Upregulation of LINC00501 by H3K27 acetylation facilitates gastric cancer metastasis through activating epithelial-mesenchymal transition and angiogenesis.

BACKGROUND: The molecular mechanism of the significant role of long noncoding RNAs (lncRNAs) in the progression and metastasis of gastric cancer (GC) remains largely elusive. Our objective is to detect overexpressed lncRNA in GC and investigate its role in promoting epithelial-mesenchymal transition and tumour microenvironment remodel. METHODS: LncRNA differential expression profile in GC was analysed using RNA microarrays. The level of LINC00501 was evaluated in both GC patient tissues and GC cell lines by quantitative reverse transcription PCR and large-scale (n = 304) tissue microarray. To explore the biological role and regulatory driver of LINC00501 in GC, various experimental techniques including Chromatin isolation by RNA purification (ChIRP), RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP) assay, dual luciferase assays were performed. RESULTS: Clinically, it was observed that LINC00501 level was abnormal overexpression in GC tissue and was associated with GC progression and distant metastasis. Gain and loss molecular biological experiments suggested that LINC00501, promoted EMT process and angiogenesis of GC. Mechanically, the enrichment of H3K27 acetylation in LINC00501 promoter region contributed to the increase of LINC00501 in GC. LINC00501 transactivated transcription of SLUG, by recruiting hnRNPR to its promoter. The growth of GC was inhibited both in vitro and in vivo by suppressing the level of LINC00501 using pharmacological intervention from the histone acetyltransferase (HAT) inhibitor -C646. CONCLUSIONS: This study suggests that LINC00501 promotes GC progression via hnRNPR/SLUG pathway, which indicates a promising biomarker and target for GC.

Identification and validation of a novel prognosis model based on m5C-related long non-coding RNAs in colorectal cancer.

BACKGROUND: The prognosis of tumor patients can be assessed by measuring the levels of lncRNAs (long non-coding RNAs), which play a role in controlling the methylation of the RNA. Prognosis in individuals with colorectal adenocarcinoma (CRC) is strongly linked to lncRNA expression, making it imperative to find lncRNAs that are associated with RNA methylation with strong prognostic value. METHODS: In this study, by analyzing TCGA dataset, we were able to develop a risk model for lncRNAs that are associated with m5C with prognostic significance by employing LASSO regression and univariate Cox proportional analysis. There were a number of methods employed to ensure the model was accurate, including multivariate and univariate Cox regression analysis, Kaplan analysis, and receiver operating characteristic curve analysis. The principal component analysis, GSEA and GSVA analysis were used for risk model analysis. The CIBERSORT instrument and the TIMER database were used to evaluate the link between the immune cells that infiltrate tumors and the risk model. In vitro experiments were also performed to validate the predicted m5C-related significant lncRNAs. RESULTS: The m5c regulators were differentially expressed in colorectal cancer and normal tissue. Based on the screening criteria and LASSO regression, 11 m5c-related lncRNAs were identified for developing the prognostic risk model. Multivariate and univariate Cox regression analysis showed the risk score is a crucial prognostic factor in CRC patients. The 1-year, 3-year, and 5-year AUC curves showed the risk score was higher than those identified for other clinicopathological characteristics. A nomogram using the risk score as a quantitative tool was developed for predicting patients' outcomes in clinical settings. In addition, the risk profile of m5C-associated lncRNAs can discriminate between tumor immune cells' characteristics in CRC. Mutation patterns and chemotherapy were analyzed between high- and low- risk groups of CRC patients. Moreover, TNFRSF10A-AS1 was chosen for the in vitro verification of the m5C-connected lncRNA to demonstrate impressive effects on the proliferation, migration and invasion of CRC cells. CONCLUSION: A risk model including the prognostic value of 11 m5C-associated lncRNAs proves to be a useful prognostic tool for CRC and improves the care of patients suffering from CRC based on these findings.

LINC00543 promotes colorectal cancer metastasis by driving EMT and inducing the M2 polarization of tumor associated macrophages.

