Characteristics of soil nitrogen and phosphorus fractions and microbial traits with increasing stand age in two-layered Cunninghumia lanceolata + Phoebe bournei plantations.

Soil nitrogen and phosphorus are two key elements limiting tree growth in subtropical areas. Understanding the regulation of soil microorganisms on nitrogen and phosphorus nutrition is beneficial to reveal maintenance mechanism of soil fertility in plantations. We analyzed the characteristics of soil nitrogen and phosphorus fractions, soil microbial community composition and function, and their relationship across three stands of two-layered Cunninghumia lanceolata + Phoebe bournei with different ages (4, 7 and 11 a) and the pure C. lanceolata plantation. The results showed that the contents of most soil phosphorus fractions increased with increasing two-layered stand age. The increase in active phosphorus fractions with increasing stand age was dominated by the inorganic phosphorus (9.9%-159.0%), while the stable phosphorus was dominated by the organic phosphorus (7.1%-328.4%). The content of soil inorganic and organic nitrogen also increased with increasing two-layered stand age, with NH4+-N and acid hydrolyzed ammonium N contents showing the strongest enhancement, by 152.9% and 80.2%, respectively. With the increase of stand age, the composition and functional groups of bacterial and fungal communities were significantly different, and the relative abundance of some dominant microbial genera (such as Acidothermus, Saitozyma and Mortierella) increased. The relative abundance of phosphorus solubilization and mineralization function genes, nitrogen nitrification function and aerobic ammonia oxidation function genes tended to increase. The functional taxa of fungi explained 48.9% variation of different phosphorus fractions. The conversion of pure plantations to two-layered mixed plantation affected soil phosphorus fractions transformation via changing the functional groups of saprophytes (litter saprophytes and soil saprophytes). Changes in fungal community composition explained 45.0% variation of different nitrogen fractions. Some key genera (e.g., Saitozyma and Mortierella) play a key role in promoting soil nitrogen transformation and accumulation. Therefore, the conversion of pure C. lanceolata plantation to two-layered C. lanceolata + P. bournei plantation was conducive to improving soil nitrogen and phosphorus availability. Bacteria and fungi played important roles in the transformation process of soil nitrogen and phosphorus forms, with greater contribution of soil fungi.

Bortezomib inhibits growth and sensitizes glioma to temozolomide (TMZ) via down-regulating the FOXM1-Survivin axis.

BACKGROUND: High-grade glioma (HGG) is a fatal human cancer. Bortezomib, a proteasome inhibitor, has been approved for the treatment of multiple myeloma but its use in glioma awaits further investigation. This study aimed to explore the chemotherapeutic effect and the underlying mechanism of bortezomib on gliomas. METHODS: U251 and U87 cell viability and proliferation were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, tumor cell spheroid growth, and colony formation assay. Cell apoptosis and cell cycle were detected by flow cytometry. Temozolomide (TMZ)-insensitive cell lines were induced by long-term TMZ treatment, and cells with stem cell characteristics were enriched with stem cell culture medium. The mRNA levels of interested genes were measured via reverse transcription-quantitative polymerase chain reaction, and protein levels were determined via Western blotting/immunofluorescent staining in cell lines and immunohistochemical staining in paraffin-embedded sections. Via inoculating U87 cells subcutaneously, glioma xenograft models in nude mice were established for drug experiments. Patient survival data were analyzed using the Kaplan-Meier method. RESULTS: Bortezomib inhibited the viability and proliferation of U251 and U87 cells in a dose- and time-dependent manner by inducing apoptosis and cell cycle arrest. Bortezomib also significantly inhibited the spheroid growth, colony formation, and stem-like cell proliferation of U251 and U87 cells. When administrated in combination, bortezomib showed synergistic effect with TMZ in vitro and sensitized glioma to TMZ treatment both in vitro and in vivo. Bortezomib reduced both the mRNA and protein levels of Forkhead Box M1 (FOXM1) and its target gene Survivin. The FOXM1-Survivin axis was markedly up-regulated in established TMZ-insensitive glioma cell lines and HGG patients. Expression levels of FOXM1 and Survivin were positively correlated with each other and both related to poor prognosis in glioma patients. CONCLUSIONS: Bortezomib was found to inhibit glioma growth and improved TMZ chemotherapy efficacy, probably via down-regulating the FOXM1-Survivin axis. Bortezomib might be a promising agent for treating malignant glioma, alone or in combination with TMZ.

