[目的]研究shRNA抑制eEF1A1表达对肝癌Hea-P细胞凋亡、增殖、迁移的影响,并通过分别下调eEF1A1和ANXA7的表达,探讨肝癌Hca-P细胞中eEF1A1、eEF1 A2与ANXA7表达的相关性及潜在的相互作用.[方法]构建靶向eEF1A 1基因的shRNA质粒表达载体,瞬时转染至小鼠肝癌Hca-P细胞中,设无处理组(CON) 、eEF1A1-shRNA组(eEF1A 1-shRNA)、转染pGPU/GFP/Neo-NC的阴性埘照组(NC)三组细胞进行实验.采用实时荧光定量PCR和Western blot技术检测eEF1A1的抑制效果,并分别检测各组eEF1 A2、ANXA7在mRNA和蛋白水平的表达.CCK-8法、流式细胞仪、Transwell小室分别检测对细胞增殖、凋亡、迁移的影响.此外,复苏已稳定低表达ANXA7的Hca-P细胞,通过实时荧光定量PCR和Western blot技术检测无处理组(CON)、ANXA7-shRNA转染组(ANXA7-shRNA)、阴性对照组(NC)三组Hca-P细胞中eEF1A1、eEF1A2在mRNA和蛋白水平的表达.[结果]eEF1A1靶向shRNA成功下调小鼠肝癌Hca-P细胞中eEF1A1基因的表达.流式细胞仪检测显示,CON组、eEF1A1-shRNA组、NC组细胞凋亡率分别为2.85％±0.37％、4.71％±0.21％和3.01％±0.33％,eEF 1A1-shRNA组的细胞凋亡较CON组和NC组明显增加(P＜0.05):CCK-8实验发现,eEF1 A1-shRNA转染细胞后细胞增殖能力较CON组和NC组明显下降;Transwell实验显示,CON组、eEF1A1-shRNA组、NC组迁移细胞数目为123.75±9.26、66.84±17.47、115.31±12.59,eEF1A 1-shRNA组细胞迁移能力较CON组和NC组显著减弱(P＜0.05).qRT-PCR和Western blot检测显示,eEF1A1-shRNA组eEF1A2、ANXA7在mRNA和蛋白水平的表达量较CON组和NC组明显降低(P＜0.05),相较其无处理组的蛋白表达水平,eEF1A2和ANXA7分别下降了57％和21％;ANXA7-shRNA组中eEF1A1、eEF1A2在mRNA和蛋白水平的表达量较CON组和NC组亦明显降低(P＜0.05).[结论]通过靶向eEF1A1的shRNA使eEF1A1低表达,可诱导细胞凋亡,抑制细胞的增殖能力及迁移能力.eEF1A1 、eEF1A2与ANXA7的表达密切相关,且eEF1A1与ANXA7存在相互作用,可能共同参与调控肿瘤的发生与发展.

Objective To investigate the radioprotective function and its mechanism of Sulforaphane (SF) in mice acute radiation-induced lung injury.Methods Totally 40 female C57BL/6J mice were equally divided into 5 groups randomly.Group A,treated by SF 3 mg/kg plus radiation;group B,treated by SF 5 mg/kg plus radiation;group C,treated by SF 10 mg/kg plus radiation;radiation group with a single dose of 12 Gy in 6 MV X-ray by a linear accelerator,and control group with sham radiation.The mice in drug group were administered intraperitoneally with different concentration of SF every other day from 7 d before irradiation to 7 d after irradiation,while the same volume of DMSO plus physiological saline solvent was given in the control and radiation groups.After being sacrificed at 14 d of SF administration,the pathomorphological changes of mice were observed in trauma lung tissue,the positioning and expression of NLRP3 was observed by immunohistochemical staining,the levels of IL-6,TNF-α and TGF-β1 in bronchoalveolar lavage fluid (BALF) were measured by ELISA,the expressions of NLRP3 and IL-1β mRNA in lung tissue were assayed by qRT-PCR,the expressions of NF-κB p65,NLRP3 and IL-1β proteins in lung tissue were assayed by Western blot,the activity of NF-κB was detected by EMSA.Results In comparison with radiation group,there was an obvious amelioration in pathological injury of lung tissue in the treatment groups:the expression of NLRP3 in lung tissue decreased;the concentration of NLRP3 in the drug intervention group (SF 10 mg / kg) markedly decreased (F =42.750,P ＜ 0.05).the IL-6,TNF-a and TGF-β1 levels in BALF decreased (tIL-6 =-62.65-21.00;tTNF-α =-32.18-16.57;tTGF-β1 =-58.22-46.11,P ＜ 0.05);the expressions of NLRP3 and IL-1β mRNA markedly decreased (tNLRP3 =-6.56-5.68;tIL-1β =-29.75--21.20,P ＜ 0.05),and the expressions of NF-κB p65,NLRP3 and IL-1β proteins decreased (tNF-κB p65 =-34.00--1.71,tNLRP3 =-25.01--16.91,tIL-1β =-73.70--55.14,P ＜ 0.05);the relative expressions of NF-κB p65 and NLRP3 were reduced in a dose-dependent manner (r =0.945,0.926);and the activity of NF-κB were obviously reduced (tNF-κB =-38.68,-614.82,-2 831.40,P ＜ 0.05).Conclusions Sulforaphane effectively alleviates the RILI in lung of mice by downregulating the expressions of inflammatory factor NLRP3.