BACKGROUND: The interaction between the tumor-microenvironment (TME) and the cancer cells has emerged as a key player in colorectal cancer (CRC) metastasis. A small proportion of CRC cells which undergo epithelial-mesenchymal transition (EMT) facilitate the reshaping of the TME by regulating various cellular ingredients. METHODS: Immunohistochemical analysis, RNA immunoprecipitation (RIP), RNA Antisense Purification (RAP), dual luciferase assays were conducted to investigate the biological function and regulation of LINC00543 in CRC. A series in vitro and in vivo experiments were used to clarify the role of LINC00543 in CRC metastasis. RESULTS: Here we found that the long non-coding RNA LINC00543, was overexpressed in colorectal cancer tissues, which correlated with advanced TNM stage and poorer prognosis of CRC patients. The overexpression of LINC00543 promoted tumorigenesis and metastasis of CRC cells by enhancing EMT and remodeling the TME. Mechanistically, LINC00543 blocked the transport of pre-miR-506-3p across the nuclear-cytoplasmic transporter XPO5, thereby reducing the production of mature miR-506-3p, resulting in the increase in the expression of FOXQ1 and induction of EMT. In addition, upregulation of FOXQ1 induced the expression of CCL2 that accelerated the recruitment of macrophages and their M2 polarization. CONCLUSIONS: Our study showed that LINC00543 enhanced EMT of CRC cells through the pre-miR-506-3p/FOXQ1 axis. This resulted in the upregulation of CCL2, leading to macrophages recruitment and M2 polarization, and ultimately stimulating the progression of CRC.

Proposal of an automated tumor-stromal ratio assessment algorithm and a nomogram for prognosis in early-stage invasive breast cancer.

BACKGROUND: The tumor-stromal ratio (TSR) has been verified to be a prognostic factor in many solid tumors. In most studies, it was manually assessed on routinely stained H&E slides. This study aimed to assess the TSR using image analysis algorithms developed by the Qupath software, and integrate the TSR into a nomogram for prediction of the survival in invasive breast cancer (BC) patients. METHODS: A modified TSR assessment algorithm based on the recognition of tumor and stroma tissues was developed using the Qupath software. The TSR of 234 invasive BC specimens in H&E-stained tissue microarrays (TMAs) were assessed with the algorithm and categorized as stroma-low or stroma-high. The consistency of TSR estimation between Qupath prediction and pathologist annotation was analyzed. Univariable and multivariable analyses were applied to select potential risk factors and a nomogram for predicting survival in invasive BC patients was constructed and validated. An extra TMA containing 110 specimens was obtained to validate the conclusion as an independent cohort. RESULTS: In the discovery cohort, stroma-low and stroma-high were identified in 43.6% and 56.4% cases, respectively. Good concordance was observed between the pathologist annotated and Qupath predicted TSR. The Kaplan-Meier curve showed that stroma-high patients were associated with worse 5-DFS compared to stroma-low patients (p = 0.007). Multivariable analysis identified age, T stage, N status, histological grade, ER status, HER-2 gene, and TSR as potential risk predictors, which were included in the nomogram. The nomogram was well calibrated and showed a favorable predictive value for the recurrence of BC. Kaplan-Meier curves showed that the nomogram had a better risk stratification capability than the TNM staging system. In the external validation of the nomogram, the results were further validated. CONCLUSIONS: Based on H&E-stained TMAs, this study successfully developed image analysis algorithms for TSR assessment and constructed a nomogram for predicting survival in invasive BC.

Comprehensive transcriptomic profiling and mutational landscape of primary gastric linitis plastica.

BACKGROUND: Primary gastric linitis plastica (GLP) is a distinct phenotype of gastric cancer with poor survival. Comprehensive molecular profiles and putative therapeutic targets of GLP remain undetermined. METHODS: We subjected 10 tumor-normal tissue pairs to whole exome sequencing (WES) and whole transcriptome sequencing (WTS). 10 tumor samples were all GLP which involves 100% of the gastric wall macroscopically. TCGA data were compared to generate the top mutated genes and the overexpressed genes in GLP. RESULTS: Our results reveal that GLP has distinctive genomic and transcriptomic features, dysfunction in the Hippo pathway is likely to be a key step during GLP development. 6 genes were identified as significantly highly mutated genes in GLP, including AOX1, ANKRD36C, CPXM1, PTPN14, RPAP1, and DCDC1). MUC6, as a previously identified gastric cancer driver gene, has a high mutation rate (20%) in GLP. 20% of patients in our GLP cohort had CDH1 mutations, while none had RHOA mutations. GLP exhibits high immunodeficiency and low AMPK pathway activity. Our WTS results showed that 3 PI3K-AKT pathway-related genes (PIK3R2, AKT3, and IGF1) were significantly up-regulated in GLP. Two genes were identified using immunohistochemistry (IHC), IGF2BP3 and MUC16, which specifically expressed in diffuse-type-related gastric cancer cell lines, and its knockdown inhibits PI3K-AKT pathway activity. CONCLUSIONS: We provide the first integrative genomic and transcriptomic profiles of GLP, which may facilitate its diagnosis, prognosis, and treatment.

