Ech1 is a potent suppressor of lymphatic metastasis in hepatocarcinoma

We have previously demonstrated that Ech1 is involved in the lymphatic metastasis of tumors in vitro. Here, we gain an insight into the role that Ech1 is playing in Hca-F cell. The expression of Annexin A7, Gelsolin and Clic1 genes, which were also relevant to tumor lymphatic metastasis, had been inhibited due to downregulation Ech1 gene by Western blot analysis. And downregulated of Ech1 inhibits the metastasic capability of Hca-F cells to peripheral lymph nodes in vivo. Our work indicates although the involvement of Ech1 in tumor metastasis development and progression, but the subcellular location of Ech1 has not much contribution to that.     

Evaluation of Annexin A7, Galectin-3 and Gelsolin as possible biomarkers of hepatocarcinoma lymphatic metastasis

We have previously demonstrated that Annexin A7 is involved in the lymphatic metastasis of hepatocarcinoma in vitro. The expression of Galectin-3 and Gelsolin, which were also relevant to tumor lymphatic metastasis, had shown the same tendency concordantly with the expression of Annexin A7 alteration by qRT-PCR and Western blot analysis. Here, we gain an insight into the role that Annexin A7 is playing in Hca-P, P_{Anxa7-upregulated} and P_{Anxa7-downregulated} cells in vivo. Then, Hca-P, P_{Anxa7-upregulated} and P_{Anxa7-downregulated} cells were injected into a mouse footpad to establish primary tumors in mice. On the fourth week after HCC cells inoculation, the mice were sacrificed for inspection the expression of Annexin A7, Galectin-3 and Gelsolin in primary tumors and in serum. Our work indicates that Annexin A7 and Gelsolin are both valuable in tumors and in serum evaluating lymph node metastasis in mice with hepatocarcinoma; Galectin-3 in tumors is significant but no much contribution in serum.     

Annexin A7 gene is an important factor in the lymphatic metastasis of tumors

In the tumor malignancy progression, lymph node metastasis (LNM) is recognized as an important factor. In this study, RNA interference (RNAi) was employed to down-regulate ANXA7 gene in Hca-F cells, a hepatocarcinoma cell line with high LNM rate. There was no significant effect on cell proliferation ability, but cell division, motility, and invasion abilities were markedly inhibited. By contrast, up-regulating the expression of ANXA7 gene in Hca-P cells with lower LNM rate, cell migration ability was improved and the percentage of cells in S phase was significantly decreased in vitro. Here, we reported that the expression of Ech1, GSN and JNK1 genes, which were relevant to tumor lymphatic metastasis, had been inhibited due to down-regulation ANXA7 gene and promoted due to up-regulation ANXA7 gene by western blot analysis. These results indicated that ANXA7 is a critical factor in the development of lymphatic metastasis in hepatocarcinoma progression.     

Anxa5 mediates the in vitro malignant behaviours of murine hepatocarcinoma Hca-F cells with high lymph node metastasis potential preferentially via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) and E-cadherin

ObjectiveAnnexin A5 (Anxa5) is associated with the progression of some cancers, while its role and regulation mechanism in tumor lymphatic metastasis is rarely reported. This study aims to investigate the influence of Anxa5 knockdown on the malignant behaviours of murine hepatocarcinoma Hca-F cell line with high lymph node metastatic (LNM) potential and the underlying regulation mechanism.MethodsRNA interfering was performed to silence Anxa5 in Hca-F. Monoclonal shRNA-Anxa5- Hca-F cells were obtained via G418 screening by limited dilution method. Quantitative real-time RT-PCR (qRT-PCR) and Western blotting (WB) were applied to measure Anxa5 expression levels. CCK-8, Boyden transwell-chamber and in situ LN adhesion assays were performed to explore the effects of Anxa5 on the proliferation, migration, invasion and adhesion capacities of Hca-F. WB and qRT-PCR were used to detect the level changes of key molecules in corresponding signal pathways.ResultsWe obtained two monoclonal shRNA-Anxa5-transfected Hca-F cell lines with stable knockdowns of Anxa5. Anxa5 knockdown resulted in significantly reduced proliferation, migration, invasion and in situ LN adhesion potentials of Hca-F in proportion to its knockdown extent. Anxa5 downregulation enhanced E-cadherin levels in Hca-F. Moreover, Anxa5 affected Hca-F behaviours specifically via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) instead of p38MAPK/c-Jun, Jnk/c-Jun and AKT/c-Jun pathways.ConclusionsAnxa5 mediates the in vitro malignant behaviours of murine hepatocarcinoma Hca-F cells via ERK2/c-Jun/p-c-Jun(Ser73) and ERK2/E-cadherin pathways. It is an important molecule in metastasis (especially LNM) and a potential therapeutic target for hepatocarcinoma.     

