[Effects of Aeration on Surface Water Nutrient Dynamics and Greenhouse Gas Emission in Different Straw Returning Paddy Fields].

Straw returning is of great significance for improving soil structure, soil fertility, crop yield, and quality. However, straw returning causes environmental problems such as increased methane emission and non-point source pollutant emission risk. How to reduce the negative effects of straw returning is an urgent problem to be solved.In this study, the effects of aerobic treatment on carbon and nitrogen concentration in surface water and greenhouse gas emissions in paddy fields with different treatments of straw returning were systematically compared.The results showed that different treatments of straw returning significantly increased chemical oxygen demand (COD) in the surface water of the paddy field and significantly promoted the methane emission of the rice field and the global warming potential (GWP), although it slightly reduced N2O emission. The increasing trends showed that wheat straw returning>rape straw>broad bean straw returning.Straw returning increased rice yield when compared with the control without straw returning, but the difference was not significant. Aerobic treatment reduced the COD in surface water by 15%-32%, the methane emission of the paddy field by 10.4%-24.8%, and the GWP of paddy field by 9.7%-24.4% under different straw returning treatments, without affecting the rice yield. The mitigation effect of aerobic treatment with wheat straw returning was the best. The results indicated the potential of oxygenation measures in greenhouse gas emission mitigation and COD emission risk reduction in straw returning paddy fields, especially in wheat straw returning paddy fields.

[Effects of Warming and Fertilization on Soil Organic Carbon and Its Labile Components in Rice-wheat Rotation].

Farmland is the important soil carbon pool of terrestrial ecosystems and organic nutrient pool for crop growth. To clarify the impact of climate warming on the soil carbon pool, this study analyzed the effects of warming and fertilization on soil organic carbon and its labile components under rice-wheat rotation using a free-air temperature increase system. The variation in soil carbon pool management index (CPMI) was also evaluated. The results showed that the combined effects of warming and fertilization on soil organic carbon content and labile organic carbon components were insignificant. Warming increased the soil organic carbon (SOC) content, and the differences between warming and the ambient control in total organic carbon (TOC) and recalcitrant organic carbon (ROC) reached a statistically significant level. Compared with those under the ambient control, the contents of TOC, ROC, and labile organic carbon (LOC) subjected to warming increased by 7.72%, 7.42%, and 10.11%, respectively. The increased microbial biomass carbon (MBC) content (20.4%) and decreased particulate organic carbon (POC) content (36.51%) may have been the main reason for the variation in SOC. Warming showed no significant effect on soil dissolved organic carbon (DOC) content, whereas it markedly reduced its soluble microbial by-product components (41.89%). The results also showed that fertilization had no significant effect on soil TOC, ROC, and LOC, but it notably reduced the contents of DOC and POC and increased the MBC content. Compared with those under the control without fertilization, the contents of DOC and POC subjected to fertilization decreased by 35.44% and 28.33%, respectively, and the MBC content increased by 33.38%. Additionally, fertilization tended to increase the anthropogenic humus component (5.13%) and soluble microbial by-product component (29.41%) in dissolved organic matter and reduce the terrestrial humus component (13.33%). Warming and fertilization both tended to improve soil CPMI. Affected by SOC and LOC, the increase in soil carbon pool index and soil lability index were the main reason for the increase in soil CPMI under warming and fertilization, respectively. Overall, the results revealed that climate warming can affect the soil carbon pool by changing soil labile carbon components, which are not affected by fertilization.

[Effect of Deep Fertilization with Slow/Controlled Release Fertilizer on N Fate in Clayey Soil Wheat Field].