Portal hypertension can lead to ectopic varices, which occur most frequently in the rectum. Rectal variceal bleeding in patients with portal hypertension is rare but can be life-threatening if not diagnosed and treated in a timely manner. However, no specific treatment guidelines have been established for rectal variceal bleeding. We herein report a case involving a woman with portal hypertension due to autoimmune liver disease who was successfully treated with a transjugular intrahepatic portosystemic stent shunt and variceal embolization. We recommend treatment of refractory ectopic variceal bleeding with a transjugular intrahepatic portosystemic stent shunt in combination with embolization.

Background SDF1/CXCR4 and AnnexinA7 play important roles in many physiological and pathological conditions, but the molecular association between them in cancer cells has not been studied thus far. Methods The expression changes of SDF1/CXCR4 were detected by gene transcriptome sequencing, qRT-PCR, Western blotting, cytoimmunofluorescence and immunohistochemistry in mouse hepatocarcinoma F/P cells, AnnexinA7 downregulated expression F (F A7DOWN ) cells, AnnexinA7 overexpression P (P A7UP ) cells, AnnexinA7 unrelated sequence F (F SHUS ) cells, empty vector P (P NCEV ) cells and normal liver cells in vitro and in vivo . Results SDF1 and CXCR4 were co-expressed in hepatocarcinoma cells. SDF1 was localized mainly in the cytoplasm of cells, while CXCR4 was mainly localized in the cell membrane. Both in vitro and in vivo , expression levels of SDF1/CXCR4 in F and P cells were higher than in normal liver cells, and expression levels of SDF1/CXCR4 in F cells with high lymphatic metastatic potential were higher than those in P cells with low lymphatic metastatic potential . Expression of SDF1 was higher than that of CXCR4 in P cells and normal liver cells, while expression of CXCR4 was higher than that of SDF1 in F cells. Expression levels of SDF1/CXCR4 were completely consistent with AnnexinA7 regulation. After the AnnexinA7 gene was downregulated or upregulated, expression levels of SDF1/CXCR4 in F A7DOWN /P A7UP cells were lower or higher than those in F SHUS /P NCEV cells . Furthermore, CXCR4 was more sensitively modulated by AnnexinA7 regulation than SDF1. Conclusions High co-expression of SDF1/CXCR4 is a molecular characteristic of hepatocarcinoma cells, especially those with high lymphatic metastatic potential. AnnexinA7 positively regulates expression levels of SDF1/CXCR4, in particular CXCR4, and AnnexinA7 is a functional regulator of SDF1/CXCR4 .

