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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.
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Objective:To explore a method for detecting recombinant human Annexin A11 (ANXA11) in serum exosomes of pancreatic cancer patients, and then primarily evaluate the clinical value of ANXA11 in pancreatic cancer patients.Methods:A prospective study was conducted and serum specimens from 70 patients diagnosed with PC, 15 patients diagnosed with benign pancreatic mass and 70 patients diagnosed with pancreatitis from the Affiliated Hospital of Nantong University were collected from December 2016 to July 2019. 70 healthy subjects during the same period were selected as control group. The abundance of ANXA11 in serum and exosomes-free serum were detected through parallel reaction monitoring (PRM) basing on high-resolution, high-precision mass spectrometer. Dot immunoblotting created by ourselves for detecting ANXA11 in exosomes and then the methodological evaluation were carried out. Levels of ANXA11 in exosomes in all subjects were statistically analyzed. Moreover, the areas under the curve (AUC) of receiver operating characteristic (ROC) curves were adopted to evaluate the diagnostic efficacy of ANXA11, CA19-9, CEA on PC. The relationship between ANXA11 and clinicopathological parameters as well as prognosis of PC patients was analyzed in the next moment. For analysis, the Mann-Whitney U test was used for comparing between either two groups, and the kruskal-wallis test was used for comparison among four groups. Results:The detection of serum exosome ANXA11 has high sensitivity and repeatability by the method of self-established dot immunoblotting. ANXA11 increased most significantly in the PC group, and the difference was statistically significant ( Hc=58.079, P<0.01) compared with other three groups. ROC curve analysis showed that the diagnostic performance of ANXA11(area under the curve (AUC=0.836) was higher than CEA (AUC=0.656) and equal to CA19-9 (AUC=0.870). The combination of ANXA11 and CA19-9 could improve the sensitivity of diagnosing PC and maintain good specificity. The level of serum exosome ANXA11 before treatment in PC patients was not related to age, gender, tumor size, tumor growth site, lymph node metastasis, distant metastasis and TNM stage ( Z values are 0.052,-0.285,-0.402,0.324,0.888,0.658,1.734, P>0.05). Furthermore, during the 10th day after surgical treatment, the level of ANXA11 showed no statistical difference compared with that before surgery ( Z value is -1.569, P=0.12). The survival time of PC patients was related to the presence of lymph node metastasis, distant metastasis, TNM staging and treatment protocols (χ 2 values are 9.354,6.086,9.389,16.998, P<0.05), while had no correlation with levels of CEA, CA19-9 and ANXA11 (χ 2 values are 1.516, 0.011, 0.159, P>0.05). Conclusions:This study successfully established an original method for detecting ANXA11 levels in serum exosomes of human. Serum exosomes ANXA11 combined with CA19-9 could improve the diagnostic sensitivity of PC.
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Background:Recurrent acute pancreatitis(RAP)is a special type of acute pancreatitis(AP). Finding the cause is the key to avoid the recurrence of RAP. Aims:To investigate the risk factors of RAP. Methods:The clinical data of 43 patients with RAP and 130 patients with only one time AP(control group)were retrospectively analyzed. Risk factors of recurrence of RAP were analyzed. Results:Univariate analysis showed that no significant differences in etiology,severity, serum levels of cholesterol,calcium,white blood cell count,amylase,ALT,AST,total bilirubin,direct bilirubin and C-reactive protein were found between RAP group and control group(P > 0. 05). Serum triglyceride level,blood glucose level and CT score in RAP group were significantly higher than those in control group(t = 3. 260,P < 0. 05;t = 2. 720, P < 0. 05;t = 2. 162,P < 0. 05). Logistic regression analysis demonstrated that serum triglyceride level,blood glucose level and CT score were risk factors of RAP(oR = 1. 86,95% CI:1. 05-3. 68,P = 0. 03;oR = 1. 23,95% CI:1. 01-1. 50,P = 0. 04;oR = 2. 46,95% CI:1. 00-6. 03,P = 0. 04). Conclusions:The recurrence of RAP is related with serum triglyceride level,blood glucose level and CT score.