Deciphering nitrous oxide emissions from tropical soils of different land uses.

Tropical regions are hotspots of increasing greenhouse gas emissions associated with land-use change. Although many field studies have quantified soil fluxes of nitrous oxide (N2O; a potent greenhouse gas) from various land uses, the driving mechanisms remain uncertain. Here, we used tropical soils of diverse land uses and actively manipulated the soil moisture (35%, 60%, and 95% water-filled pore space [WFPS]) and substrate supply (control, nitrate, and nitrate plus glucose) to investigate the responses of N2O emissions with short-term incubations. We then identified key factors regulating N2O emissions out of a series of soil physicochemical and biological factors and explored how these factors interacted to drive N2O emissions. Land-use changes from primary forest to oil palm or Acacia plantation risks emitting more N2O, whereas low emissions could be maintained by conversion to Macaranga forest or Imperata grassland; these laboratory observations were corroborated by a literature synthesis of field N2O measurements across tropical regions. Soil redox potential (Eh) and labile organic nitrogen (LON; amino acid mixture, arginine, and urea) mineralization were among the factors with greatest influence on N2O emissions. In contrast to common understandings, the control of WFPS over N2O emissions was largely indirect, and acted through Eh. The mineralization of LON, particularly arginine, potentially played multiple roles in N2O production (e.g., bottlenecks of nitrifier-denitrification or simultaneous nitrification-denitrification versus substrate competition for co-denitrification). Structural equation models suggest that soil-environmental factors of different levels (from distal including land use, soil moisture, and pH to proximal such as LON mineralization) drive N2O emissions through cascading interactions. Overall, we show that, despite identical initial soil conditions, land conversion can substantially alter the N2O emission potential. Also, collectively considering soil-environmental regulators and their interactions associated with land conversion is crucial to predict and design mitigation strategies for N2O emissions from land-use change.

Cropland intensification mediates the radiative balance of greenhouse gas emissions and soil carbon sequestration in maize systems of sub-Saharan Africa.

Sub-Saharan Africa (SSA) must undertake proper cropland intensification for higher crop yields while minimizing climate impacts. Unfortunately, no studies have simultaneously quantified greenhouse gas (GHG; CO2 , CH4 , and N2 O) emissions and soil organic carbon (SOC) change in SSA croplands, leaving it a blind spot in the accounting of global warming potential (GWP). Here, based on 2-year field monitoring of soil emissions of CO2 , CH4 , and N2 O, as well as SOC changes in two contrasting soil types (sandy vs. clayey), we provided the first, full accounting of GWP for maize systems in response to cropland intensifications (increasing nitrogen rates and in combination with crop residue return) in SSA. To corroborate our field observations on SOC change (i.e., 2-year, a short duration), we implemented a process-oriented model parameterized with field data to simulate SOC dynamic over time. We further tested the generality of our findings by including a literature synthesis of SOC change across maize-based systems in SSA. We found that nitrogen application reduced SOC loss, likely through increased biomass yield and consequently belowground carbon allocation. Residue return switched the direction of SOC change from loss to gain; such a benefit (SOC sequestration) was not compromised by CH4 emissions (negligible) nor outweighed by the amplified N2 O emissions, and contributed to negative net GWP. Overall, we show encouraging results that, combining residue and fertilizer-nitrogen input allowed for sequestering 82-284 kg of CO2 -eq per Mg of maize grain produced across two soils. All analyses pointed to an advantage of sandy over clayey soils in achieving higher SOC sequestration targets, and thus call for a re-evaluation on the potential of sandy soils in SOC sequestration across SSA croplands. Our findings carry important implications for developing viable intensification practices for SSA croplands in mitigating climate change while securing food production.

MiR-192-5p/RB1/NF-κBp65 signaling axis promotes IL-10 secretion during gastric cancer EMT to induce Treg cell differentiation in the tumour microenvironment.