Our previous study (Oncotarget 2016; 7:46) demonstrated that the over-expression of sulfatase-1 in murine hepatocarcinoma Hca-F cell line (a murine HCC cell with lymph node metastatic [LNM] rate of >75%) downregulates mesothelin and leads to reduction in lymphatic metastasis, both in vitro and in vivo. In current work, we investigated the effects of Sulf-1 knockdown on mesothelin (Msln) and it’s effects on the in vitro cell proliferation, migration, invasion, and in vivo tumor growth and LNM rate for Hca-P cells (a murine HCC cell with LNM rate of <25%). Western blotting and qRT-PCR assay indicated that both in vitro and in vivo Sulf-1 was down-regulated by 75% and 68% and led to up regulation of Msln by 55% in shRNA-transfected-Sulf-1-Hca-P cells compared with Hca-P and nonspecific sequence control plasmid transfected Hca-P cell (shRNA-Nc-Hca-P). The in vitro proliferation, migration and invasion potentials were significantly enhanced following Sulf-1 stable down-regulation. In addition, Sulf-1 knock-down significantly promoted tumor growth and increased LNM rates of shRNA-Sulf-1-Hca-P-transplanted mice by 78.6% (11 out of 14 lymph nodes were positive of cancer). Consistent with our previous work, we confirmed that Sulf-1 plays an important role in hepatocarcinoma cell proliferation, migration, invasion and metastasis. The interaction between Sulf-1 and Msln is a potential therapeutic target in the development of liver cancer therapy.

Neuropilin-2 (NRP-2) not only functions as a receptor for semaphorins, a family of neural axon guidance factors, but also interacts with VEGFs, a family of vascular endothelial growth factors. As an independent receptor or a co-receptor, NRP-2 binds to ligands VEGF-C/D, activates the VEGF-C/D-NRP-2 signaling axis, and further regulates lymphangiogenesis-associated factors in both lymphatic endothelial cells (LECs) and some tumor cells during tumor progression. Via VEGF-C/D-NRP-2 axis, NRP-2 induces LEC proliferation, reconstruction and lymphangiogenesis and subsequently promotes tumor cell migration, invasion and lymphatic metastasis. There are similarities and differences among NRP-1, NRP-2 and VEGFR-3 in chemical structure, ligand specificity, chromosomal location, soluble protein forms, cellular functions and expression profiles. High expression of NRP-2 in LECs and tumor cells has been observed in different anatomic sites, histological patterns and progression stages of various tumors, especially during tumor lymphangiogenesis and lymphatic metastasis, and therefore the NRP-2 and VEGF-C/D-NRP-2 axis are closely related to tumor development, progression, invasion, and metastasis. In addition, it is important for prognosis of tumor. The studies on NRP-2 targeted therapy have recently achieved some successes, utilizing NRP-2 blocking antibodies, NRP-2 inhibitory peptides, soluble NRP-2 antagonists, small molecule inhibitors and various NRP-2 gene therapeutic strategies.

Hepatocellular carcinoma (HCC) ranks in the top of cancers leading to death. Early diagnosis is the big challenge in the case of HCC. Our in vitro study showed that Ezrin expression in lymphatic metastasis hepatocellular carcinoma (LNM-HCC) was associated with the metastatic rate. Here we aim to evaluate Ezrin expression as diagnostic and/or prognostic biomarker of LNM-HCC in mice. Chinese inbred 615 mice, Hca-F and Hca-P cell lines were used in the study. Histological changes were determined by Hematoxylin and Eosin, while Ezrin expression was assessed by qRT-PCR, western blot, immunohistochemistry, and enzyme-linked immunosorbent assay. Ezrin expression in this study gives credit to our in vitro study which Ezrin expression was positively correlated with LNM-HCC and negatively with Annexin7 (A7) expression. The highest histological changes were observed in high metastatic primary/secondary tumors combined with high Ezrin expression. Ezrin and A7 are higher in total primary tumors than in total secondary tumors (P = 0.0001, P = 0.021), respectively. Ezrin expression was enhanced in Hca-P A7 down-regulated primary/secondary tumors (P = 0.004), whereas, Ezrin expression was suppressed in Hca-F A7 upregulated primary/secondary tumors. Serum ELISA indicated differential expression of Ezrin among the study groups (P ≤ 0.0001). Ezrin expression was higher in NC-Hca-F than NC-Hca-P (P ≤ 0.0001), suppressed in Hca-F A7 upregulation (P ≤ 0.0001) and in enhanced in Hca-P A7 down-regulation (P = 0.0001). In conclusion, Ezrin level may serve as a differential diagnostic and/or prognostic biomarker for high and low LNM-HCC and may be beneficial in the diagnosis of HCC disease. © 2017 BioFactors, 43(5):662-672, 2017.

Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that up regulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 down regulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its over expression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy.