In vivo and in vitro effect of hepatocarcinoma lymph node metastasis by upregulation of Annexin A7 and relevant mechanisms.

We unveiled the association of Annexin A7 with vascular endothelial growth factor-C (VEGF-C) and the effect of upregulation of Annexin A7 in Hca-F and Hca-P cells on inhibiting hepatocarcinoma (HCC) lymph node metastasis (LNM) in vitro and in vivo. A total of 200 inbred 615 mice were randomly divided into four equal groups inoculated with Hca-F, Hca-P, FAnxa7-upregulated, and PAnxa7-upregulated cells, respectively. The primary tumor, popliteal, inguinal, and iliac lymph nodes were prepared for immunohistochemical (IHC) staining, real-time quantitative polymerase chain reaction (qRT-PCR) analysis, Western blot, and hematoxylin-eosin (H&E) staining. There was over 50 % increase both in the number of FAnxa7-upregulated and PAnxa7-upregulated cells migrated through the filter compared to their controls (FAnxa7-control, Hca-F and PAnxa7-control, Hca-P). However, no significant differences were noted in invasion ability between them (all P > 0.05). Tumor lymph vessels were significantly reduced in FAnxa7-upregulated and PAnxa7-upregulated tumors when compared with Hca-F and Hca-P tumors (all P < 0.05). Blood vessel density did not differ significantly between FAnxa7-upregulated and PAnxa7-upregulated tumors and Hca-F and Hca-P tumors. Enzyme-linked immunosorbent assay (ELISA) for VEGF-C showed that upregulating Annexin A7 decreased VEGF-C secretion in FAnxa7-upregulated and PAnxa7-upregulated cells (P < 0.05). The IHC staining result showed that the level of serum Annexin A7 was found to be statistically higher in all experimental groups than that in the control group (P < 0.05). The present results indicated that alterations in serum Annexin A7 expression may be of prognostic relevance in HCC lymphatic metastasis.

[Apoptosis of MDA-MB-231 cells induced by berberine alpha-hydroxy beta-decanoylethyl sulfonate].

To investigate the cell proliferation inhibition and apoptosis induced by berberine a-hydroxy f-decanoylethyl sulfonate (HB) on MDA-MB-231 cells in vitro, and the inhibitory effect of HB on the expression of poly adenosine diphosphate RNA polymerase (PARP), MTT assay was used to detect the viability of MDA-MB-231 cells and cell cycle was examined by flow cytometry. The results showed that HB could significantly inhibit the proliferation of MDA-MB-231 cells, and mildly arrested cell cycle progression at S phase. The IC50S for 24, 48 and 72 h treatment were 4.65, 1.46 and 0.75 mg.L-1 (7.55, 2.37 and 1.22 micromol.L-1), respectively. Annexin V-FITC/PI double staining assay showed that HB increased apoptotic ratio of MDA-MB-231 cells. Western blotting analysis showed the expressions of procaspase-3, procaspase-9 and PARP were decreased after HB treatment, while their fragment increased. The results suggest that HB can inhibit the growth and induce apoptosis of MDA-MB-231 cells, which may be associated with inhibition of the expression of procaspase-3, procaspase-9 and PARP.

[Effect of Ech1 overexpression on biological behavior of mouse hepatocarcinoma Hca-P cells in vitro].