Medulloblastoma stem cells: Promising targets in medulloblastoma therapy.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite great improvements in the therapeutic regimen, relapse and leptomeningeal dissemination still pose great challenges to the long-term survival of MB patients. Developing more effective strategies has become extremely urgent. In recent years, a number of malignancies, including MB, have been found to contain a subpopulation of cancer cells known as cancer stem cells (CSCs), or tumor initiating/propagating cells. The CSCs are thought to be largely responsible for tumor initiation, maintenance, dissemination, and relapse; therefore, their pivotal roles have revealed them to be promising targets in MB therapy. Our growing understanding of the major medulloblastoma molecular subgroups and the derivation of some of these groups from specific stem or progenitor cells adds additional layers to the CSC knowledge base. Herein we review the current knowledge of MB stem cells, highlight the molecular mechanisms relating to MB relapse and leptomeningeal dissemination, and incorporate these with the need to develop more effective and accurate therapies for MB patients.

Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide (TMZ) treatment.

PURPOSE: The aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms. METHODS: U251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry. RESULTS: We successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O(6)-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways. CONCLUSION: HIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation.

Microarray analysis of the aberrant microRNA expression pattern in gliomas of different grades.

Previous studies have focused on miRNA expression in brain gliomas. However, both the expression pattern of miRNAs in gliomas of different grades and various miRNAs involved in malignant progression of gliomas are poorly understood. In the present study, we used miRNA microarray-based screening to investigate the miRNA expression profile in gliomas, which was further verified by qRT-PCR in selected miRNAs. In total, we found 13 differentially expressed miRNAs between gliomas and their matched surrounding tissues. Among them, 12 miRNAs were upregulated and only one (miR-4489) was downregulated compared with the control. Furthermore, the lower expression level of miR-4489 was confirmed by qRT-PCR in 26 glioma samples. Our microarray result revealed 8, 9 and 15 aberrantly expressed miRNAs in gliomas of World Health Organization (WHO) grade II-IV, respectively. Gene Ontology (GO) and Pathway analysis indicated that target genes of the 13 miRNAs were significantly enriched in central nervous system- and tumor-related biological processes and signaling pathways. The dysregulated miRNAs identified in the present study contribute to the tumorigenesis and malignant progression of gliomas and may serve as useful markers for advanced glioma pathological grading and prognosis.

MiR-22 is frequently downregulated in medulloblastomas and inhibits cell proliferation via the novel target PAPST1.

Medulloblastoma is the most frequent malignant central nervous system tumor in children. MicroRNAs (miRs) are small, non-coding RNAs that target protein-coding and non-coding RNAs, and play roles in a variety of cellular processes through regulation of multiple targets. In the present study, we analyzed miR-22 expression and its effect in cell proliferation and apoptosis in medulloblastomas. Quantitative reverse transcription PCR (RT-PCR) revealed significantly lower expression of miR-22 in 19 out of 27 (70%) medulloblastomas, D341, DAOY, ONS-76 medulloblastoma cell lines, compared with normal cerebellum. Forced expression of miR-22 by lentiviral vector transfection reduced cell proliferation and induced apoptosis, while knockdown of miR-22 increased proliferative activity in DAOY and ONS-76 cells. DAOY cells with miR-22 overexpression in nude mice yielded tumors smaller than those originated from control DAOY cells. Microarray analysis in DAOY cells with forced miR-22 expression showed significant changes in expression profiles, PAPST1 being the most significantly (10 folds) downregulated gene. Quantitative RT-PCR revealed PAPST1 mRNA upregulation in 18 out of 27 (67%) medulloblastomas. In addition, a luciferase reporter assay in ONS-76 and DAOY cells suggested that miR-22 directly targets the PAPST1 gene, and lentivirus-mediated knockdown of PAPST1 suppressed proliferation of DAOY and ONS-76 medulloblastoma cells. These results suggest that frequently downregulated miR-22 expression is associated with cell proliferation in medulloblastomas, and this may be at least in part via PAPST1, which is a novel target of miR-22.

[The developmental changes of GHR and IGF-1R gene expressions in porcine hypothalamus and pituitary].