Clayey soil seriously affects water-holding capacity and nutrient movement. Adopting appropriate agronomic measures to optimize the distribution of soil inorganic nitrogen (SIN) and reduce the nitrogen (N) loss in this soil is the key to agricultural sustainable development. To clarify the effect of deep fertilization of slow/controlled release fertilizer with sowing on N loss in a clayey soil wheat field, two types of fertilizers, conventional fertilizer (CN) and slow/controlled release fertilizer (RCU), were selected in this study. Here, we evaluated the effects of these two fertilizer types on wheat yield, seasonal N runoff loss, ammonia volatilization, and N2O emissions in wheat fields in two typical fertilization modes (manual surface sowing and spreading (B) and belowground fertilization of slow/controlled release urea with mechanized strip sowing (D)). The temporal and spatial distribution characteristics of SIN in topsoil were also analyzed. The results showed that under the same fertilizer type, the wheat yield of D treatment was significantly higher than that of B treatment, whereas the yield of RCU was notably higher than that of CN under the same fertilization mode. D-RCU achieved the highest yield of 6.97 t·hm-2. The seasonal N losses from runoff and ammonia volatilization were higher than that from N2O emissions, and the responses of different N loss pathways to fertilizer types and fertilization methods were diverse. Fertilizer type and runoff occurrence time were the main influencing factors of N runoff loss, and N runoff loss of the RCU treatment was higher in the non-fertilization period. Unfortunately, affected by annual rainfall pattern, the seasonal N runoff loss of the RCU treatment (20.35 kg·hm-2) was significantly higher than that of the CN treatment (10.49 kg·hm-2). The late growth period was the main phase of ammonia volatilization, and the later period was jointly affected by fertilization modes and fertilizer types. The B-CN treatment induced the highest seasonal ammonia volatilization (18.15 kg·hm-2), which was significantly higher than that of the other treatments (7.31-8.38 kg·hm-2). Additionally, the D-RCU treatment (2.41 kg·hm-2) tended to reduce the N2O emissions in comparison to that in the B-CN treatment (4.02 kg·hm-2). The results also indicated that the horizontal movement of SIN was higher than the vertical movement. Deep fertilization of RCU was conducive to optimizing the spatial and temporal distribution of SIN, which was the main reason for the increase in wheat yield and the control of N loss from wheat fields. These results suggest that RCU is a suitable alternative fertilizer for increasing yield and reducing N loss in clayey soil wheat fields; D-RCU can increase the wheat yield and reduce ammonia volatilization and N2O emissions in wheat fields by optimizing the spatial and temporal distribution of SIN, and its increasing effect on N runoff loss in the non-fertilization period deserves attention.

Effectiveness of intraoperative cell salvage combined with a modified leucocyte depletion filter in metastatic spine tumour surgery.

BACKGROUND: To compare the effectiveness of intraoperative cell salvage (IOCS) combined with a modified leucocyte depletion filter (MLDF) with IOCS combined with a regular leucocyte depletion filter (RLDF) in eliminating tumour cells from blood salvage during metastatic spine tumour surgery (MSTS). METHODS: Patients with a known primary epithelial tumour who underwent MSTS were recruited for this study. Blood samples were collected in 5 stages: from the patients' vein before anaesthesia induction (S1), from the operative field at the time of maximum tumour manipulation (S2), and from the operative blood after IOCS processing (S3) and after IOCS+RLDF (S4) and IOCS+MLDF (S5) processing. The polyploids of tumour cells in the blood samples were collected and counted with immunomagnetic separation enrichment and fluorescence in situ hybridization. RESULTS: We recruited 20 patients. Tumour cells were detected in 14 patients (70%) in S1, 16 patients (80%) in S2, 13 patients (65%) in S3, and 12 patients (60%) in S4. MLDF was added in 8 patients. Tumour cells were detected in only 1 of 8 patients in S5 (12.5%). There were significantly fewer tumour cells in the samples collected after MLDF processing (S5) than in the samples collected after RLDF (S4) and around the tumour (S2) (P = 0.016 and P = 0.039, respectively). Although no significant difference was observed between S4 and S1, a downward trend was observed after IOCS+RLDF processing. CONCLUSIONS: Tumour cells could be removed by IOCS combined with RLDF from blood salvaged during MSTS, but residual tumour cells remained. The findings support the notion that MLDF eliminates tumour cells more effectively than RLDF. Hence, this technique can be applied to MSTS. TRIAL REGISTRATION: ChiCTR1800016162 Chinese Clinical Trial Registry.

Fatty Acids Metabolism: The Bridge Between Ferroptosis and Ionizing Radiation.

