Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

DLC1 inhibits colon adenocarcinoma cell migration by promoting secretion of the neurotrophic factor MANF.

DLC1 (deleted in liver cancer-1) is downregulated or deleted in colorectal cancer (CRC) tissues and functions as a potent tumor suppressor, but the underlying molecular mechanism remains elusive. We found that the conditioned medium (CM) collected from DLC1-overexpressed SW1116 cells inhibited the migration of colon adenocarcinoma cells HCT116 and SW1116, but had no effect on proliferation, which suggested DLC1-mediated secretory components containing a specific inhibitor for colon adenocarcinoma cell migration. Analysis by mass spectrometry identified mesencephalic astrocyte-derived neurotrophic factor (MANF) as a candidate. More importantly, exogenous MANF significantly inhibited the migration of colon adenocarcinoma cells HCT116 and SW1116, but did not affect proliferation. Mechanistically, DLC1 reduced the retention of MANF in ER by competing the interaction between MANF and GRP78. Taken together, these data provided new insights into the suppressive effects of DLC1 on CRC, and revealed the potential of MANF in the treatment of CRC.

MicroRNA-1254 inhibits the migration of colon adenocarcinoma cells by targeting PSMD10.

OBJECTIVE: MicroRNA-1254 (miR-1254) has not been studied in colorectal cancer (CRC) to date. This study aimed to investigate the inhibitory mechanism of miR-1254 in CRC tumorigenesis. METHODS: MiR-1254 expression was examined using real-time polymerase chain reaction in CRC and adjacent non-tumorous tissues. The correlation between miR-1254 expressions and proliferation and migration of cancer cells was determined using the CCK-8 and transwell assays. RNA sequencing was used to identify differentially expressed genes downstream from miR-1254. A luciferase reporter assay was used to confirm the direct interaction between miR-1254 and its predicted target gene, PSMD10. Moreover, PSMD10 was either overexpressed or silenced in colon carcinoma cells overexpressing miR-1254 to determine whether their interaction contributed to CRC migration and epithelial-mesenchymal transition (EMT). RESULTS: Significantly lower miR-1254 expressions were observed in CRC tissues than in adjacent non-tumorous tissues. Exogenous miR-1254 expression suppressed the migration of colon carcinoma cell lines SW1116 and HCT116. RNA sequencing and luciferase assays revealed that miR-1254 directly binded to the 3'-untranslated region of PSMD10, an important regulator of EMT and cell migration. PSMD10 knockdown inhibited EMT and colon cancer cell migration, whereas PSMD10 overexpression reversed the inhibition of EMT and cell migration caused by miR-1254. CONCLUSION: MiR-1254 may act as a tumor suppressor in CRC and may inhibit CRC migration by directly targeting PSMD10 to suppress the EMT process.

Slit2­Robo1 signaling promotes the adhesion, invasion and migration of tongue carcinoma cells via upregulating matrix metalloproteinases 2 and 9, and downregulating E­cadherin.

Whether Slit homologue 2 (Slit2) inhibits or promotes tumor cell migration remains controversial, and the role of Slit2­Roundabout 1 (Robo1) signaling in oral cancer remains to be fully elucidated. The aim of the present study was to investigate the role of Slit2­Robo1 signaling in the adhesion, invasion and migration of tongue carcinoma cells, and the mechanism by which Slit2­Robo1 signaling inhibits or promotes tumor cell migration. Tca8113 tongue carcinoma cells were treated with the monoclonal anti­human Robo1 antibody, R5, to inhibit the Slit2­Robo1 signaling pathway, with immunoglobulin (Ig)G2b treatment as a negative control. The expression levels of Slit2 and Robo1 were determined using flow cytometry. The effects of R5 on the adhesion, invasion and migration of Tca8113 tongue carcinoma cells were investigated. Gelatin zymography was used to investigate the activity of matrix metalloproteinase 2 (MMP2) and MMP9. Western blot analysis was used to evaluate the expression levels of E­cadherin in Tca8113 cells treated with 10 µg/ml of either R5 or IgG2b. Slit2 and Robo1 proteins were found to be expressed in the Tca8113 cells. R5 significantly inhibited the adhesion, invasion and migration of Tca8113 cells in vitro. R5 also inhibited the activities of MMP2 and MMP9, and increased the expression of E­cadherin in the Tca8113 cells. These results suggested that Slit2­Robo1 signaling promoted the adhesion, invasion and migration of tongue carcinoma cells by upregulating the expression levels of MMP2 and MMP9 and, downregulating the expression of E­cadherin.