Inhibition of annexin A7 gene and protein induces the apotosis and decreases the invasion,migration of the hepatocarcinoma cell line

Our previous studies have shown that annexin A7 (ANXA7) gives different expressions in the mouse hepatocarcinoma cell lines with low or high lymphatic metastatic potential in both gene and protein levels. In this study, whether by using RNA interference (RNAi) technique downregulating ANXA7 in the gene level or by using antibody against ANXA7 in the protein level, the depressed expression of ANXA7 could induce apotosis and decrease the invasion, migration capacities of the Hca-P cell, a hepatocarcinoma cell line with low lymphatic metastatic potential in vitro. The results indicate that ANXA7 is an important factor in tumors with the lymphatic metastasis.     

Annexin A7 and its binding protein galectin-3 influence mouse hepatocellular carcinoma cell line in vitro

Lymph node metastasis is recognized as an important mode of liver cancer metastasis. Our previous study has built two hepatocarcinoma cell lines, Hca-F with high (75%) and Hca-P with low (25%) incidences of lymph node metastasis, and has indicated that annexin A7 is an important factor in the lymphatic metastasis of tumors. There is evidence that galectin-3 is the binding protein of annexin A7 and works in protein complexes. Our current study shows that both annexin A7 and galectin-3 express higher in Hca-F than Hca-P. Annexin A7 was successfully down-regulated in Hca-P by RNA interference, and this resulted in concomitant reduction of galactin 3 expression in annexin A7 down regulated compared to the control and N-control cells. Using CCK-8 assay, the expression level of annexin A7 and galectin-3 were found to have correlation with the proliferation ability; Transwell assay showed annexin A7 and galectin-3 are involved in cell migration and invasion regulation in mouse hepatocellular carcinoma cell lines, immunofluorescence assay indicate annexin A7 and galectin-3 were co-located annexin A7 and galectin-3 played roles in DNA damage and cell proliferation cycle checkpoint arrest pathway. Those phenomena indicated that annexin A7 influences lymphatic metastasis of tumors by interacting with galectin-3 through the regulation of tumor cell proliferation, attachment, migration and invasion.     

Enoyl coenzyme A hydratase 1 is an important factor in the lymphatic metastasis of tumors

We have previously demonstrated that enoyl coenzyme A hydratase 1 (Ech1) is involved in the lymphatic metastasis of tumors. In this study, RNAi was used to investigate the role of Ech1 in Hca-F, a hepatocarcinoma cell line with high rates of lymphatic metastasis. The downregulation of Ech1 inhibited proliferation of the Hca-F cells, increased the ratio of Hca-F cells in S phase to G₁ phase and decreased the adhesion and migration capacities of Hca-F cells. A higher expression level of Ech1 was confirmed in tissue from patients with gastric carcinoma (GC) with lymph node metastases (LNM), indicating the clinical association with tumor metastasis. The results indicate that Ech1 is a critical factor in the development of lymphatic metastasis in these tumors.     

CRKL knockdown promotes in vitro proliferation,migration and invasion,in vivo tumor malignancy and lymph node metastasis of murine hepatocarcinoma Hca-P cells

Our previous study (Biomed Pharmacother 2015;69:11) demonstrated that the over-expression of CRKL, a chicken tumor virus number 10 regulator of kinase-like protein, suppresses in vitro proliferation, invasion and migration of murine hepatocarcinoma Hca-P cell, a murine HCC cell with lymph node metastatic (LNM) rate of ∼25%. In current work, we investigated the effects of CRKL knockdown on the in vitro cell proliferation, migration and invasion, and on the in vivo tumor malignancy and LNM rate and level for Hca-P cells. Western blotting assay indicated that CRKL was down-regulated by ∼90% in a monoclonal CrkL-shRNA-transfected Hca-P cells. Compared with Hca-P and unrelated-shRNA-transfected Hca-P cell, the in vitro proliferation, migration and invasion potentials were significantly enhanced following CRKL stable deregulation. CRKL knock-down significantly promoted the tumorigenicity malignancy, LNM rates and level of Hca-P-transplanted mice. Consistent with our previous work, it can be concluded CRKL plays an important role in hepatocarcinoma cell proliferation, invasion and migration as well hepatocarcinoma malignancy and metastasis. It functions as a potential tumor suppressor in hepatocarcinoma.     

