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Objective To investigate the abnormal expression of Golgi protein 73(GP73) in CD4+ T lymphocytes of the pa-tients with hepatocellular carcinoma (HCC) and its influence of Th1/Th2/Th17 subtype differentiation .Methods Fifty cases of HCC hospitalized in this hospital from May 2015 to February 2016 and 50 healthy volunteers as controls were selected .Peripheral blood was collected and CD4+ T lymphocytes were isolated ;then the expression levels of GP73 and nuclear factor kappa-light-chain-enhancer (NF-κB) in CD4+ T lymphocytes were determined by using RT-qPCR and Western blotting methods ;furthermore ,the se-cretion levels of IL-4 ,IL-17 and IFN-γin the supernatants were examined by using ELISA method .Results GP73 mRNA expres-sion in peripheral blood CD4+ T lymphocytes in the HCC patients were significantly up-regulated compared with the healthy volun-teers ,the difference was statistical difference (P<0 .05) .The expression level of in GP73 overexpression group was significantly in-creased(P<0 .05) ,while which in the GP73 interference group was significantly decreased (P<0 .05) .Over-expression of GP73 in-duced significant increase of IL-4 and IL-17 levels and significant decrease of IFN-γ(P<0 .05);silencing GP73 induced marked de-crease in the expression of IL-4 and IL-17 in CD4+ cells and obvious increase of IFN-γ(P<0 .05) .Conclusion GP73 is over-ex-pressed in peripheral blood CD4+ T cells of HCC patients ,moreover GP73 is very likely to participate in the inflammatory reaction by activating NF-κB to cause the unbalance of Th1/Th2/T17 and promote the occurrence and development of HCC .
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Objective To explore the mechanism of mTOR-mediated liver cancer cell invasion.Methods q-PCR was used to check the expression of miR-27a and GP73;miR-27a mimics were transfected into GP73-high expressing M97H cells and miR-27a inhibitors were transfected into GP73-low expressing HepG2 cells,q-PCR and Western blot were performed to observe the expression of GP73;Dual-luciferase assay was also performed to verify the binding sites of miR-27a in GP73 3'UTR;miR-27a mimics were transfected into M97H cells and miR-27a inhibitors were transfected into HepG2 cells,Transwell assay was used to measure cell invasion.Results mTOR downregulated miR-27a and upregulated GP73;GP73 was downregulated by miR-27a and upregulated by miR-27a suppression;GP73 was a target gene of miR-27a;miR-27a inhibited the invasion of M97H cells rather than HepG2 cells.Conclusions miR-27a is negatively regulated by mTOR and inhibits liver cancer cell invasion via targeting GP73.
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Objective@#To investigate the expression of miR-212 and miR-132 in the serum of patients with primary liver cancer and their targeted regulation of GP73.@*Methods@#The patients with liver cancer, chronic hepatitis B, or liver cirrhosis who were hospitalized in Taizhou People’s Hospital from January 2015 to December 2016 were enrolled, and healthy volunteers were also enrolled as controls. Quantitative real-time PCR was used to measure the serum levels of miR-212 and miR-132, and the association between the expression of serum miR-212 and miR-132 and the clinicopathological features of patients with liver cancer was analyzed. A Spearman’s rank correlation analysis was used to analyze the correlation between serum miR-212/miR-132 and GP73. Western blot was used to measure the protein expression of GP73, and MTT assay was used to measure the survival rate of cells. The Levene’s homogeneity of variance test was used for data analysis. The independent samples t-test was used for comparison of means between two samples, and ANOVA was used for comparison of means between multiple samples.@*Results@#A total of 90 patients with liver cancer, 60 with chronic hepatitis B, 68 with liver cirrhosis, and 100 healthy volunteers were enrolled. The relative expression levels of miR-212 and miR-132 in serum were 0.046 6 ± 0.024 7 and 0.005 9 ± 0.003 0 in the patients with liver cancer, 0.979 7 ± 0.259 5 and 1.001 8 ± 0.249 9 in the healthy volunteers, 0.588 2 ± 0.216 5 and 0.345 7 ± 0.233 8 in the patients with hepatitis, and 0.313 8 ± 0.153 3 and 0.080 1 ± 0.042 66 in the patients with liver cirrhosis. Compared with the normal controls, all patients had significant reductions in the expression of serum miR-212 (t = 10.26, 20.86, and 35.80, all P < 0.01) and miR-132 (t = 16.55, 36.09, and 39.85, all P < 0.01). In the patients with liver cancer, the relative expression of miR-212 and miR-132 was negatively correlated with alpha-fetoprotein (miR-212: t = -4.46, P < 0.01; miR-132: t = -4.83, P < 0.01), TNM stage (miR-212: t = 6.569, P < 0.01; miR-132: t = 7.31, P < 0.01), degree of tumor differentiation (miR-212: t = 5.268, P < 0.01; miR-132: t = 5.914, P < 0.01), and presence of portal vein tumor thrombus (miR-212: t = 5.16, P < 0.01; miR-132: t = 3.681, P < 0.01), while it was not correlated with tumor size (miR-212: t = 0.687, P > 0.05; miR-132: t = 0.887, P > 0.05). In addition, serum miR-212 and miR-132 were negatively correlated with GP73 in the patients with liver cancer (miR-212: rs = -0.709, P < 0.01; miR-132: rs = -0.877, P < 0.01). Overexpression of miR-212 or miR-132 in HepG2 cells significantly inhibited the activity and expression of 3’-UTR, and interference of miR-212 or miR-132 significantly increased the activity and expression of 3’-UTR in GP73. Overexpression of GP73 reversed the reduction in survival rate of hepatoma cells induced by the overexpression of miR-212 or miR-132.@*Conclusion@#Patients with liver cancer have a significant reduction in the expression of miR-212 and miR-132 in serum, which is closely associated with the development, progression, and metastasis of liver cancer, and miR-212 and miR-132 in hepatoma cells inhibit the growth of liver cancer by targeted regulation of GP73 expression.