GH and IGF-1 may serve as negative feedback factors to regulate GH secretion from pituitary by binding to their respective receptors in hypothalamus and/or pituitary. In order to evaluate the line-specific developmental patterns of negative feedback regulation of GH secretion, Erhualian (EHL) and Large White (LW) pigs with significant difference in growth rate were employed in present study to investigate the developmental changes of GH receptor (GHR) mRNA and type-1 IGF receptor (IGF-1R) mRNA in hypothalamus and pituitary from birth till 180 days of age by relative quantitative RT-PCR. Pigs were sampled at birth, 3 , 20, 30, 90, 120 and 180 days of age respectively. Hypothalamic GHR mRNA was expressed according to an age-dependent manner, being low at birth, then increased steadily till day 120, followed by a decrease (P < 0.05) at the age of 180 days, suggesting that the sensitivity of hypothalamus to the GH negative feedback influence increase steadily during fast-growing period. LW boars expressed higher level of GHR mRNA than EHL boars (P < 0. 05) in hypothalamus. In pituitary, however, the GHR mRNA level was not significantly correlated with the breeds and age. The results suggested that GH might act mainly at the level of hypothalamus to regulate GH secretion. In contrast, the expression of IGF-1R mRNA exhibited line-specific developmental patterns in pituitary but not in hypothalamus. Hypothalamic expression of IGF-1R mRNA was abundant but did not show significant differences between ages, groups or lines. In pituitary, however, the IGF-1R mRNA expression was found to be high at birth both in EHL and LW pigs, subsequently declined till day 20, then followed by a slow rise reaching the second peak at the age of 90 days. At the age of 180 days, the pituitary IGF-1R mRNA level was higher in EHL pigs than that in LW pigs (P < 0.05), but the opposite was true at the age of 30 and 90 days. These results suggest that the site for receiving the feedback signal of IGF-1 is more likely in pituitary rather than in hypothalamus in the pig.

[Developmental changes of myoglobin gene expression in longissimus dorsi muscle of Erhualian and large white pigs].

In present study, the developmental patterns of myoglobin (MB) mRNA expression were compared between Erhualian and Large White boars, Gender difference was also examined in Erhualian pigs. Semi-quantitative RT-PCR was applied to determine the level of MB mRNA. Different developmental patterns were observed in two breeds of pigs. MB mRNA expression was low in both breeds at D3, while divergent trends were followed by different breed of pigs thereafter. No significant changes in MB mRNA expression were observed in Large White boars over the period of investigation, although a higher level was seen at D120. In Erhualian boars, however, the level of MB mRNA increased significantly (P<0.01) from D3 to D20 and stayed high consistently afterwards. As a result, Erhualian boars expressed higher level of MB mRNA in Longissimus Dorsi muscle compared with Large White boars on 20, 90, 120 and 180 days of age. Similar patterns of MB mRNA expression were found in both sexes of Erhualian pigs, except at D180, a remarkable decrease occurred to females (P<0.01), resulting in a significant (P<0.01) gender difference at D180 with higher level of MB mRNA expressed in Erhualian boars.

[The developmental patterns of GH-R, IGF-1 and IGF-IR gene expression in adipose tissue of Erhualian and large white pigs].

Growth hormone (GH) is essential for postnatal growth in animal, it regulates numerous cellular functions by direct effect on its receptor (GH-R) in many different tissues. In present experiment single tube semi-quantitative RT-PCR was applied to investigate the developmental patterns of GH-R, IGF-1 and IGF-I R mRNA expression in dorsal subcutaneous adipose tissue of Erhualian and Large White pigs, and 18S internal standards were used as control. Eight Erhualian pigs were sampled at birth, 3, 20, 30, 45, 90, 120, 180 days of age respectively, four Large White boars were sampled at 20, 30, 90, 120, 180 days of age respectively. The results showed that GH-R mRNA levels in adipose tissue were changed with age (P < 0.05). The expression of GH-R mRNA in Erhualian gilts was low at birth but reached a peak at 45 days of age, and kept constant thereafter. The expression of GH-R mRNA in Erhualian boars was high at birth and reached a peak at 45 day, then decreased remarkably. It had no differences between sexes in general. The expression of GH-R mRNA in Large White boars was lower than that in Erhualian boars (P < 0.01). The IGF-1 mRNA expression in adipose tissue was changed with age. The IGF-1 mRNA expression in Erhualian gilts was lower than that in Erhualian boars (P < 0.05), and the IGF-1 mRNA expression in Large White boars was lower than that in Erhualian boars (P < 0.01). The IGF-I R mRNA expression in adipose tissue was changed with aging, but it had no differences between sexes and breeds in general. The results suggest that the GH-R, IGF-1 and IGF-I R mRNA expression in adipose tissue follow specific developmental pattern, and the different patterns of IGF-1 gene expression may contribute to different sediment of adipose tissue between two breeds.

