[The effect of HOXC10 gene on biological behaviors of glioma cells and mechanism in tumor microenvironment].

Objective: To study the effects of Homeobox C10 (HOXC10) on biological characteristics such as migration, invasion and proliferation of glioma cancer cells and to explore the role of HOXC10 gene in glioma microenvironment. Methods: The expression level of HOXC10 in high grade glioma (glioblastoma) and low grade glioma and its effect on patient survival were analyzed by using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) database. Hoxc10-siRNA-1, HOXC10-siRNA-2 and siRNA negative control (NC) were transfected into U251 cells according to the operation instructions of HOXC10-siRNA transfection. 100 ng/ mL recombinant protein chemokine ligand 2 (reCCL2) was added into the transfection group, and was labeled as HOXC10-siRNA-1+ reCCL2 and HOXC10-siRNA-2+ reCCL2 groups. The expressions of HOXC10 mRNA and target protein in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot. The proliferation ability of cells in each group was detected by cell counting kit 8 (CCK8) method. The migration ability of cells was detected by Transwell assay and Nick assay, and cell apoptosis was detected by flow cytometry. The expression of chemokines in each group was detected by multiple factors. Co-incubation assays were performed to determine the role of HOXC10 and chemokine ligand 2 (CCL2) in recruiting and polarizing tumor-associated macrophages (M2-type macrophages). Results: The median expression level of HOXC10 in high grade gliomas was 8.51, higher than 1.00 in low grade gliomas (P<0.001) in TCGA database. The median expression level of HOXC10 in high grade gliomas was 0.83, higher than 0.00 in low grade gliomas (P=0.002) in CGGA database. The 5-year survival rate of patients with high HOXC10 expression in TCGA database was 28.2%, lower than 78.7% of those with low HOXC10 expression (P<0.001), and the 5-year survival rate of patients with high HOXC10 expression in CGGA database was 20.3%, lower than 58.0% of those with low HOXC10 expression (P<0.001). The numbers of cell migration in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (45±3) and (69±4) respectively, lower than (159±3) in NC group (P<0.05). The cell mobility of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group at 48 hours were (15±2)% and (28±4)% respectively, lower than (80±5)% of NC group (P<0.05). The expressions of vimentin in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (141 740.00±34 024.56) and (94 655.00±5 687.97), N-cadherin were (76 810.00±14.14) and (94 254.00±701.45), ß-catenin were (75 786.50±789.84) and (107 296.50±9 614.53), lower than (233 768.50±34 114.37), (237 154.50±24 715.50) and (192 449.50±24 178.10) of NC group (P<0.05). The A value of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.44±0.05) and (0.32±0.02) at 96 hours, lower than 0.92±0.12 of NC group (P<0.05). The apoptosis rates of HOXC10-siRNA-1 group and HOXC10 siRNA-2 group were (10.23±1.24)% and (13.81±2.16)%, higher than (4.60±0.07)% of NC group (P<0.05). The expression levels of CCL2 in U251 cells in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (271.63±44.27) and (371.66±50.21), lower than (933.93±29.84) in NC group (P<0.05). The expression levels of CCL5 (234.81±5.95 and 232.62±5.72), CXCL10 (544.13±48.14 and 500.87±15.65) and CXCL11 (215.75±15.30 and 176.18±16.49) in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were higher than those in NC group (9.98±0.71, 470.54±18.84 and 13.55±0.73, respectively, P<0.05). The recruited numbers of CD14(+) THP1 in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (159.33±1.15) and (170.67±1.15), respectively, lower than (360.00±7.81) in NC group (P<0.05), while addition of reCCL2 promoted the recruitment of CD14(+) THP1 cells (287.00±3.61 and 280.67±2.31 in HOXC10-siRNA-1+ reCCL2 group and HOXC10-siRNA-2+ reCCL2 group, respectively, P<0.05). The expressions level of M2-type macrophage-related gene TGF-ß in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.30±0.02) and (0.28±0.02), respectively, lower than (1.06±0.10) in NC group (P<0.05). The expressions level of M1-related gene NOS2 in HOXC10-siRNA-1 and HOXC10-siRNA-2 were (11 413.95±1 911.85) and (5 894.00±945.21), respectively, higher than (13.39±4.32) in NC group (P<0.05). Conclusions: The expression of HOXC10 in glioma is high and positively correlated with the poor prognosis of glioma patients. Knockdown of HOXC10 can inhibit the proliferation, migration and metastasis of human glioma U251 cells. HOXC10 may play an immunosuppressive role in glioma microenvironment by promoting the expression of CCL2 and recruiting and polarizing tumor-associated macrophages (M2 macrophages).

KDM2B/FBXL10 targets c-Fos for ubiquitylation and degradation in response to mitogenic stimulation.

KDM2B (also known as FBXL10) controls stem cell self-renewal, somatic cell reprogramming and senescence, and tumorigenesis. KDM2B contains multiple functional domains, including a JmjC domain that catalyzes H3K36 demethylation and a CxxC zinc-finger that recognizes CpG islands and recruits the polycomb repressive complex 1. Here, we report that KDM2B, via its F-box domain, functions as a subunit of the CUL1-RING ubiquitin ligase (CRL1/SCF(KDM2B)) complex. KDM2B targets c-Fos for polyubiquitylation and regulates c-Fos protein levels. Unlike the phosphorylation of other SCF (SKP1-CUL1-F-box)/CRL1 substrates that promotes substrates binding to F-box, epidermal growth factor (EGF)-induced c-Fos S374 phosphorylation dissociates c-Fos from KDM2B and stabilizes c-Fos protein. Non-phosphorylatable and phosphomimetic mutations at S374 result in c-Fos protein which cannot be induced by EGF or accumulates constitutively and lead to decreased or increased cell proliferation, respectively. Multiple tumor-derived KDM2B mutations impaired the function of KDM2B to target c-Fos degradation and to suppress cell proliferation. These results reveal a novel function of KDM2B in the negative regulation of cell proliferation by assembling an E3 ligase to targeting c-Fos protein degradation that is antagonized by mitogenic stimulations.

