VIRMA facilitates intrahepatic cholangiocarcinoma progression through epigenetic augmentation of TMED2 and PARD3B mRNA stabilization.

BACKGROUND: N6-methyladenine modification of RNA, a critical component of the regulatory role at the post-transcriptional level, has a crucial effect on tumor development and progression. vir-Like m6A methyltransferase associated (VIRMA) has been recently discovered as an N6-methyladenine methyltransferase; however, its specific role in intrahepatic cholangiocarcinoma (ICC) remains to be investigated in-depth. METHODS: VIRMA expression and its association with clinicopathological characteristics were evaluated using The Cancer Genome Atlas (TCGA) dataset and tissue microarrays. In vivo and in vitro assays were performed to determine the role of VIRMA in ICC proliferation and metastasis. The underlying mechanism by which VIRMA influences ICC was clarified by RNA sequencing (RNA-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), SLAM sequencing (SLAM-seq), RNA immunoprecipitation, a luciferase reporter assay, and chromatin immunoprecipitation assay. RESULTS: VIRMA showed high expression in ICC tissues, and this finding predicted a dismal prognostic outcome. The high expression of VIRMA in ICC was due to the demethylation of H3K27me3 modification in the promoter region. Functionally, VIRMA is required for the endothelial-mesenchymal transition (EMT) process in ICC cells, as shown by multiple ICC models in in vitro and in vivo experiments. Mechanistically, multi-omics analysis using ICC cells demonstrated that TMED2 and PARD3B were the direct downstream target of VIRMA. The methylated TMED2 and PARD3B transcripts were directly recognized by HuR, which exerted stabilizing effects on its bound RNA. VIRMA-induced expression of TMED2 and PARD3B activated the Akt/GSK/ß-catenin and MEK/ERK/Slug signaling pathways, thereby promoting ICC proliferation and metastasis. CONCLUSIONS: The present study showed that VIRMA plays a critical role in ICC development by stabilizing TMED2 and PARD3B expression through the m6A-HuR-mediated mechanism. Thus, demonstrating VIRMA and its pathway as candidate therapeutic targets for ICC treatment.

WTAP Is Correlated With Unfavorable Prognosis, Tumor Cell Proliferation, and Immune Infiltration in Hepatocellular Carcinoma.

WTAP is involved in various pathological and physiological processes, but its function in hepatocellular carcinoma (HCC) remains elusive. In this study, we investigated the role of WTAP in HCC. Firstly, the mRNA and protein of WTAP were expressed highly in HCC tissue, which reflected clinicopathological characteristics of HCC patients. Then, an interactive analysis of genetic profiles and Kaplan-Meier curves was performed to show that WTAP was an independent predictor of survival of HCC patients. Meanwhile, genes co-expressed with WTAP, potential protein-protein interactions, related signaling pathways, and immune cell infiltration were identified. It was found that high WTAP expression correlated with enhanced interactions between cytokines and their receptors, cell cycle, and chemokine signaling pathways, as well as increased immune cell infiltration. At last, WTAP knockdown experiments in vitro indicate that the WTAP silencing inhibited HCC proliferation and aggressiveness. We conclude that WTAP may be a novel biomarker for prognosis and a therapeutic target for HCC.

Preparation and Photocatalysis of CuO/Bentonite Based on Adsorption and Photocatalytic Activity.

A CuO/bentonite composite photocatalyst was prepared to fully utilize the adsorption capacity of bentonite and the photocatalytic activity of CuO. CuO and bentonite were chosen as a photocatalyst due to the excellent optical property of CuO and large specific surface area of bentonite, together with their high stability and low production cost. The sample was characterized by XRD, SEM, and BET. The effects of several factors on degradation process were investigated such as dosage of H2O2, irradiation time, pH of the solution, and dosage of catalyst. The optimum conditions for decolorization of methylene blue solution by CuO/bentonite were determined. Under optimal conditions, the decolorization efficiency of methylene blue by a 1.4% CuO/bentonite (400 °C) composite photocatalyst under visible irradiation at 240 min reached 96.98%. The degradation process follow edpseudo-second-order kinetics. The photocatalytic mechanism is discussed in detail. This composite structure provides a new solution to the cycle and aggregation of the photocatalyst in water.