BACKGROUND: Regulatory T (Treg) cells are important components of the tumour microenvironment (TME) that play roles in gastric cancer (GC) metastasis. Although tumour cells that undergo epithelial-mesenchymal transition (EMT) regulate Treg cell function, their regulatory mechanism in GC remains unclear. METHODS: The miR-192-5p was identified by examining three Gene Expression Omnibus GC miRNA expression datasets. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were conducted to identify interactions between miR-192-5p and RB1. The role of miR-192-5p/RB1 in GC progression was evaluated based on EdU incorporation, wound healing and Transwell assays. An in vitro co-culture assay was performed to measure the effect of miR-192-5p/RB1 on Treg cell differentiation. In vivo experiments were conducted to explore the role of miR-192-5p in GC progression and Treg cell differentiation. RESULTS: MiR-192-5p was overexpressed in tumour and was associated with poor prognosis in GC. MiR-192-5p bound to the RB1 3'-untranslated region, resulting in GC EMT, proliferation, migration and invasion. MiR-192-5p/RB1 mediated interleukin-10 (IL-10) secretion by regulating nuclear factor-kappaBp65 (NF-κBp65), affecting Treg cell differentiation. NF-κBp65, in turn, promoted miR-192-5p expression and formed a positive feedback loop. Furthermore, in vivo experiments confirmed that miR-192-5p/RB1 promotes GC growth and Treg cell differentiation. CONCLUSION: Collectively, our studies indicate that miR-192-5p/RB1 promotes EMT of tumour cells, and the miR-192-5p/RB1/NF-κBp65 signaling axis induces Treg cell differentiation by regulating IL-10 secretion in GC. Our results suggest that targeting miR-192-5p/RB1/NF-κBp65 /IL-10 may pave the way for the development of new immune treatments for GC.

LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis.

Gastric cancer is anatomically proximal to peritoneum. Gastric cancer peritoneal metastasis is a complex biological process which is corresponded with disharmony within dysfunctional adipose tissue and metabolism reprogramming. Laminin gamma 1 (LAMC1) is highly expressed in cancer cells of peritoneal metastatic sites, however, the mechanism of LAMC1-metiated gastric cancer metastases to adipose tissue-rich peritoneum remains unclear. In our study, immunohistochemical staining, single cell sequencing, a co-culture model, luciferase reporter, RNA immunoprecipitation (RIP), Chromatin immunoprecipitation (CHIP) and single-molecular magnetic tweezers assays were conducted, and our results showed that LAMC1 related to Perilipin-1 content was highly expressed in peritoneal metastatic sites and mainly secreted by tumor cells. Gastric cancer cells secreted LAMC1 in an autocrine manner to detached from the primary site and promoted preadipocytes mature, rupture and release of free fatty acids (FFAs) in the peritoneal microenvironment to form pre-metastatic niche by the paracrine pathway. Reversely, differentiated preadipocyte-derived conditioned medium inhibited glycolysis and enhanced fatty acid oxidation (FAO) rate to promote cell proliferation, mesenchymal-epithelial transformation which led to tumor peritoneal colonization. In terms of biological mechanisms, one of differentiated preadipocyte-derived FFAs, palmitic acid-activated STAT3 inhibited miR-193a-3p by binding to its promoter directly; Using single-molecular magnetic tweezers, this binding manner was proved to be stable, reversable and ATP-dependent. Moreover, miR-193a-3p regulated LAMC1 in a post-translational manner. Furthermore, high LAMC1 expression in serum predicted a higher risk of peritoneal metastasis. In conclusion, our results illustrated that palmitic acid/p-STAT3/miR-193a-3p/LAMC1 pathway promotes preadipocyte differentiation, pre-metastatic niche formation and gastric cancer cell colonization to peritoneum.

Hypoxia-induced HIF-1α/lncRNA-PMAN inhibits ferroptosis by promoting the cytoplasmic translocation of ELAVL1 in peritoneal dissemination from gastric cancer.

Peritoneal metastasis (PM) is the main site of gastric cancer (GC) distant metastasis and indicates an extremely poor prognosis and survival. Hypoxia is a common feature of peritoneal metastases and up-regulation of hypoxia inducible factor 1 alpha (HIF-1α) may be a potential driver in the occurrence of PM. Ferroptosis is a recently discovered form of regulated cell death and closely related to the occurrence and development of tumors. However, the underlying mechanism link HIF-1α to ferroptosis in PM of GC remains unknown. Here, lncRNA-microarrays and RNA library construction/lncRNA-seq results shown that lncRNA-PMAN was highly expressed in PM and significantly modulated by HIF-1α. Upregulation of PMAN is associated with poor prognosis and PM in patients with GC. PMAN was up-regulated by HIF-1α and improved the stability of SLC7A11 mRNA by promoting the cytoplasmic distribution of ELAVL1, which was identified in RNA-pulldown/mass spectrometry results. Accumulation of SLC7A11 increases the level of l-Glutathione (GSH) and inhibits the accumulation of reactive oxygen species (ROS) and irons in the GC cells. Finally protect GC cells against ferroptosis induced by Erastin and RSL3. Our findings have elucidated the effect of HIF-1α/PMAN/ELAVL1 in GC cells ferroptosis and provides theoretical support for the potential diagnostic biomarkers and therapeutic targets for PM in GC.