OBJECTIVE: To investigate the effect of enoyl coenzyme A hydratase-1 (Ech1) on the proliferation and invasion ability of mouse hepatocarcinoma Hca-P cells in vitro. METHODS: Recombinant pcDNA3.1(+)-Ech1 gene and pcDNA3.1(+) were transfected into Hca-P cells by cationic liposomes introduction. Clone of PEch1 cells that stably expressing Ech1 and clone of control Pvector cells were screened by G418. The Ech1 expression was identified subsequently by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The malignant behaviors of the cell lines were compared by proliferation, invasion and migration test. RESULTS: The cell line Hca-P cells stably expressing Ech1 gene was constructed. The relative expression of Ech1 mRNA in the PEch1 group was 3.21 ± 0.43 and in the Pvector group was 1.44 ± 0.03, with a significant difference between the two groups (P = 0.029). The results of ELISA revealed that the expression of Ech1 protein was 0.140 ± 0.005 in the PEch1 group, 0.088 ± 0.003 in the Pvector group, and 0.078 ± 0.006 in the Hca-P group, showing a significant difference between the PEch1 group and the Pvector and Hca-P groups (P < 0.05). Transwell migration test showed that the number of penetrated cells in the PEch1 group was 143.00 ± 7.25 cells, significantly higher than that of the Pvector group (95.73 ± 3.88 cells) and un-treated Hca-1 group (106.67 ± 3.54 cells, both P < 0.05). The Transwell invasion assay showed that the number of penetrated cells was 77.20 ± 5.46 cells in the PEch1 group, significantly higher than 46.34 ± 4.35 cells in the Pvector group and 49.80 ± 5.21 cells in the un-treated Hca-1 group (both P < 0.05). CONCLUSIONS: The results showed that overexpressed Ech1 in Hca-P cells may significantly increase the cell proliferation in a time-dependent manner. The up-regulation of Ech1 may increase to some extent the migration and invasion capacity of Hca-P cells. The efforts aiming at up-regulation of Ech1 expression may become a therapeutic target in the treatment of hepatocarcinoma.

[Down-regulation of Ech1 decreases the adhesion ability of mouse hepatocarcinoma Hca-F cells].

To examine the differential expression pattern of Ech1 protein in mouse Hca-F and Hca-P hepatocarcinoma cell lines with high and low rates of lymphatic metastasis, respectively, and to investigate the relationships between Ech1 expression and adhesion of Hca-F cells. Fluorescence two-dimensional difference in-gel electrophoresis (2D DIGE) and mass spectrometry were used to detect Ech1 expression. Ech1 gene silencing was achieved by stable transfection of Hca-F cells with a plasmid vector harboring short hairpin RNA (shRNA) targeting Ech1, pGPU6/GFP/Neo-shRNA-Ech1. Ech1 mRNA and protein expressions were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting analysis, respectively. Adhesive properties of cells were assessed by hematoxylin-eosin staining and fluorimetric detection of extracellular matrix (ECM) proteins. Endogenous Ech1 protein level was remarkably higher in the highly metastatic Hca-F cell line than in the Hca-P cell line (2.7-fold by 2D DIGE; 1.5-fold by Western blotting). shRNA-induced silencing of Ech1 significantly reduced the adhesion ability of Hca-F cells, as evidenced by decreased absorbance values of fibronectin and collagen I (Hca-F cells vs. pGPU6/GFP/Neo-shRNA-Ech1 cells: 1.42+/-0.26 vs. 1.01+/-0.27 and 1.14+/-0.07 vs. 0.90+/-0.09, respectively; P less than 0.05). Down-regulation of Ech1 can inhibit the adhesive capacity of metastatic Hca-F cells.

Retraction Notice to: A Novel Mechanism of Doxorubicin Resistance and Tumorigenesis Mediated by MicroRNA-501-5p-Suppressed BLID.

[This retracts the article DOI: 10.1016/j.omtn.2018.06.011.].

[Expression of enoyl CoA hydratase 1 reduces cell proliferation and migration in mouse hepatocarcinoma cells].

OBJECTIVE: To study the expression of enoyl CoA hydratase 1 (ECH1) and the effect when down-regulation of ECH1 gene expression in mouse hepatocarcinoma cell. METHODS: Immunofluorescence was used for detecting the expression of ECH1, and stably transfected Hca-F cells with pGPU6/GFP/Neo-shRNA-ECH1 expression plasmids. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay. The Boyden-transwell assay (8 µm pore size) was performed to analyze the inhibitory effect of shRNA on Hca-F cell migration and invasion. RESULTS: ECH1 expression was obtained in the cytoplasm and upregulated expression in Hca-F cells than that in Hca-P cells. The down-regulation of ECH1 could inhibit the cell proliferation of Hca-F cells, decrease the number of cell pass through Transwell (27.07 ± 17.49) compared with scramble-negative (72.38 ± 18.83) and Hca-F controls (59.06 ± 30.33), decrease the migration capacities of Hca-F cells, increase the ratio of Hca-F cells in S phase (86.1%) compared with scramble-negative (75.8%) and Hca-F controls (66.2%) and decrease the ratio of G(1) phase (9.4%) compared with scramble-negative (24.2%) and Hca-F controls (30.3%). CONCLUSION: ECH1 serves as a potential critical factor attributes to tumor lymphatic metastasis.