Exposure of tumor cells to ionizing radiation (IR) alters the microenvironment, particularly the fatty acid (FA) profile and activity. Moreover, abnormal FA metabolism, either catabolism or anabolism, is essential for synthesizing biological membranes and delivering molecular signals to induce ferroptotic cell death. The current review focuses on the bistable regulation characteristics of FA metabolism and explains how FA catabolism and anabolism pathway crosstalk harmonize different ionizing radiation-regulated ferroptosis responses, resulting in pivotal cell fate decisions. In summary, targeting key molecules involved in lipid metabolism and ferroptosis may amplify the tumor response to IR.

Circular RNA expression in isoproterenol hydrochloride-induced cardiac hypertrophy.

Circular RNA (circRNA) is a novel class of noncoding RNAs, and the roles of circRNAs in the development of cardiac hypertrophy remain to be explored. Here, we investigate the potential roles of circRNAs in cardiac hypertrophy. By circRNA sequencing in left ventricular specimens collected from 8-week-old mice with isoproterenol hydrochloride-induced cardiac hypertrophy, we found 401 out of 3323 total circRNAs were dysregulated in the hypertrophic hearts compared with the controls. Of these, 303 circRNAs were upregulated and 98 were downregulated. Moreover, the GO and KEGG analyses revealed that the majority of parental gene of differentially expressed circRNAs were not only related to biological process such as metabolic process and response to stimulus, but also related to pathway such as circulatory system and cardiovascular diseases. On the other hand, total 1974 miRNAs were predicted to binding to these differentially expressed circRNAs, and the possible target mRNAs of those miRNAs were also predicted and analyzed in terms of functional annotation. Finally, we identified that ANF and miR-23a are downstream targets of circRNA wwp1, suggesting that circRNA wwp1 exerts inhibitory roles of cardiac hypertrophy via down-regulation of ANF and miR-23a, which underlying the potential mechanisms whereby circRNA regulates cardiac hypertrophy.

MicroRNA-9 functions as a tumor suppressor and enhances radio-sensitivity in radio-resistant A549 cells by targeting neuropilin 1.

Radiotherapy is commonly used to treat lung cancer but may not kill all cancer cells, which may be attributed to the radiotherapy resistance that often occurs in non-small cell lung cancer (NSCLC). At present, the molecular mechanism of radio-resistance remains unclear. Neuropilin 1 (NRP1), a co-receptor for vascular endothelial growth factor (VEGF), was demonstrated to be associated with radio-resistance of NSCLC cells via the VEGF-phosphoinositide 3-kinase-nuclear factor-κB pathway in our previous study. It was hypothesized that certain microRNAs (miRs) may serve crucial functions in radio-sensitivity by regulating NRP1. Bioinformatics predicted that NRP1 was a potential target of miR-9, and this was validated by luciferase reporter assays. Functionally, miR-9-transfected A549 cells exhibited a decreased proliferation rate, increased apoptosis rate and attenuated migratory and invasive abilities. Additionally, a high expression of miR-9 also significantly enhanced the radio-sensitivity of A549 cells in vitro and in vivo. These data improve understanding of the mechanisms of cell radio-resistance, and suggest that miR-9 may be a molecular target for the prediction of radio-sensitivity in NSCLC.

Enhancing chemotherapy sensitivity by targeting PcG via the ATM/p53 pathway.

Histone modification and chromatin remodeling are important events in response to DNA damage, and Polycomb group (PcG) proteins, catalyzing H3K27 methylation, are involved. However, the biological function and mechanism of PcG in DNA damage are not fully understood. Additionally, downstream effectors in hepatocellular carcinoma (HCC) remain unclear. The present study investigated the biological and mechanistic roles of PcG in the DNA damage response induced by chemotherapeutic drugs in HCC. It was found that chemotherapy drugs, such as epirubicin (EPB) and mitomycin C (MMC), effectively blocked expression of PcG in p53-wild-type HepG2 cells but not in PLC/PRF5 and Hep3B cells with p53 mutation or deletion. PcG-related target genes involved in DNA damage were identified, including p53, Ataxia telangiectasia mutated (ATM) and Forkhead box O3 (FOXO3). Moreover, targeting PcG-induced p53 expression was associated with increased drug sensitivity in HCC cells. shRNA targeting enhancer of zeste homolog 2 (EZH2) or its inhibitor GSK126 significantly promoted chemotherapeutic drug-induced genotoxicity and increased HepG2 cell chemosensitivity. Mechanistically, chromatin immunoprecipitation (ChIP) assays confirmed that PcG binds to the ATM promoter and inhibits its expression through covalent modification of H3K27me3. Herein, we establish a potential chemotherapy association with GSK126, and the findings suggest this link might represent a new strategy for increasing the sensitivity of HCC to chemotherapeutic agents.