Role of B7-H4 siRNA in Proliferation, Migration, and Invasion of LOVO Colorectal Carcinoma Cell Line.

OBJECTIVES: Colorectal cancer is one of the most common malignancies. Recent studies investigated that B7-H4 is highly expressed in various cancers. We aimed at exploring the effect of B7-H4 siRNA on proliferation, invasion, and migration of LOVO cells which expressed B7-H4 notably. DESIGN AND METHODS: Colon adenocarcinoma dataset was downloaded from The Cancer Genome Atlas. 35 colorectal cancer patients admitted to Shanghai Tongren Hospital were enrolled in this study. Cell proliferation and cell cycle distribution were identified by CCK8 and flow cytometry, respectively. Transwell assay was performed to detect the invasion and migration of LOVO cells. CXCL12/CXCR4 expression and JAK2/STAT3 phosphorylation were determined by real-time PCR and western blot. RESULTS: B7-H4 expressed is elevated in colorectal cancer tissues than in the adjacent normal tissues. B7-H4 siRNA effectively inhibited the proliferation at 24 h and 48 h, arrested cell cycle at G0/G1, and suppressed cell invasion and migration. Gene set enrichment analysis showed that CXCL12/CXCR4 and JAK/STAT were correlative with the B7-H4 expression. Additionally, CXCL12/CXCR4 expression and JAK2/STAT3 phosphorylation were reduced. CONCLUSIONS: B7-H4 siRNA can effectively inhibit proliferation, invasion, and migration of LOVO cells by targeting CXCL12/CXCR4 and JAK2/STAT3 signaling, which can serve as a new target for colorectal carcinoma treatment.

Cloning and transcription analysis of the Candida rugosa propionyl-CoA dehydrogenase gene and its expression in Pichia pastoris.

The propionyl-CoA dehydrogenase (PACD) gene was cloned from Candida rugosa by the cDNA RACE technique. The full cDNA of the PACD gene has a length of 1408 bp, which contains a complete open reading frame (ORF) of 1329 bp, coding for 442 amino acids. The cDNA of PACD was cloned into the expression plasmid pPIC9K and transformed into Pichia pastoris GS115. The recombinant protein was purified by Ni-NTA affinity chromatography, and its size was observed to be approximately 49 kDa as estimated by SDS-PAGE. Anti-His antibodies were used to characterise the recombinant PACD by western-blot analysis. The recombinant protein retained the activity of catalysing propionyl-CoA to acryloyl-CoA. The results of dot-blotting hybridisation using a PACD cDNA probe indicated that the PACD mRNA level was modified at different stages: mRNA levels were low for the first 36 h, then increased through 48 h and eventually reached a stable level. These results indicate that propionate induction could significantly activate PACD mRNA expression. Information from this study will be helpful in elucidating the metabolic pathway for 3-hydroxypropionic acid production in C. rugosa.

Expression and characterization of a novel propionyl-CoA dehydrogenase gene from Candida rugosa in Pichia pastoris.

The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6× His-tagged recombinant PACD gene was expressed in Pichia pastoris GS115 and purified with Ni-NTA affinity chromatography. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified PACD was 49 kDa. The results showed that the recombinant protein had the activity of catalyzing propionyl-CoA to acrylyl-CoA. The K (m), k (cat), and V (max) values of the purified PACD were calculated to be 40.86 µM, 0.566 s(-1) and 0.693 U mg(-1) min(-1). The optimal temperature and pH of the purified PACD were 30 °C and 7.0, respectively. The recombinant PACD maintained 76.3%, 30.1%, and 4.3% of its original activity after 2 h incubation in standard buffer at 30, 40, and 50 °C, respectively. Mg(2+) had an activating effect on the enzyme, while Mn(2+), Ca(2+), Zn(2+), and Cu(2+) had weak inhibition. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified ß-oxidation pathway from glucose to 3-hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit the engineered strains for effective production of 3-HP from the most abundant biomass-sugars.