Developmental patterns of GHr and SS mRNA expression in porcine gastric tissue.

AIM: The present study was aimed to investigate the developmental patterns of growth hormone receptor (GHr) and somatostatin (SS) mRNA expression in porcine gastric tissue and its relationship with gastric growth and gastric functional development. METHODS: Erhualian and Large White boars were selected randomly and sampled at birth (D0), 3, 20, 30, 90, 120 and 180 days of age respectively, meanwhile the bodyweight and gastric weight were recorded. The single tube semi-quantitative RT-PCR was applied in this experiment to investigate the developmental patterns of gastric GHr and SS mRNA expression, the correlations between the patterns of mRNA expression and the relative gastric weight (ratio of gastric weight to bodyweight) and the pepsin contents in gastric mucous membrane were analyzed. In order to further elucidate the effect of GH on gastric function, the primary cultures of gastric fundic mucosal cells were treated with 2 ng/ml, 20 ng/ml and 200 ng/ml of rpGH for 18 hrs respectively, and the pepsin contents in culture medium were measured. RESULTS: The expression of GHr mRNA was high at birth, followed by a significant decrease at 3 days of age (D3) in both breeds. In Large White boars, the expression of GHr mRNA reached a peak at D90 and remained a plateau afterward. In Erhualian pigs, however, a slight yet significant increase occurred at D30 reaching a level that was kept constant thereafter. From birth to D30, the expression of GHr mRNA in gastric tissue was higher in Erhualian boars than that in Large White boars, but from D90 to D180, the higher expression of GHr mRNA was found in Large White boars. The gastric GHr mRNA expression was significantly correlated with the relative gastric weight (r=0.541, P<0.05) and pepsin content in gastric mucosa (r=0.625, P<0.05) respectively. The gastric SS mRNA expression was high at birth (Erhualian 1.59, Large White 0.80), but dropped significantly at D3 (Erhualian 0.95, Large White 0.19, P<0.05), a stepwise increase was followed thereafter until a peak at D30 (Erhualian 1.71, Large White 0.95) In general, Erhualian pigs expressed higher levels of SS mRNA in gastric tissue as compared with Large White pigs at the same age (P<0.05). No significant correlations between SS mRNA and relative gastric weight or pepsin content were found. In vitro, 2 ng/ml of rpGH elicited significant increase in pepsin secretion from primary cultures of gastric mucosal cells (P<0.05), and no responses were observed at 20 ng/ml and 200 ng/ml. CONCLUSION: The results suggested that: 1, GHr and SS mRNA in porcine stomach are expressed according to strain specific developmental patterns; 2, GH acts directly at the gastric tissue and regulates the structural and functional growth of stomach.

[The developmental patterns of GHr gene expression in gastric tissue of Erhualian and large white pigs].

Pituitary growth hormone (GH) is essential for postnatal growth in animal, it regulates numerous cellular functions by direct effect on it's receptor (GHr) in many different tissues. And it is believed that the abundance of GHr in different tissue determines the tissue sensitivity to GH. However, the regulation of GH on porcine stomach development is still unknown. So, in present experiment single tube semi-quantitative RT-PCR was applied to investigate the developmental patterns of gastric GHr mRNA expression in Large White and Erhualian Boars, and the Classic 18S internal standards (Ambion Inc. USA) was used. Pigs were sampled at birth, and 3, 20, 30, 45, 90, 120, 180 days of age respectively, meanwhile the bodyweight and gastric weight were recorded. The results indicated that the expression of gastric GHr mRNA was high at birth, followed by a significantly decrease (P < 0.05) at 3 days of age (D3) in both breeds. In Erhualian boars, GHr mRNA expression reached a peak at D45, then remarkablly declined till D90 (P < 0.05), and kept constant thereafter. In Large White boars, however, the expression of GHr mRNA reached a peak at D90 and remained pleateau afterwards. From birth to D30, the GHr mRNA expression was higher in Erhualian than that in Large White, the significant strain difference were founded at D20 (P < 0.05). But from D30 to D180, the higher expression was found in Large white, the significant strain difference was especially at D90 (P < 0.05). Significant positive correlation was found between the expression of GHr mRNA and the ratio of gastric weight to bodyweight from birth to D180 (r = 0.54, P < 0.05). The results suggest that, (1) GHr mRNA in porcine stomach is expressed according a strain specific development pattern; (2) GH directly acts at the gastric tissue and regulates it's growth. The relationship between GH and the development of gastric function is to be elucidated.