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Objective To study the effect of Golgi protein 73(GP73) on inflammation, and to reveal the effect of GP73 on tumorigenesis and metastasis.Methods The transcriptional activity of NF-κB and the expression of IL-1β, IL-6 and TNF-αwith GP73 overexpression or knockdown were detected to illuminate the role of GP73 in inflammation.According to the TCGA database, the correlation between the transcriptional activity of GP73 and the expression of NF-κB, IL-1β, IL-6 and TNF-αwas analyzed to determine the role of GP73 in tumor inflammation.Results Correlative analysis showed that there was a positive correlation between the expression of GP73 with NF-κB, IL-1β, IL-6 and TNF-α.The transcriptional activity of NF-κB was upregulated by GP73 overexpression, but downregulated by GP73 knockdown.The expression of IL-1β, IL-6 and TNF-αwas upregulated by GP73 overexpression.Ammonium pyrrolidinedithiocarbamate ( PDTC ) was in-volved in inflammation reaction induced by GP73.Conclusion GP73 is possibly involved in inflammation and promotes tu-morigenesis and metastasis.
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Objective To knock out the GP73 gene in H22 cells originating in mice using CRISPR/Cas9 gene editing system and construct H22 GP73 gene knockout stable strain for identification of its functions .Methods Two pairs of sgRNAs that could specifically identify the upstream and downstream of GP 73 gene first promoter were designed before a recombinant eukaryotic expressional plasmid was constructed using pX 459 .After enzyme digestion and sequencing , two pairs of recombinant plasmids were co-transfected into H22 cells before puromycin was used to screen positive cells and monoclonal cells which stably knocked out GP 73 gene were developed .The knockout effect was measured by Western blotting.Cell Titer 96? AQueous One Solution Assay was used to detect the effect on cell reproductive capacity when the GP73 was knocked out .The transferability was detected through wound healing test .Results The result of Western blotting suggested that GP73 protein was undetected in the construction of H22 GP73 knockout gene stable strain after transfection.The transfer and reproduction slowed down .Conclusion H22 GP73 gene knockout stable strain can be successfully built using CRISPR/Cas9 gene editing system ,thus facilitating studies on the function of GP 73 in hepatocarcinogenesis .
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Objective To investigate changes of GP73 after hepatectomy and its correlations with hepatocellular carcinoma (HCC) recurrence. Methods Perioperative serum GP73 was monitored in hepatic hemangioma and HCC patients undergoing hepatectomy. Clinicopathologic features and follow-up results were collected to evaluate the relationship between serum GP73 level and patients' prognosis.Results There was no statistical difference between preoperative GP73 and postoperative GP73 in hepatic hemangioma group.While preoperative GP73 in HCC group was 9.9(3.7 - 15.8) relative unit (RU),and that on POD3 (postoperative day 3 ) was 9.1 ( 3.4 - 13.3 ) RU,on POD7 was 74.3 ( 1.7 - 9.0) RU,on POD14 was 3.3(2.1 -5.4) RU ( F =72.606,P ＜ 0.001 ).HCC recurred in 21 cases during follow-up,GP73 in recurrent cases [ 11.0 (8.4 - 13.8 ) RU ] was significantly higher than postoperative trough values while it was not different from their preoperative GP73 level [ 9.9 ( 2.9 - 15.0) RU ] ( Z =1.185,P ＞0.05). The preoperative GP73 level between recurrent subgroup and nonrecurrent subgroup was not significantly different (Z =- 1.546,P ＞ 0.05 ).Preoperative GP73 did not correlate to patients' survival.Conclusions Hepatectomy for HCC leads to a significant decrease of GP73 and postoperative HCC recurrence accompanies reelevation of GP73. GP73 could be used as a postoperative monitor for HCC recurrence.
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Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with the highest cause of death and increasing incidence worldwide, and the annual incidence rate is rising. The early diagnosis of HCC is very essential to its prognosis. At present, AFP has been widely used in the survey of HCC diagnosis, therapeutic effect and predict recurrence. However, the sensitivity and specificity of AFP is not satisfactory. In recent years a variety of new serum tumor markers have emerged one after another. A new serum marker-Golgi glycoprotein-73(GP-73) in early diagnosis of HCC is expected to become the new target maker,its sensitivity and specificity are better than AFP.
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Hepatocellular carcinoma(HCC)is one of the malignant tumors with the highest cause of death and increasing incidence worldwide.Accurate diagnosis in early stage is vital for the treatment of patients.Presently,routine screening strategies including ?-fetoprotein(AFP)and ultrasound every 6 months have been recommended for early detection in patients with liver cirrhosis to detect HCC at earlier stages.However,the sensitivity and specificity of AFP are far from satisfaction.With the development of recently techniques such as proteomics,it is possible for new HCC-specific markers being available in the near future.Golgi Protein 73(GP73)is most likely to be a promising serum marker.In a few available literatures,GP73 has sensitivity and specificity of 69% and 90%.And fucose-studded GP73 has sensitivity and specificity of 90% and 100%.Apart from GP73,lens culinaris agglutinin-reactive AFP,des-gamma carboxyprothrombin,?-L-fucosidase,glypican-3,hepatocyte growth factor,transforming growth factor-?1,vascular endothelial growth factor,and mucin 1 have been proposed as markers for HCC detection.Clinical trials are undergoing to estimate the value of the above markers,and may change the status in quo ofthe diagnosis of HCC.We have started a multi-centeral trial to investigate GP73 in HBV and HBV related HCC patients with positive preliminary results.