Surface charge transition nano-theranostics based on ultra-small Fe3O4 nanoparticles for enhanced photodynamic and photothermal therapy against nasopharyngeal carcinoma.

Platinum-based concurrent chemo-radiotherapy is the most common strategy for the treatment of Nasopharyngeal carcinoma. However, low efficacy and side effects are the two major problems associated with this approach. Therefore, it is urgent need to explore novel therapeutic modalities to meet clinically standards. Photothermal therapy (PTT) and photodynamic therapy (PDT) are non-invasive and light trigger modalities received great attention to overcome the limitations and significantly improved cancer therapy. Here, we developed acidity surface charge transformable nanocluster (NCs) composed of Indocyanine green (ICG), Fe3O4, and Palmitoyl ascorbic acid (PA) with pH-responsive PEG-b-PAEMA-PDMA for enhanced synergistic PDT/PTT. NCs has the neutral hydrophilic surface helps to prolong blood circulation and instantly transformed to positively charged surface at tumoral acidic pH (6.5), which promoted the cellular uptake. Under laser irradiation (808 nm, 1 W/cm2), NCs produced PTT effect, concurrently it converts singlet oxygen (1O2) into H2O2, which can be further involved in Fenton reaction and produce toxic hydroxyl radical (•OH) enhances therapy efficacy. In vitro experiments on HNE-1 cancer cells showed improved intracellular uptake of NCs at low pH and simultaneously induced higher cytotoxicity medicated by synergetic PDT/PTT effect. In vivo therapeutic study revealed that NCs treatment under laser irradiation showed superior inhibition of tumor growth in HNE-1 tumor bearing mice model. Taken together, the present findings suggest that NCs could be used as "all in one" nano theranostic agent for enhanced PDT/PTT of cancer therapy.

GSH triggered intracellular aggregated-cisplatin-loaded iron oxide nanoparticles for overcoming cisplatin resistance in nasopharyngeal carcinoma.

Platinum-based combined chemo-radiotherapy is the most commonly used approach against Nasopharyngeal carcinoma (NPC). However, off target effect and poor efficiency are the two main concerns regarding this approach. Therefore, it is an urgent need to explore novel therapeutic modalities to meet clinically standards. In this work we have established a new anti-cancer drug delivery system, composed of cisplatin (CDDP)-loaded magnetic iron oxide nanoparticles (Fe3O4), further functionalized with surface modification of folic acid (FA) and intracellular aggregation ability peptide (Cys(StBu)-Lys-CBT), named as (FA-MNP-CDDP-CBT). FA-MNP-CDDP-CBT was much more effective on the reversal of CDDP resistance with an average reduction in half maximal inhibitory concentration (IC 50) of 40.9% and 59.1% in HNE-1 cells and HNE-1/DDP resistant cells respectively compared to CDDP alone. Moreover, FA-MNP-CDDP-CBT had also shown a superior targeted uptake effect and higher ROS generation. Convincingly, we observed a remarkable increase in the apoptosis rate of NPC cells by using western blot and flow cytometry. Thus, this newly design nano-system provides a facile approach to enhance the antitumor activity by reducing the side effects of chemotherapy, minimizing systemic toxicity, and reversing CDDP treatment resistance, which could be proposed for NPC patients with primary or secondary chemo-resistance in the future.

Interactome analysis of gene expression profiles identifies CDC6 as a potential therapeutic target modified by miR-215-5p in hepatocellular carcinoma.