Reclamation-induced tidal restriction increases dissolved carbon and greenhouse gases diffusive fluxes in salt marsh creeks.

Intertidal creeks play an important role in transporting nutrients between coastal ecosystems and ocean. Reclamation is a predominant anthropogenic disturbance in coastal regions; however, the influence of reclamation on carbon and nitrogen species and greenhouse gas (GHG) fluxes in creek remains unclear. In a subtropical salt marsh of eastern China, the seasonal patterns of dissolved carbon (DOC, DIC, CO2, and CH4) and inorganic nitrogen (NH4+-N, NO2--N, and NO3--N and N2O) species, and the diffusive fluxes of CO2, CH4, and N2O, were compared between the natural tidal creeks and the reclaimed creeks. Due to notably changed hydrological and biological conditions in the reclaimed creeks, concentrations of all dissolved carbon species, NH4+-N and NO2--N increased significantly by 60.2-288.2%, while NO3--N and N2O decreased slightly, compared to the natural tidal creeks. DIC and NO3--N were the primary components of the total dissolved carbon and inorganic nitrogen in both creek types; however, their proportions decreased as a result of elevated DOC, CO2, CH4, NH4+-N, and NO2--N following reclamation. Significantly higher global warming potential (0.58 ± 0.15 g CO2-eq m-2 d-1) was found in the reclaimed creeks, making them hotspot of greenhouse effects, compared to the natural tidal creeks. Our results indicated that changes in flow velocity, salinity, Chlorophyll a, and pH were the main factors controlling the dissolved carbon and nitrogen and consequent GHG emissions, due to reclamation. This study is helpful in understanding of carbon and nitrogen sink-source shifts resulting from land use changes in coastal wetlands.

[Localization and expression of CLIC1 in hepatocarcinoma ascites cell lines with high or low potentials of lymphatic spread].

OBJECTIVE: To study the localization and expression of CLIC1 in mouse hepatocarcinoma ascites cell lines with different metastatic potentials. METHODS: Mouse hepatocarcinoma ascites models (a high potential of lymphatic metastasis cell line-Hca-F, and a low potential of lymphatic metastasis cell line-Hca-P) were investigated using fluorescent two-dimensional difference-gel electrophoresis (2-D DIGE) and mass spectrometry for detecting the localization and expression of CLIC1. Immunofluorescence, immunocytochemistry and Western blot were used to assess CLIC1 protein status in the two cell lines. RESULTS: CLIC1 expression was obtained in the cytoplasm and plasma membrane of cells in both cell lines. 2-D DIGE showed that CLIC1 was overexpressed in Hca-F cells, 1.6 folds higher than that of the Hca-P cells. Hca-F cells also had a higher integral membrane CLIC1 in the Hca-P cells. CONCLUSIONS: Although CLIC1 expression is detected in both Hca-F and Hca-P cell lines, a higher protein expression level is present in Hca-F cells. CLIC1 may play an important role in tumor metastasis.

[Effects of silencing chloride intracellular channel 1 gene expression on the proliferation and invasion of mouse hepatocellular carcinoma cell lines].

