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
10.3760/cma.j.cn311367-20220504-00207
- VernacularTitle: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
- Author:
Liyang WANG
1
;
Wei HUANG
;
Shuzhen WU
;
Tao MA
;
Zhaoxiu LIU
;
Cuihua LU
Author Information
1. 南通大学附属医院消化内科，南通 226001
- Keywords:
Carcinoma, hepatocellular;
TMED4;
Cell proliferation;
Cell migration
- From:
Chinese Journal of Digestion
2022;42(10):667-674
- CountryChina
- Language:Chinese
-
Abstract:
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( χ2=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. 1 741.62 mm 3(1 783.39 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.