Background: Illustrating the pathogenesis of hepatocellular carcinoma (HCC) pathogenesis as well as identifying specific biomarkers are of great significance. Methods: The original CEL files were obtain from Gene Expression Omnibus, then affymetrix package was used to preprocess the CEL files, the function of DEGs were investigated by multiple bioinformatics approach. Finally, typical HCC cell lines and tissue samples were using to validate the role of CDC6 in vitro. Bioinformatics software was used to predict potential microRNA of CDC6. Luciferase assay was used to verify the interactions between CDC6 and microRNA. Results: A total of 445 DEGs were identified in HCC tissues based on two GEO datasets. GSEA results showed that the significant enriched gene sets were only associated with cell cycle signaling pathway. In the co-expression analysis, there were 370 hub genes from the blue modules were screened. We integrated DEGs, hub genes, TCGA cohort and GSEA analyses to further obtain 10 upregulated genes for validation. These genes were overexpressed in HCC tissues and negatively associated with overall and disease-free survival in HCC patients and related to immune cell infiltration in HCC microenvironments. Finally, Cell Division Cycle 6 (CDC6) was highlighted as one of the most probable genes among the 10 candidates participating in cancer process. The expression of CDC6 either in public datasets and HCC tissues sample were commonly high than the non-cancerous counterpart. Furthermore, we recognized that miR-215-5p, could directly bind to the 3'UTR of CDC6. In addition, CDC6 promoted proliferation via regulation of G1 phase checkpoint and was negative regulated by miR-215-5p to involve in the proliferation of HCC. Conclusion: Our study suggested that CDC6 served as a potential therapeutic target for HCC.

Experimental study on the influence of locked-in stress on the uniaxial compressive strength and elastic modulus of rocks.

The rock contains many inclusions which produce high locked-in stress under the ground stress. In order to study the influence of locked-in stress on the mechanical properties of rocks, the rock-like materials and nitrile rubber particles are used to make a test block of the rock-like model which contains inclusions. The rubber particles will expand as the test block is heated, which creates locked-in stress in the inclusions. Uniaxial compression tests of similar model blocks with different locked-in stresses and different inclusion contents were performed by using a water bath and MTS-5T uniaxial compression testing machine. The results show that the peak strength and elastic modulus decreased with the increasement of locked-in stress and inclusion content. In the meantime, the relationship among the peak strength, the elastic modulus of the test piece, the locked-in stress and the inclusion content were obtained with the help of a mathematical fitting analysis of the quantitative formula. Furthermore, the expression and value curve of the joint impact factor are calculated. This paper evaluates the importance of the locked-in stress in the mechanical properties of the rock-like material and provide a guide for other researchers to further investigate the locked-in stress in rocks.

SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma.

Rationale: Hepatocellular carcinoma (HCC) is one of the leading causes of mortality worldwide. Methyltransferase-like 3 (Mettl3), an RNA N6-methyladenosine (m6A) methyltransferase, has been shown to act as an oncogene in several human cancers. However, the regulatory role of posttranslational modifications of Mettl3 in liver cancer remains elusive. Methods: SUMOylation was analyzed using immunoprecipitation and western blot assays. In vitro and in vivo biological functions were examined using MTS, colony formation, wound healing, transwell, apoptosis, and viability assays and the BALB/c nude mouse model, respectively. Immunohistochemistry was conducted to evaluate the prognostic value of Mettl3 expression in HCC. The regulatory mechanism of Mettl3 in HCC was investigated by m6A dot blot, immunofluorescence, dual luciferase reporter, protein stability, and RNA stability assays. Results: Mettl3 was found to be SUMOylated by a small ubiquitin-like modifier SUMO1. Further, SUMOylation of Mettl3 was increased upon mitogen stimulation, which correlated with UBC9 upregulation, and was positively correlated with high metastatic potential of liver cancer. Finally, SUMOylation of Mettl3 was found to regulate HCC progression via controlling Snail mRNA homeostasis in an m6A methyltransferase activity-dependent manner. Conclusions: This study revealed a novel mechanism of SUMOylated Mettl3-mediated Snail mRNA homeostasis, identifying the UBC9/SUMOylated Mettl3/Snail axis as a novel mediator of the SUMO pathway involved in HCC progression.