OBJECTIVE: To study the effects of silencing CLIC1 gene expression on the proliferation and invasion of Hca-F cells. METHODS: The mouse CLIC1 cDNA sequence was retrieved from NCBI. Three shRNA sequences were designed and cloned into pGPU6/GFP/Neo plasmids. The plasmids were transfected into Hca-F cells with Lipofectamine 2000. Cell Counting-8 (CCK-8) kit and transwell chamber were used to study the effects of CLIC1 on the proliferation and invasion of Hca-F cells. RESULTS: The pGPU6/GFP/Neo-shRNA-3 plasmid effectively repressed the expression of CLIC1 mRNA. Inhibition of CLIC1 gene expression led to decreased cell proliferation and reduced invasion. CONCLUSION: CLIC1 is essential for the proliferation and invasion of Hca-F cells.

Sea-level rise will reduce net CO2 uptake in subtropical coastal marshes.

Coastal marshes have a significant capacity to sequester carbon; however, sea-level rise (SLR) is expected to result in prolonged flooding and saltwater intrusion in coastal regions. To explore the effects of SLR projections on net CO2 uptake in coastal marshes, we conducted a "double-check" investigation, including the eddy covariance (EC) measurements of the CO2 fluxes in subtropical coastal marshes along inundation and salinity gradients, in combination with a mesocosm experiment for analyzing CO2 flux components under waterlogging and increased salinity conditions. During the same measurement periods, the net ecosystem CO2 exchange (NEEEC based on the EC dataset) in an oligohaline marsh was higher than that in a low-elevation mesohaline marsh, whereas the NEEEC was lower than that in a high-elevation freshwater marsh. The declines in NEEEC between the marshes could be attributed to a greater decrease in gross primary production relative to ecosystem respiration. Waterlogging slightly increased the NEEms (NEE based on the mesocosms) because of inhibited soil respiration and slight changes in plant photosynthesis and shoot respiration. However, the NEEms measured during the drainage period decreased significantly due to the stimulated soil respiration. The NEEms decreased with increasing salinity (except under mild salinity), and waterlogging exacerbated the adverse impacts of salinity. The amplificatory effect of decreases in both leaf photosynthesis and growth under hydrological stresses contributed more to reduce the NEEms than to respiratory effluxes. Both waterlogging and increased salinity reduced the root biomass, soil microbial biomass, and activities of assayed soil enzymes (except for cellulase under waterlogging conditions), leading to limited soil respiration. The declines in plant growth, photosynthesis, and soil respiration could also be attributed to the decrease in soil nutrients under waterlogging and increased salinity conditions. We propose that the coupling of SLR-driven hydrological effects lowers the capacity of CO2 uptake in subtropical coastal marshes.

[Expression of COX-2 and pregnancy associate plasma protein A in coronary arteries and their relationship with acute coronary syndrome: an autopsy study of 42 cases].

OBJECTIVE: To study the expression of COX-2 and pregnancy associate plasma protein A (PAPP-A) in coronary arteries and their relationship with acute coronary syndrome. METHODS: Twenty-one autopsy cases with acute coronary syndrome encountered during the period from 2002 to 2007 were enrolled into the study. Another 21 autopsy cases without evidence of acute coronary syndrome were used as the controls. The right and left coronary arteries of each group were dissected, embedded and processed as paraffin sections. Immunohistochemical study for CD68 and alpha-actin was performed to highlight the presence of macrophages and smooth muscle cells, respectively. The expression of COX-2 and PAPP-A was evaluated. RESULTS: In the acute coronary syndrome group, COX-2 was localized mainly in the cytoplasm of endothelial cells, macrophages and smooth muscle cells. COX-2 expression in the cytoplasm of smooth muscle cells (28.60%) was significantly higher than that in the control group (4.76%, chi(2) = 14.13, P< 0.05). There was a positive correlation on COX-2 and PAPP-A expression in smooth muscle cells of the media layer of coronary arteries in acute coronary syndrome group (r = 0.88, P < 0.05). The expression of PAPP-A in smooth muscle cells of the media layer in coronary arteries not associated with plaque formation, was higher than that when there were atherosclerotic plaques (chi(2) = 10.36, P < 0.05). CONCLUSION: In coronary arteries, COX-2 and PAPP-A play certain roles in the pathogenesis of acute coronary syndrome.