[Control Effect of Side Deep Fertilization with Slow-release Fertilizer on Ammonia Volatilization from Paddy Fields].

In order to reduce the ammonia volatilization in paddy fields, seven treatments were evaluated. These included three slow-release nitrogen fertilizers[sulfur-coated urea (SCU); resin-coated urea (RCU); release bulk blending fertilizer (RBB)], two fertilization modes[single base fertilization (B) and combined with panicle fertilizer (BF)], and conventional split fertilization (CN). The effects of side deep fertilization for slow-release nitrogen fertilizers on ammonia volatilization and surface water nitrogen dynamics were examined using a rice transplanter with a fertilizer sowing mechanism in the Taihu Lake region. The results showed that total nitrogen and ammonium nitrogen concentration in the surface water of the SCU treatment in the base period were higher, and those for RCU and RBB were lower than in the CN treatment. The cumulative ammonia volatilization during the whole rice season varied among different types of slow-release nitrogen fertilizers from 3.84% to 28.17% of the total N applied. The nitrogen loss from ammonia volatilization using the three slow-release nitrogen fertilizers was decreased when compared with conventional split fertilization. The ammonia volatilization loss exhibited the following relationship for the treatments:CN, B-SCU > BF-SCU, BF-RBB, BF-RCU, B-RBB, and B-RCU. When the slow-release nitrogen fertilizers were applied in single base fertilization, the total ammonia volatilization for the SCU was significantly higher than those for the RCU and RBB, while no significant differences were detected when these three slow-release fertilizers were combined with panicle fertilizer. Moreover, although the ammonia volatilization of BF-SCU was lower than that of B-SCU, those of BF-RCU and BF-RBB were higher than those with the B-RCU and B-RBB treatments, respectively. There are no significant differences for nitrogen volatilization when any of these three different fertilizers are applied as B or BF. The results for the emissions during ammonia volatilization during different stages indicated that the ammonia volatilization of SCU at the basal-tillering fertilization stage (7.54%) and the tillering-panicle fertilization stage (16.04%) were higher than those of the panicle fertilization-mature stage. The N loss from ammonia volatilization for RBB in the base-tillering fertilization stage (2.91%) increased more than in the tillering-panicle fertilization stage and panicle fertilization-mature stage. For RCU treatment, the highest rate for ammonia volatilization was detected at the panicle fertilization-mature stage (2.75%). Compared with the single base fertilization mode, ammonia volatilization during the panicle fertilization-mature stage was increased when combined with panicle fertilizer (BF) for the slow-release fertilizer. There was no obvious correlation between the N loss with ammonia volatilization for the three slow-release nitrogen fertilizers and the concentration of ammonium nitrogen in surface water during the panicle fertilization-mature stage.

Genetic alteration and misexpression of Polycomb group genes in hepatocellular carcinoma.

Although the abnormal expression of Polycomb-group (PcG) proteins is closely associated with carcinogenesis and the clinicopathological features of hepatocellular carcinoma (HCC), the genetic mutation profile of PcG genes has not been well established. In this study of human HCC specimens, we firstly discovered a highly conserved mutation site, G553C, in the Polycomb Repressive Complex 2 (PRC2) gene enhancer of zeste homolog 2 (EZH2). This site also harbors a single nucleotide polymorphism (SNP), rs2302427, which plays an important antagonistic role in HCC. Kaplan-Meier survival curves showed that the tumor-free and overall survival of patients with EZH2 G553C were superior to those without the mutation. The G allele frequencies in patients and healthy subjects were 0.2% and 0.122%, respectively, with significant differences in distribution. The individuals carrying the GG and the GC genotypes at rs2302427 showed 3.083-fold and 1.827-fold higher risks of HCC, respectively, compared with individuals carrying the wild-type allele. Furthermore, Immunohistochemical staining revealed that the expression levels of CBX8 (in 53/123 samples) and BMI1 (in 60/130 samples) were markedly increased in human HCC specimens. Importantly, the overall and tumor-free survival rates were significantly reduced in the group of patients who simultaneously expressed PRC1 and PRC2. These results argue that a combination of PRC1 and PRC2 expression has a significant predictive/prognostic value for HCC patients. Taken together, our results indicate the abnormal expression and genetic mutation of PcG members are two independent events; cumulative genetic and epigenetic alterations act synergistically in liver carcinogenesis.