Zinc finger protein 703 induces EMT and sorafenib resistance in hepatocellular carcinoma by transactivating CLDN4 expression.

Metastasis is one of the most common reasons of hepatocellular carcinoma (HCC) death; however, the molecular mechanism underlying HCC metastasis remains incompletely defined. Here we report a new function of Zinc Finger Protein 703 (ZNF703), a member of the NET/NlZ family of zinc finger transcription factors, in promoting hepatocellular carcinoma metastasis. We demonstrated that the overexpression of ZNF703 in human HCC tissue is correlated with tumor metastasis and recurrence, it is also related with the prognosis and survival rate of patients. ZNF703 overexpression promotes HCC progression in vitro and in vivo, whereas ZNF703 knockdown has the opposite effect. In addition, ZNF703 induces epithelialmesenchymal transition (EMT) via directly binding to the CLDN4 promoter and transactivating CLDN4 expression. Downregulation of CLDN4 can attenuate ZNF703-mediated HCC metastasis, whereas upregulation of CLDN4 can reverse the decreased metastasis induced by ZNF703 knockdown. Our data revealed that ZNF703 expression is correlated with CLDN4 level, the overexpression of both ZNF703 and CLDN4 are leaded to poorer prognosis of patients with HCC. Moreover, ZNF703 knockdown can enhance the sensitivity of HCC cell to sorafenib, whereas ZNF703 overexpression has the opposite effect. These results suggested that ZNF703 might be a potential target for cancer therapies and a candidate prognostic biomarker for predicting whether patients with HCC are befitting for sorafenib treatment.

POPDC3 is a potential biomarker for prognosis and radioresistance in patients with head and neck squamous cell carcinoma.

Radiotherapy is the primary means of treatment for patients with head and neck squamous cell carcinoma (HNSCC); however, radioresistance-induced recurrence is the primary cause of HNSCC treatment failure. Therefore, identifying specific predictive biomarkers of the response to radiotherapy may improve prognosis. In the present study, to identify the potential candidate genes associated with radioresistance in patients with HNSCC, the microarray datasets GSE9716, GSE61772 and GSE20549 were downloaded from the Gene Expression Omnibus database. The original CEL files were preprocessed using the Affymetrix package and quantile normalization and background correction were conducted using the Core package in Bioconductor. The GSE9716 dataset, consisting of 18 irradiated and 16 non-irradiated samples, was divided into two groups according to their exposure to irradiation: i) Non-irradiation group, which included 8 radioresistant samples and 8 radiosensitive samples; and ii) post-irradiation group, which included 9 radioresistant samples and 9 radiosensitive samples. The two groups were treated as separate datasets and screened. A total of 86 differentially expressed genes (DEGs) were identified in the non-irradiation group and 405 DEGs in the post-irradiation group. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis detected several significant functions associated with the DEGs. In the co-expression analysis, 76 hub genes in the light green module and 917 hub genes with a high connectivity were selected for further analysis. Finally, overlapping the DEGs and hub genes from the two groups yielded a map of 13 shared differentially expressed genes. The 13 genes showed significantly different expression in radioresistant samples compared with the radiosensitive samples before and after irradiation. Out of these genes, popeye domain-containing protein 3 (POPDC3) was highly expressed in the post-irradiation group compared with the non-irradiation group. In survival analysis, high POPDC3 expression correlated with poor a prognosis for patients with HNSCC. The independent prognostic factors were identified using univariate and multivariate Cox analyses based on The Cancer Genome Atlas database. These were incorporated into a nomogram to predict 3- and 5-year overall survival. Receiver operating characteristic curves were used to estimate the accuracy of the nomogram. Together these studies suggest that POPDC3 may serve as a potential predictive biomarker for prognosis and radioresistance of patients with HNSCC as well as clinical diagnosis and treatment of patients.