EMT-cancer cells-derived exosomal miR-27b-3p promotes circulating tumour cells-mediated metastasis by modulating vascular permeability in colorectal cancer.

BACKGROUND: Metastasis is the main cause of death in colorectal cancer (CRC). Circulating tumour cells (CTCs) are regarded as the precursor cells of metastasis. The CTCs, which underwent epithelial-mesenchymal transition (EMT), are associated with metastasis and responsible for poor prognosis. EMT cancer cells modulate endothelial permeability in the invasive front and facilitate cancer cell intravasation, resulting in CTCs-mediated distant metastasis. Exosomes derived from cancer cells are key mediators of cancer-host intercommunication. However, the mechanism by which EMT-tumour cells-derived exosomes modulate vascular permeability and promote CTCs generation has remained unclear. METHODS: Exosomes isolation and purification were conducted by ultra-centrifugation. Exosomal miRNA was identified by sequencing followed by quantitative PCR. In vitro co-culture assay experiments were conducted to evaluate the effect of exosomal miR-27b-3p on the permeability of blood vessel endothelium. Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) were performed to investigate the underlying mechanism by which miR-27b-3p is packaged into exosomes. A mouse model was established to determine the role of exosomal miR-27b-3p in blood vessel permeability modulation in vivo. RESULTS: We found that EMT-CRC cells attenuate the blood vessel barrier by transferring miR-27b-3p to human umbilical vein endothelial cells (HUVECs) in exosomes. Mechanically, miR-27b-3p atteuated the expression of vascular endothelial cadherin (VE-Cad) and p120 at the post-transcriptional level by binding to 3'-untranslated region of VE-Cad and p120 directly. The packaging of miR-27b-3p into exosomes was induced by heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which activated by STAT3. Clinically, miR-27b-3p up-regulated in CRC tissues. Plasma exosomal miR-27b-3p was positively correlated with malignant progression and CTC count in CRC patients. Our study reveals a novel mechanism by which EMT-CRC cells promote metastasis, increasing blood vessel permeability and facilitating the generation of CTCs. CONCLUSION: Exosomal miR-27b-3p secreted by EMT-CRC cells increases blood vessel permeability and facilitates the generation of CTCs. Exosomal miR-27b-3p may become a promising biomarker for CRC metastasis.

SDC2 and TFPI2 Methylation in Stool Samples as an Integrated Biomarker for Early Detection of Colorectal Cancer.

BACKGROUND: Detection of aberrant methylated DNA in the stool is an effective early screening method for colorectal cancer (CRC). Previously, reporters identified that syndecan-2 (SDC2) and tissue factor pathway inhibitor 2 (TFPI2) were aberrantly methylated in most CRC tissues. However, the combined diagnostic role of them remains undefined. Our research aimed at probing the role and efficiency of the methylation status of SDC2 and TFPI2 in CRC early screening by using bioinformatics analysis and clinical stool sample validation. METHODS: The promoter and CpG site methylation levels of SDC2 and TFPI2 and their correlation with clinicopathological characteristics of CRC were analyzed using UALCAN, Methsurv, and Wanderer. UCSC Xena was used to perform survival analyses. LinkedOmics was used to do functional network analysis. DNA was isolated and purified from stool, and quantitative methylation-specific PCR (qMSP) was applied to detect methylatedSDC2 and TFPI2. RESULTS: The results showed that promoter and most CpG site methylation levels of SDC2 and TFPI2 were significantly higher in CRC than in normal tissues. Moreover, SDC2 and TFPI2 methylation showed a positive correlation. Functional network analysis suggested that both methylated SDC2 and TFPI2 were involved in tumor cells' metabolic programs. Besides, there was a higher positive integrated detection rate in CRC (n=61) with a sensitivity of 93.4% and in adenoma (Ade) (n=16) with a sensitivity of 81.3% than normal with a specificity of 94.3% in stool samples. What is more, integration of methylated SDC2 and TFPI2 showed a higher sensitivity and Youden index than a single gene in detecting Adeor CRC. CONCLUSION: Our data indicate that SDC2 and TFPI2 were hypermethylated in CRC, and integrated detection of methylated SDC2 and TFPI2 in stool has the potential to be an effective and noninvasive tool of CRC early screening.