Receptor interacting protein 3-induced RGC-5 cell necroptosis following oxygen glucose deprivation.

BACKGROUND: Necroptosis is a type of regulated form of cell death that has been implicated in the pathogenesis of various diseases. Receptor-interacting protein 3 (RIP3), a member of the RIP family of proteins, has been reported as an important necroptotic pathway mediator in regulating a variety of human diseases, such as myocardial ischemia, inflammatory bowel disease, and ischemic brain injury. Our previous study showed that RIP3 was expressed in rat retinal ganglion cells (RGCs), where it was significantly upregulated during the early stage of acute high intraocular pressure. Furthermore, RIP3 expression was co-localized with propidium iodide (PI)-positive staining (necrotic cells). These results suggested that RIP3 up-regulation might be involved in the necrosis of injured RGCs. In this study, we aimed to reveal the possible involvement of RIP3 in oxygen glucose deprivation (OGD)-induced retinal ganglion cell-5 (RGC-5) necroptosis. METHODS: RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8 h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. RIP3 expression was detected by western blot and flow cytometry was used to detect the effect of RIP3 on RGC-5 necroptosis following OGD in rip3 knockdown cells. Malondialdehyde (MDA) lipid peroxidation assay was performed to determine the degree of oxidative stress. RESULTS: PI staining showed that necrosis was present in the early stage of OGD-induced RGC-5 cell death. The presence of RGC-5 necroptosis after OGD was detected by flow cytometry using necrostatin-1, a necroptosis inhibitor. Western blot demonstrated that RIP3 up-regulation may be involved in RGC-5 necroptosis. Flow cytometry revealed that the number of OGD-induced necrotic RGC-5 cells was reduced after rip3 knockdown. Furthermore, MDA levels in the normal RGC-5 cells were much higher than in the rip3-knockdown cells after OGD. CONCLUSIONS: Our findings suggest that RGC-5 cell necroptosis following OGD is mediated by a RIP3-induced increase in oxidative stress.

EGF Protects Cells Against Dox-Induced Growth Arrest Through Activating Cyclin D1 Expression.

It has been reported that the antitumor drug doxorubicin (Dox) exerts its toxic effects via GATA-4 depletion and that over-expression of GATA-4 reverses Dox-induced toxicity and apoptosis; however, the precise mechanisms remain unclear. In this study, we observed, for the first time, that EGF protects cells against Dox-mediated growth arrest, G2/M-phase arrest, and apoptosis. Additionally, EGF expression was down-regulated in Dox-treated cells and up-regulated in GATA-4 over-expressing cells. Utilizing real-time PCR and western blotting analysis, we found that the expression of the cell cycle-associated protein cyclin D1 was inhibited in GATA-4-silenced cells and Dox-treated cells and was enhanced in GATA-4 over-expressing cells and EGF-treated cells. Furthermore, EGF treatment reversed the inhibited expression of cyclin D1 that was mediated by GATA-4 RNAi or Dox. Our results indicate that EGF, as a downstream target of Dox, may be involved in Dox-induced toxicity as well as in the protective role of GATA-4 against toxicity induced by Dox via regulating cyclin D1 expression, which elucidates a new molecular mechanism of Dox toxicity with important clinical implications.

EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner.

The regulation of cardiac differentiation is critical for maintaining normal cardiac development and function. The precise mechanisms whereby cardiac differentiation is regulated remain uncertain. Here, we have identified a GATA-4 target, EGF, which is essential for cardiogenesis and regulates cardiac differentiation in a dose- and time-dependent manner. Moreover, EGF demonstrates functional interaction with GATA-4 in inducing the cardiac differentiation of P19CL6 cells in a time- and dose-dependent manner. Biochemically, GATA-4 forms a complex with STAT3 to bind to the EGF promoter in response to EGF stimulation and cooperatively activate the EGF promoter. Functionally, the cooperation during EGF activation results in the subsequent activation of cyclin D1 expression, which partly accounts for the lack of additional induction of cardiac differentiation by the GATA-4/STAT3 complex. Thus, we propose a model in which the regulatory cascade of cardiac differentiation involves GATA-4, EGF, and cyclin D1.