Candidate tumor suppressor gene IRF6 is involved in human breast cancer pathogenesis via modulating PI3K-regulatory subunit PIK3R2 expression.

Background/Aims: The tumor-suppressive functions of interferon regulatory factor 6 (IRF6) in some tumors have been preliminarily established, but its pathogenesis and underlying molecular mechanisms in breast cancer, the most common malignancy in women, remains poorly understood. Methods: Pairs of typical breast cancer cell lines (high- and low-aggressive) in addition to 27 breast cancer tissue samples and 31 non-cancerous breast tissues were used to investigate the expression level of IRF6 and Lentivirus-mediated gain-of-function studies, short hairpin RNA-mediated loss-of-function studies in vivo and in vitro were used to validate the role of IRF6 in breast cancer. Next, we performed RNA-Seq analysis to identify the molecular mechanisms of IRF6 involved in breast cancer progression. Results: Our findings showed that IRF6 was downregulated in highly invasive breast cancer cell lines but upregulated in poorly aggressive ones. Functional assays revealed that elevated IRF6 expression could suppress cell proliferation and tumorigenicity, and enhanced cellular chemotherapeutic sensitivity. To identify the molecular mechanisms involved, we performed a genome-wide and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in breast cancer cells using RNA sequencing of gene expression profiles following the overexpression of IRF6. Genome-wide and KEGG analyses showed that IRF6 might mediate the PI3K-regulatory subunit PIK3R2, which in turn modulated the PI3K/AKT pathway to control breast cancer pathogenesis. Conclusion: We provide the first evidence of the involvement of IRF6 in breast cancer pathogenesis, which was found to modulate the PI3K/AKT pathway via mediating PIK3R2; indicating that IRF6 can be targeted as a potential therapeutic treatment of breast cancer.

GTSE1 is involved in breast cancer progression in p53 mutation-dependent manner.

BACKGROUND: With the rapid development of the high throughput detection techniques, tumor-related Omics data has become an important source for studying the mechanism of tumor progression including breast cancer, one of the major malignancies worldwide. A previous study has shown that the G2 and S phase-expressed-1 (GTSE1) can act as an oncogene in several human cancers. However, its functional roles in breast cancer remain elusive. METHOD: In this study, we analyzed breast cancer data downloaded from The Cancer Genome Atlas (TCGA) databases and other online database including the Oncomine, bc-GenExMiner and PROGgeneV2 database to identify the molecules contributing to the progression of breast cancer. The GTSE1 expression levels were investigated using qRT-PCR, immunoblotting and IHC. The biological function of GTSE1 in the growth, migration and invasion of breast cancer was examined in MDA-MB-231, MDA-MB-468 and MCF7 cell lines. The in vitro cell proliferative, migratory and invasive abilities were evaluated by MTS, colony formation and transwell assay, respectively. The role of GTSE1 in the growth and metastasis of breast cancer were revealed by in vivo investigation using BALB/c nude mice. RESULTS: We showed that the expression level of GTSE1 was upregulated in breast cancer specimens and cell lines, especially in triple negative breast cancer (TNBC) and p53 mutated breast cancer cell lines. Importantly, high GTSE1 expression was positively correlated with histological grade and poor survival. We demonstrated that GTSE1 could promote breast cancer cell growth by activating the AKT pathway and enhance metastasis by regulating the Epithelial-Mesenchymal transition (EMT) pathway. Furthermore, it could cause multidrug resistance in breast cancer cells. Interestingly, we found that GTSE1 could regulate the p53 function to alter the cell cycle distribution dependent on the mutation state of p53. CONCLUSION: Our results reveal that GTSE1 played a key role in the progression of breast cancer, indicating that GTSE1 could serve as a novel biomarker to aid in the assessment of the prognosis of breast cancer.