RACK1 promotes the proliferation,migration and invasion capacity of mouse hepatocellular carcinoma cell line in vitro probably by PI3K/Rac1 signaling pathway

Hca-P and Hca-F is a pair of synogenetic mouse hepatocarcinoma ascites cell lines, possessing different capacity of lymphatic metastasis. Receptor of activated C-kinase 1 (Rack1), together with Jnk1 and gelsolin (Gsn) were previously identified as differentially expressed proteins for lymphatic metastatic potential between the two cell lines. As an intracellular scaffold protein, Rack1 could recruit such signaling molecules as integrins, Src, PKC which are involved in many important biological processes and play key roles in cancer progression. In our present studies, pCDNA3.1(+)-Rack1, a eukaryotic expression plasmid, was constructed and stably transfected into Hca-P cells with a low metastatic potential. CCK8 assay and transwell system were used to evaluate the effects of Rack1 on proliferation, migration and invasion of Hca-P cells in vitro. Then, LY294002, an inhibitor of PI3K, was added into the culture medium of pCDNA3.1(+)-Rack1-Hca-P cells and their biological behaviors observed further. Moreover, the expression of Jnk1, Rac1 and Gsn of pCDNA3.1(+)-Rack1-Hca-P cells were detected by western blot after pretreated with various doses of LY294002. As a result, the proliferation, migration and invasion of pCDNA3.1(+)-Rack1-Hca-P cells were significantly enhanced and could be inhibited by LY294002. In addition, the expression of Gsn, Rac1 and Jnk1 of pCDNA3.1(+)-Rack1-Hca-P cells also decreased after pretreated with LY294002. The expression of Gsn can be inhibited by NSC33766 (an inhibitor of Rac1). Taken together, Rack1/PI3K/Rac1 signaling pathway may play a crucial role in malignant biological behaviors of mouse hepatocarcinoma cells with lymphatic metastasis potential. It may be a potential target for therapy of cancer lymphatic metastasis.     

Annexin—A1在胃食管交界处腺癌中的表达及临床意义

目的研究Annexin-A1在胃食管交界处不同组织中的表达,探讨其与胃食管交界处腺癌发生、发展的关系及临床意义。方法应用免疫组化S-P法检测Annexin-A1在20例胃食管交界处正常黏膜组织、20例胃食管交界处上皮内瘤变组织、66例胃食管交界处腺癌组织中的表达情况。结果胃食管交界处腺癌中Annexin-A1蛋白的阳性表达率（71．2％）明显高于上皮内瘤变组织（55．0％）及正常黏膜组织（25．0％）,差异具有统计学意义（P〈0．05）;Annexin-A1的表达与胃食管交界处腺癌临床病理分期、组织分化程度、淋巴结转移、浸润深度密切相关（P〈0．05）,但与胃食管交界处腺癌患者的性别、年龄、及远处转移无显著关系（P〉O．05）。结论Annexin-A1的异常表达可能在胃食管交界处腺癌的发生、发展中起重要作用。     