The effect and underlying mechanism of Timosaponin B-II on RGC-5 necroptosis induced by hydrogen peroxide.

BACKGROUND: Necroptosis is an important mode of cell death, which is due to oxidant stress accumulation. Our previous study indicated that oxidant stresses could be reduced by Timosaponin B-II (TBII), a kind of Chinese herb RhizomaAnemarrhenae monomer extraction. We wonder the possible effect of Timosaponin B-II, whether it can protect cells from necroptosis via reducing the oxidant stress, in RGC-5 following hydrogen peroxide (H2O2) insult. METHODS: RGC-5 cells were grown in DMEM, the model group was exposed in H2O2 with the concentration of 300 µM, and the experimental group was pre-treated with Timosaponin B-II at different concentrations (1 µM, 10 µM, 100 µM and 1000 µM) for 24 hrs. MTT assay was carried out to measure the cytotoxicity of H2O2, MDA concentration assay was executed to evaluate the degree of oxidative stress, TNF-α ELISA Assay was used to measure the concentration of TNF-α, finally, the degree of necrosis were analyzed using flow cytometry. RESULTS: We first constructed the cell injury model of necroptosis in RGC-5 upon H2O2 exposure. Morphological observation and MTT assay were used to evaluate the degree of RGC-5 death. MDA assay were carried out to describe the degree of oxidant stress. Annexin V/PI staining was used to detect necroptotic cells pre-treated with or without Timosaponin B-II following H2O2 injury. TNF-α ELISA was carried out to detect the TNF-α accumulation in RGC-5. Upon using Timosaponin B-II with concentration of 100 µM, the percentage of cell viability was increased from 50% to 75%, and the necrosis of cells was reduced from 35% to 20% comparing with H2O2 injury group. Oxidant stress and TNF-α was reduced upon injury which decreased the ratio of RGC-5 necroptosis. CONCLUSION: Our study found out that Timosaponin B-II might reduce necroptosis via inhibition of ROS and TNF-α accumulation in RGC-5 following H2O2 injury.

MicroRNA-135a and -200b, potential Biomarkers for Alzheimer׳s disease, regulate ß secretase and amyloid precursor protein.

Amyloid precursor protein (APP) and ß-site amyloid precursor protein cleaving enzyme (BACE-1) play important roles in the generation of Alzheimer׳s disease (AD), a progressive neurodegenerative disorder. In the present study, microRNA (miR) microarray was used to analyze the miR expression profiles in the hippocampi from APP/PS1 transgenic and wild type mice. The miRs with significant alteration and putative targets on APP or BACE-1 were retrieved (miR-135a, -200b and -429). The deregulations of these miRs were confirmed in mice and further verified in AD patient samples by qPCR. Primary mouse hippocampal neurons, SH-SY5Y and HEK293 cells were used to study the function of miRs on APP and BACE-1. We found that miR-135a, which was downregulated significantly in hippocampi from APP/PS1 transgenic mice compared with the wild type control, directly interacted with the 3'-UTR of BACE-1 and repressed its expression and activity. On the other hand, miR-200b and -429, which were downregulated significantly in hippocampi from APP/PS1 transgenic mice compared with the wild type control, targeted the 3'-UTR of APP and repressed its expression. Furthermore, Aß42 could downregulate miR-200b expression which may generate a vicious cycle resulted in accumulating Aß42. The levels of miR-135a and -200b in the serum of DAT group were significantly lower than that of control groups (P<0.05). The serum miR-200b level of MCI group was higher than that of DAT group (P<0.05) and lower than that of control group (P<0.05). We also found decreased miR-135a and -200b levels in the cerebrospinal fluid of DAT group compared with the control group (P<0.05). In conclusion, these findings showed that miR-135a, -200b and -429 may take part in the progress of AD; miR-200b was of great potential as noninvasive and easily detected blood-based biomarkers of MCI and DAT patients.