Global expression profiling and pathway analysis of mouse mammary tumor reveals strain and stage specific dysregulated pathways in breast cancer progression.

It is believed that the alteration of tissue microenvironment would affect cancer initiation and progression. However, little is known in terms of the underlying molecular mechanisms that would affect the initiation and progression of breast cancer. In the present study, we use two murine mammary tumor models with different speeds of tumor initiation and progression for whole genome expression profiling to reveal the involved genes and signaling pathways. The pathways regulating PI3K-Akt signaling and Ras signaling were activated in Fvb mice and promoted tumor progression. Contrastingly, the pathways regulating apoptosis and cellular senescence were activated in Fvb.B6 mice and suppressed tumor progression. We identified distinct patterns of oncogenic pathways activation at different stages of breast cancer, and uncovered five oncogenic pathways that were activated in both human and mouse breast cancers. The genes and pathways discovered in our study would be useful information for other researchers and drug development.

Silencing of HMGA2 reverses retardance of cell differentiation in human myeloid leukaemia.

BACKGROUND: High-mobility group AT-hook 2 (HMGA2) may serve as an architectural transcription factor, and it can regulate a range of normal biological processes including proliferation and differentiation. Upregulation of HMGA2 expression is correlated to the undifferentiated phenotype of immature leukaemic cells. However, the underlying mechanism of HMGA2-dependent myeloid differentiation blockage in leukaemia is unknown. METHODS: To reveal the role and mechanism of HMGA2 in differentiation arrest of myeloid leukaemia cells, the quantitative expression of HMGA2 and homeobox A9 (HOXA9) was analysed by real-time PCR (qRT-PCR). The regulatory function of HMGA2 in blockage of differentiation in human myeloid leukaemia was investigated through in vitro assays (XTT assay, May-Grünwald-Giemsa, flow cytometry analysis and western blot). RESULTS: We found that the expression of HMGA2 and HOXA9 was reduced during the process of granulo-monocytic maturation of acute myeloid leukaemia (AML) cells, knockdown of HMGA2 promotes terminal (granulocytic and monocytic) differentiation of myeloid leukaemia primary blasts and cell lines, and HOXA9 was significantly downregulated in leukaemic cells with knockdown of HMGA2. Downregulation of HOXA9 in myeloid leukaemia cells led to increased differentiation capacity in vitro. CONCLUSIONS: Our data suggest that increased expression of HMGA2 represents a possible new mechanism of myeloid differentiation blockage of leukaemia. Aberrant expression of HMGA2 may enhance HOXA9-dependent leukaemogenesis and myeloid leukaemia phenotype. Disturbance of the HMGA2-HOXA9 pathway is probably a therapeutic strategy in myeloid leukaemia.

Relationship between estrus cycles and behavioral durations of captive female alpine musk deer.

Owing to poor breeding success in captive alpine musk deer, an understanding of the behavioral patterns of musk deer in captivity is important. This study was conducted from June 2004 to January 2005 at the Xinglongshan Musk Deer Farm, which is located within Xinglongshan National Nature Reserve, Gansu Province, China. Focal sampling and continuous recording were used to observe the behaviors of 51 female alpine musk deer (Moschus sifanicus), 42 of which had completed a single estrus cycle and nine of which had completed two or more estrus cycles. All animals were adults that had been born and raised in captivity. The durations of 12 behaviors, including environmental sniffing, moving and feeding, were recorded during the non-breeding seasons and behavioral patterns were compared. The behavioral patterns of females that had completed a single estrus cycle and females that had completed multiple estrus cycles were compared to assess potential behavioral differences. The results showed that females who had only one complete estrus cycle demonstrated more resting behavior, but less feeding and locomotor behavior than females who had completed multiple estrus cycles. Furthermore, single estrus cycle females demonstrated tail-rubbing during the breeding season. The results may yield useful information that can be used in developing better musk deer farming practices.