[Value of detecting p16 gene methylation in the diagnosis of malignant pleural effusion].

OBJECTIVE: To investigate aberrant methylation in the promoter of p16 gene in the sediment cells of pleural effusion and evaluate its clinical significance in the differentiating benign and malignant pleural effusion. METHODS: Using methylation-specific PCR (MSP), aberrant promoter methylation of p16 gene was detected in the sedimental cells of pleural effusion samples from 66 patients with pleural effusion. RESULTS: Of the 66 patients with pleural effusion, 36 had a definite diagnosis of malignant pleural effusion, and the rest were confirmed to have benign pleural effusion. The positivity rate of p16 gene promoter methylation was 69.4% (25/36) in malignant pleural effusion and 13.3% (4/30) in benign pleural effusion specimens, showing a significant difference between them (χ² = 20.915, P < 0.01). The diagnostic sensitivity, specificity and accuracy of aberrant promoter methylation of p16 gene in the 36 malignant cases were 69.4%, 86.7% and 77.3%, respectively. The positive expression of p16 gene promoter methylation in malignant pleural effusion was not correlated to the histological type or the pathological grade of the tumor (P > 0.05). CONCLUSION: Detection of aberrant methylation in p16 gene promoter in the sediment cells of pleural effusion specimens by MSP method allows differentiation between benign and malignant pleural effusion.

[The effect of recombinant panton-valentine leukocidin on the production of cytokines by human alveolar macrophages].

OBJECTIVE: To investigate the effect of recombinant panton-valentine leukocidin (rPVL) on the regulation of human alveolar macrophage CD14 and IL-10 and TNF-alpha. METHODS: Human alveolar macrophages (AM) were purified and cultured from bronchoalveolar lavage fluid. Each sample was divided into groups according to different concentrations and exposure times of rPVL. Semi-quantitative RT-PCR was used to evaluate the CD14 mRNA levels and Double-antibody-sandwich-ELISA was used to measure the IL-10 and TNF-alpha levels in AM cultures. RESULTS: CD14 mRNA decreased after rPVL treatment in time-and concentration dependent manners. There were no statistically significant differences in CD14 mRNA among the blank control groups (F = 1.708, P > 0.05). CD14 mRNA in the T6N10 group and the T6N100 group( T = time in hours, N = concentration of rPVL/nmol/L) decreased as compared to the T6N0 group (t = 4.132, 6.818, both P < 0.001), and that in the T24N10 group and the T24N100 group also decreased as compared to the T24N0 group (t = 7.401, 11.415, both P < 0.001), indicating that the expression of CD14 was downregulated by rPVL treatment. There were also statistically significant differences in CD14 mRNA between T6N10 and T24N10 groups, T6N100 and T24N100 groups (t = 4.692, 6.019, both P < 0.001), T6N10 and T6N100 groups, T24N10 and T24N100 groups (t = 2.686, 4.014, P < 0.01 respectively), indicating that the expression of CD14 decreased as the treatment time and the concentration of rPVL increased. The IL-10 concentrations of the T24N10 and T24N100 groups increased as compared to the T24N0 group (t = 4.036, 3.941, both P < 0.01) in time-dependent and concentration-dependent manners with rPVL treatment. The TNF-alpha concentration of the T24N10 group decreased while that of the T24N100 group increased as compared to the T24N0 group (t = 2. 824, 8. 468, both P < 0.01, respectively), indicating that a lower concentration of rPVL inhibited TNF-alpha release while a higher concentration of rPVL induced release of TNF-alpha. CONCLUSION: The results suggest that rPVL could reduce the expression of CD14 and induce disordered release of anti-inflammatory and proinflammatory cytokines by AMs, which may be one of the important mechanisms underlying the high mortality of infection with PVL-positive Staphylococcus aureus.