.01 vs.439.70±12.34),while the number of migrated HepG2 cells in TMED4 knockdown group was higher than that in control group(249.00±6.00 vs.160.00±6.56),and the differences were statistically significant(t=14.81 and 17.34,both P<0.001).The wound healing test showed that the healing rate of SMMC-7721 cells in TMED4 overexpression group was lower than that in control group((0.21±0.01)%vs.(0.45±0.01)%),the healing rate of HepG2 cells in TMED4 knockdown group was higher than that in control group((0.46±0.01)%vs.(0.20±0.01)%),and the differences were statistically significant(t=200.10 and 30.46,both P<0.001).The results of subcutaneous tumor formation assay in nude mice showed that the growth rate of cells in TMED4 overexpression group was slower than that in control group.After cell inoculation for 6 weeks,the subcutaneous tumor volume of mice in TMED4 overexpression group was smaller than that in control group(27.36 mm^(3)(138.70 mm^(3))vs.1741.62 mm^(3)(1783.39 mm^(3))),the tumor weight was lower than that in control group(0.06 g(0.14 g)vs.1.46 g(1.09 g)),and the differences were statistically significant(both Z=-2.31,both P<0.001).The results of Western blotting showed that the expression of Snail at protein level in SMMC-7721 cells of the TMED4 overexpression group was lower than that of the control group(0.32±0.01 vs.0.90±0.03),the protein level of Snail in HepG2 cells of TMED4 knockdown group was higher than that of control group(1.03±0.01 vs.0.97±0.01),and the differences were statistically significant(t=28.49 and 12.31,both P<0.001).The results of real time fluorescent quantitative polymerase chain reaction showed that the expression of Snail at mRNA level in SMMC-7721 cells of TMED4 overexpression group was lower than that of control group(0.13±0.05 vs.1.00±0.15),the expression of Snail at mRNA level in HepG2 cells of TMED4 knockdown group was higher than that of control group(1.25±0.32 vs.0.21±0.14),and the differences were statistically significant(t=9.62 and 5.10,P<0.001 and P=0.007).Conclusion TMED4 may affect the proliferation and migration of hepatocarcinoma cells by regulating the expression of Snail,and which is expected to become a potentially therapeutic target for hepatocellular carcinoma.