Calpain: a molecule to induce AIF-mediated necroptosis in RGC-5 following elevated hydrostatic pressure.

BACKGROUND: RIP3 (Receptor-interacting protein 3) pathway was mainly described as the molecular mechanism of necroptosis (programmed necrosis). But recently, non-RIP3 pathways were found to mediate necroptosis. We deliberate to investigate the effect of calpain, a molecule to induce necroptosis as reported (Cell Death Differ 19:245-256, 2012), in RGC-5 following elevated hydrostatic pressure. RESULTS: First, we identified the existence of necroptosis of RGC-5 after insult by using necrostatin-1 (Nec-1, necroptosis inhibitor) detected by flow cytometry. Immunofluorescence staining and western blot were used to detect the expression of calpain. Western blot analysis was carried out to describe the truncated AIF (tAIF) expression with or without pretreatment of ALLN (calpain activity inhibitor). Following elevated hydrostatic pressure, necroptotic cells pretreated with or without ALLN was stained by Annexin V/PI, The activity of calpain was also examined to confirm the inhibition effect of ALLN. The results showed that after cell injury there was an upregulation of calpain expression. Upon adding ALLN, the calpain activity was inhibited, and tAIF production was reduced upon injury along with the decreased number of necroptosis cells. CONCLUSION: Our study found that calpain may induce necroptosis via tAIF-modulation in RGC-5 following elevated hydrostatic pressure.

EZH2 represses target genes through H3K27-dependent and H3K27-independent mechanisms in hepatocellular carcinoma.

UNLABELLED: Alterations of polycomb group (PcG) genes directly modulate the trimethylation of histone H3 lysine 27 (H3K27me3) and may thus affect the epigenome of hepatocellular carcinoma (HCC), which is crucial for controlling the HCC cell phenotype. However, the extent of downstream regulation by PcGs in HCC is not well defined. Using cDNA microarray analysis, we found that the target gene network of PcGs contains well-established genes, such as cyclin-dependent kinase inhibitors (CDKN2A), and genes that were previously undescribed for their regulation by PcG, including E2F1, NOTCH2, and TP53. Using chromatin immunoprecipitation assays, we demonstrated that EZH2 occupancy coincides with H3K27me3 at E2F1 and NOTCH2 promoters. Interestingly, PcG repress the expression of the typical tumor suppressor TP53 in human HCC cells, and an increased level of PcG was correlated with the downregulation of TP53 in certain HCC specimens. Unexpectedly, we did not find obvious H3K27me3 modification or an EZH2 binding signal at the TP53 promoters, suggesting that PcG regulates TP53 expression in an H3K27me3-independent manner. Finally, the reduced expression of PcGs effectively blocked the aggressive signature of liver cancer cells in vitro and in vivo. IMPLICATIONS: Taken together, our results establish the functional and mechanistic significance of certain gene regulatory networks that are regulated by PcGs in HCC.

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose-dependent manner.

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real-time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α-MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL-2-antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA-6 or peroxisome proliferator-activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α-MHC, and GATA-4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.

Insulin induces C2C12 cell proliferation and apoptosis through regulation of cyclin D1 and BAD expression.

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.

miR-200b targets GATA-4 during cell growth and differentiation.

GATA-4 is an important transcription factor involved in several developmental processes of the heart, such as cardiac myocyte proliferation, differentiation and survival. The precise mechanisms underlying the regulation of GATA-4 remain unclear, this is especially true for the mechanisms that mediate the post-transcriptional regulation of GATA-4. Here, we demonstrate that miR-200b, a member of the miR-200 family, is a critical regulator of GATA-4. Overexpression of miR-200b leads to the downregulation of GATA-4 mRNA and a decrease in GATA-4 protein levels. Moreover, miR-200b not only inhibits cell growth and differentiation but also reverses the growth response mediated by GATA-4, whereas depletion of miR-200b leads to a slight reversal of the anti-growth response achieved by knocking down endogenous GATA-4. More importantly, the cell cycle-associated gene cyclin D1, which is a downstream target of GATA-4, is also regulated by miR-200b. Thus, miR-200b targets GATA-4 to downregulate the expression of cyclin D1 and myosin heavy chain (MHC), thereby regulating cell growth and differentiation.