Fucoidan-induced ID-1 suppression inhibits the in vitro and in vivo invasion of hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a fast growing tumor associated with a high tendency for vascular invasion and distant metastasis. Recently, we reported that fucoidan displays inhibitory effect on proliferation and invasion of HCC cells. In this study, we investigated the anti-metastatic effect of fucoidan on HCC cells and the key signal that modulates metastasis. The anti-metastatic effect of fucoidan was evaluated in vitro using an invasion assay with human HCC cells (Huh-7, SNU-761, and SNU-3085) under both normoxic (20% O₂ and 5% CO₂, at 37 °C) and hypoxic (1% O_2, 5% CO₂, and 94% N_2, at 37 °C) conditions. Complementary DNA (cDNA) microarray analysis was performed to find the molecule which is significantly suppressed by fucoidan. In vivo study using a distant metastasis model by injecting SNU-761 cells into spleen via portal vein was performed to confirm the inhibitory effect by small interfering RNA (siRNA) transfection. Immunoblot analyses were used to investigate the signaling pathway. Fucoidan significantly suppressed the invasion of human HCC cells (Huh-7, SNU-761, and SNU-3085). Using cDNA microarray analysis, we found the molecule, ID-1, which was significantly suppressed by fucoidan treatment. Downregulation of ID-1 by siRNA significantly decreased invasion of HCC cells, both in vitro and in vivo (both P < 0.05) in a NDRG-1/CAP43-dependent manner. In immunoblot assay, downregulation of ID-1 by siRNA decreased the expressions of epithelial-mesenchymal transition markers including CK19, vimentin, MMP2, and fibronectin. Immunofluorescence study also revealed that actin rearrangement was inhibited when ID-1 was down-regulated in HCC cells. Interestingly, in SNU-761 cells, the ID-1 expressions under hypoxic conditions were lower as compared to those under normoxic conditions. Under hypoxic conditions, HIF-1α up-regulated NDRG-1/CAP43, while HIF-2α down-regulated ID-1, which might be a compensatory phenomenon against hypoxia-induced HCC invasion. In conclusion, NDRG-1/CAP43-dependent down-regulation of ID-1 suppressed HCC invasion both in vitro and in vivo, which was modulated by fucoidan treatment. Moreover, the compensatory down-regulation of ID-1 against hypoxia-induced HCC invasion was observed. ID-1 is a novel therapeutic target for the treatment of metastatic HCC.     

Chloride intracellular channel 1 is an important factor in the lymphatic metastasis of hepatocarcinoma

We have previously demonstrated that chloride intracellular channel 1 (CLIC1) is involved in the lymphatic metastasis of tumors. In this study, a self-designed shRNA sequence of mouse CLIC1 gene was synthesized and inserted into a pGPU6/GFP/Neo plasmid, then stably transfected into mouse hepatic carcinoma cell line Hca-F cells to down-regulate the expression of CLIC1 gene. The levels of expression of CLIC1 mRNA and protein were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and western blot (WB) analysis, respectively. The down-regulation of CLIC1 enhanced proliferative activity, increased the ratio of G2/M and decreased percentage of apoptosis. In addition, the capability of migration and invasion decreased significantly. The results indicate that CLIC1 is a critical factor in the development of lymphatic metastasis.     

Interference of lysine-specific demethylase 1 inhibits cellular invasion and proliferation in vivo in gastric cancer MKN-28 cells

BackgroundLysine-specific demethylase 1(LSD1), the first identified histone demethylase, plays an important role in the epigenetic regulation of gene activation and repression. Up-regulated LSD1expression has been reported in several malignant tumors.Our aim, therefore, was to better understand the mechanisms underlying the upregulation of LSD1 in gastric cancer.MethodsWe used lentiviral shRNA to knockdown LSD1 in the gastric cancer MKN-28 cell line. Cell proliferation was measured by MTT assay while cell apoptosis was assessed by Annexin V-FITC/PI double staining flow cytometry. The invasive potential of gastric cancer cells was determined by matrigel invasion assay. Protein expression was detected by Western blot. In vivo, the effect of knocking down LSD1 on tumor growth and protein expression in gastric cancer cells in nude mice was investigated.ResultsLSD1 knockdown in MKN-28 cell lines resulted in increasing the activity of cisplatin in vitro and the inhibition of cancer cell proliferation and invasion, and induced cell apoptosis. The expression of TGF-β1, VEGF, Bcl-2, β-catenin, p-ERK and p-Smad 2/3 proteins was inhibited in LSD1 knockdown cells. Moreover, in an in vivo model of gastric cancer, LSD1 knockdown suppressed tumor growth and protein expression.ConclusionLSD1 knockdown affected the fuction of gastric cancer MKN-28 cell line. LSD1 may be a latent target in the diagnosis and therapy of gastric cancer.     