Regulation of glycolysis and gluconeogenesis by acetylation of PKM and PEPCK.

Glycolysis is a catabolic process of glucose hydrolysis needed for energy and biosynthetic intermediates, whereas gluconeogenesis is a glucose production process important for maintaining blood glucose levels during starvation. Although they share many enzymes, these two processes are not simply the reverse of each other and are instead reciprocally regulated. Two key enzymes that regulate irreversible steps in these two processes are pyruvate kinase (PK) and phosphoenolpyruvate carboxy kinase (PEPCK), which catalyze the last and first step of glycolysis and gluconeogenesis, respectively, and are both regulated by lysine acetylation. Acetylation at Lys305 of the PKM (muscle form of PK) decreases its activity and also targets it for chaperone-mediated autophagy and subsequent lysosome degradation. Acetylation of PEPCK, on the other hand, targets it for ubiquitylation by the HECT E3 ligase, UBR5/EDD1, and subsequent proteasomal degradation. These studies established a model in which acetylation regulates metabolic enzymes via different mechanisms and also revealed cross talk between acetylation and ubiquitination. Given that most metabolic enzymes are acetylated, we propose that acetylation is a major posttranslational modifier that regulates cellular metabolism.

[Triterpenoid saponins from Eclipta prostrata L].

AIM: To study the triterpenoid saponins in the Chinese traditional medicine Eclipta prostrata L.. METHODS: Column chromatography with silica gel and HPLC were employed for the isolation and purification. The molecular structures were determined on the basis of spectral analysis (IR, MS, 1HNMR, 13CNMR, HMQC and HMBC). RESULTS: Two new triterpenoid saponins, named eclalbasaponins XI (4) and XII (5), were obtained and their structures were elucidated as 3-O-[beta-D-glucopyranosyl(1-->2)-beta-D-glucopyranosyl]-16 alpha-ethoxy-olean-12-ene-28-oic acid-28-O-beta-D-glucopyranoside and 3-O-[(2-O-sulfuryl-beta-D-glucopyranosyl) (1-->2)-beta-D-glucopyranosyl]-echinocystic acid-28-O-beta-D-glucopyranoside, respectively, along with three known saponins, eclalbasaponins II (1), I (2) and III (3). CONCLUSION: Compounds 4 and 5 are new compounds, 1 and 5 induced morphological deformation of Pyricularia oryzae mycelia.

TGF-beta 1 modulated the expression of alpha 5 beta 1 integrin and integrin-mediated signaling in human hepatocarcinoma cells.

Integrins are a family of cell surface adhesion molecules which mediate cell adhesion and initiate signaling pathways that regulate cell spreading, migration, differentiation, and proliferation. TGF-beta is a multifunctional factor that induces a wide variety of cellular processes. In this study, we show that, TGF-beta 1 treatment enhanced the amount of alpha 5 beta 1 integrin on cell surface, the mRNA level of alpha 5 subunit, and subsequently stimulated cell adhesion onto a fibronectin (Fn) and laminin (Ln) matrix in SMMC-7721 cells. TGF-beta 1 could also promote cell migration. Furthermore, our results showed that TGF-beta1 treatment stimulated the tyrosine phosphorylation level of FAK, which can be activated by the ligation and clustering of integrins. PTEN can directly dephosphorylate FAK, and the results that TGF-beta 1 could down-regulate PTEN at protein level suggested that TGF-beta 1 might stimulate FAK phosphorylation through increasing integrin signaling and reducing dephosphorylation of FAK. These studies indicated that TGF-beta 1 and integrin-mediated signaling act synergistically to enhance cell adhesion and migration and affect downstream signaling molecules of hepatocarcinoma cells.

[TGF-beta 1 rapidly reduced hepatocarcinoma cells adhesion on to fibronectin and stimulated FAK dephosphorylation].

In order to investigate whether TGF-beta 1 could rapidly regulate integrin induced signaling, we treated SMMC-7721 human hepatocellular carcinoma cells with human recombinant TGF-beta 1 for 10 min, and examined cell adhesion, integrin amount and FAK tyrosine phosphorylation. We used cell adhesion assay to estimate the affinity of alpha 5 beta 1 integrin with fibronectin, and analyzed the amount of integrin alpha 5 and beta 1 subunits by performing FACS analysis. Then western blot analysis was carried out to examine tyrosine phosphorylation level of FAK. Our results showed that TGF-beta 1 could rapidly attenuated cell adhesion onto Fn without changing the expression of alpha 5 beta 1 integrin, and at the meantime dephosphorylated FAK. It suggested that TGF-beta 1 rapidly regulated the activation of integrin, and stimulated FAK dephosphorylation, which might induce depolarization in SMMC-7721 hepatocellular carcinoma cells, then facilitates the detachment of tumor cells at early stages of migration.