[Differences and Relationship Between Rhizosphere Characteristics and Methane Emissions of Double-cropping Rice Variety].

Six field varieties of early rice and late rice were selected as test materials for field experiments to explore the difference in CH4 emissions among different rice varieties, and Static Obscura-Gas Chromatography was used to determine the CH4 gas. The results demonstrated a significant difference in the CH4 emissions flux between early and late rice varieties. The average yield of total fertility CH4 emissions was highest in Xiangzaoxian 24 and lowest in Zhuliangyou 819, with a difference of 34.6%. Of the late rice varieties, Tyou 15 was the highest and the Ziyou 299 was the lowest, with a difference of 33.9%. Differences in CH4 emissions and the greenhouse effect of unit yields between different double cropping rice varieties differed significantly. The cumulative CH4 emissions from early rice varieties ranged from 198.3-303.44 kg·hm-2, and the lowest emissions were from Zhuliangyou 819. The greenhouse effect per yield ranged from 0.67 to 1.40 kg·kg-1, and Luliangyou 996 had the lowest emission value. The late-season rice varieties exhibited significantly higher cumulative CH4 emissions compared to early rice, ranging from 291.93 to 388.28 kg·hm-2, and Ziyou 299 had the lowest emission value. The greenhouse effect of per yields rice varieties, while the late rice varieties were contrary to early rice. Reducing carbon and nitrogen concentrations in the rhizosphere and increasing Eh values could reduce CH4 emissions.

A Novel Mechanism of Doxorubicin Resistance and Tumorigenesis Mediated by MicroRNA-501-5p-Suppressed BLID.

Doxorubicin is a widely used anthracycline-based anti-tumor agent for both solid and liquid tumors. Mounting evidence has demonstrated that microRNAs (miRNAs) are involved in chemoresistance and tumorigenesis. However, the roles of microRNA-501-5p (miR-501) in doxorubicin resistance and gastric cancer cell proliferation and invasion are still not fully understood. In this study, we identified that BLID (BH3-like motif-containing protein, cell death inducer) was directly regulated by miR-501 at the post-transcriptional level in multiple gastric cancer cell lines. Endogenous miR-501 was higher, whereas BLID was lower, in doxorubicin-resistant gastric cancer SGC7901/ADR cells compared with their parental SGC7901 cells. miR-501 suppressed gastric cancer cell apoptosis, induced resistance to doxorubicin, and enhanced cell proliferation, migration, and invasion. Subcutaneous injection of miR-501 lentivirus-infected SGC7901 cells resulted in rapid growth of xenograft tumors and resistance to doxorubicin treatment, unlike injection of negative miRNA lentivirus-infected SGC7901 cells. This is achieved at least partially by directly targeting BLID and subsequent inactivation of caspase-9 and caspase-3 and phosphorylation of Akt. Taken together, miR-501 induces doxorubicin resistance and enhances the tumorigenesis of gastric cancer cells by suppressing BLID. miR-501 might be a potential target for doxorubicin resistance and gastric cancer therapy.

Identification of differentially expressed genes in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis.

AIM: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lympho-genous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes, 4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology. CONCLUSION: Tumor metastasis is an incident involving multiple genes. SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis.

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.

Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed. RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes. CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip.

AIM: In order to obtain lymphogenous metastasis-associated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential. METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip(r) MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics. RESULTS: Out of the 14 500 known genes investigated, 110 (0.8%) were up regulated at least 2(3) fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 2(3) fold. According to the Gene Ontology and TreeView analysis, the 110 genes were further classified into two groups: differential biological process profile and molecular function profile. CONCLUSION: Using high-throughput gene chip method, a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

[Screening of differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis].

OBJECTIVE: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastasis Hca-F and its synogenetic cell line Hca-P with low metastatic potential was constructed by suppression subtractive hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Ten differentially expressed cDNA fragments of Hca-F with high potential of lymphatic spreading were obtained, two of which were newly identified ones. CONCLUSION: SSH is a useful technique to detect genes of differential expression and an effective method to clone novel genes.