Inhibitory effects of shRNA on expression of JNK1 and migration and invasion in mouse hepatocellular carcinoma cell lines mediated by ultrasound-targeted microbubble destruction

AimThe inhibitory effects on expression of JNK1 in mouse hepatocellular carcinoma cell lines and cell migration and invasion were mediated by ultrasound-targeted microbubble destruction (UTMD).Materials and methodsThe best shRNA vector was built and screened. The hepatocellular carcinoma cell lines were cultured in vitro and divided into five groups: the group of normal Hca-F cells, the group of shRNA plasmid (already selected from the above procedure), the group of Lipofectamine, the group of UTMD (ultrasound microbubbles combined with ultrasound exposure) and the group of Lipofectamine and UTMD. The transfection rate was observed by inverted fluorescence microscope. The expression levels of JNK1 mRNA and protein were evaluated by fluorescence quantitative PCR and Western Blot respectively. The cell proliferation was detected by CCK-8. The ability of migration and invasion in vitro was detected by transwell assay.ResultsThe best shRNA vector was established. The comparison of the transfection rate: The group of Lipofectamine and UTMD was larger than that of the groups of shRNA plasmid, Lipofectamine lipofection and UTMD (all P < 0.05). There was no significant difference between the group of Lipofectamine and the group of UTMD (P > 0.05). The comparison of the expression levels of JNK1 mRNA and protein: Both of the mRNA and protein expression levels were lowest in the group of Lipofectamine and UTMD (all P < 0.05). CCK-8 showed that cell viability decreased most in the group of Lipofectamine and UTMD (all P < 0.05); Transwell assay showed that the abilities of migration and invasion decreased most in the group of Lipofectamine and UTMD (all P < 0.05).ConclusionThe transfection rate of JNK1 shRNA can be improved through the combination of lipofection and UTMD in mouse hepatocellular carcinoma cell lines, therefore enhancing the inhibitory effects of gene expression. The inhibitory effects of cell proliferation, migration and invasion can also be enhanced.     

The microRNA-217 functions as a tumor suppressor and is frequently downregulated in human osteosarcoma

ObjectiveDysregulation of miRNA is always associated with cancer development and progression. Aberrant expression of miR-217 has been found in some types of cancer. However, its expression and function in osteosarcoma remain unclear. The aim of this study was to explore the effects of miR-217 in osteosarcoma tumorigenesis and development.MethodsThe expression level of miR-217 was quantified by real-time RT-PCR in human osteosarcoma cell lines and tissues. MTT, flow cytometric, transwell invasion and migration assays, and tumorigenicity in vivo were adopted to observe the effects of miR-217 on MG-63 cell phenotypes.ResultsMiR-217 was significantly downregulated in osteosarcoma cell lines and clinical specimens. Decreased miR-217 expression was significantly associated with large tumor size, positive distant metastasis, and advanced clinical stage. Low miR-217 expression in osteosarcoma was an independent predictor of poor survival. Overexpression of miR-217 can inhibit the proliferation, invasion, migration and promoted apoptosis of MG-63 cells in vitro and in vivo.ConclusionsThese findings indicate that miR-217 may act as a tumor suppressor in osteosarcoma and would serve as a novel therapeutic agent for miRNA-based therapy.     

DICER-AS1 functions as competing endogenous RNA that targets CSR1 by sponging microRNA-650 and suppresses gastric cancer progression

ObjectiveThis study explored the functional interactions between the long non-coding RNA DICER-AS1 and the cellular stress response 1 (CSR1) gene in gastric cancer.MethodsQuantitative polymerase chain reaction (qPCR) and western blotting were used to measure DICER-AS1, CSR1, and miR-650 expression levels. Gastric cancer cell line proliferation and migration abilities were analyzed using the MTT and transwell migration and invasion assays, respectively. Bioinformatic analysis and dual luciferase reporter assays were employed to study the functional interactions among miR-650, DICER-AS1, and CSR1.ResultsDICER-AS1 and CSR1 expression levels were significantly decreased in gastric cancer tissues compared with normal tissues, and qPCR analysis showed that miR-650 was upregulated in gastric cancer tissues. Bioinformatic analysis and dual luciferase reporter assays revealed that DICER-AS1 functioned as a competing endogenous RNA that sponged miR-650, which in turn regulated CSR1 expression. Importantly, ectopic DICER-AS1 and CSR1 expression inhibited cell proliferation and migration in vitro and suppressed xenograft tumorgenicity in vivo.ConclusionsThese results suggest that DICER-AS1 functions as a competing endogenous RNA that regulates miR-650 to suppress proliferation and migration of gastric cancer cells by targeting CSR1. These findings indicate that targeting DICER-AS1 and miR-650 could be a novel treatment for gastric cancer.