Exploring the autophagy-related pathogenesis of active ulcerative colitis.

BACKGROUND: The pathogenesis of ulcerative colitis (UC) is complex, and recent therapeutic advances remain unable to fully alleviate the condition. AIM: To inform the development of novel UC treatments, bioinformatics was used to explore the autophagy-related pathogenesis associated with the active phase of UC. METHODS: The GEO database was searched for UC-related datasets that included healthy controls who met the screening criteria. Differential analysis was conducted to obtain differentially expressed genes (DEGs). Autophagy-related targets were collected and intersected with the DEGs to identiy differentially expressed autophagy-related genes (DEARGs) associated with active UC. DEARGs were then subjected to KEGG, GO, and DisGeNET disease enrichment analyses using R software. Differential analysis of immune infiltrating cells was performed using the CiberSort algorithm. The least absolute shrinkage and selection operator algorithm and protein-protein interaction network were used to narrow down the DEARGs, and the top five targets in the Dgree ranking were designated as core targets. RESULTS: A total of 4822 DEGs were obtained, of which 58 were classified as DEARGs. SERPINA1, BAG3, HSPA5, CASP1, and CX3CL1 were identified as core targets. GO enrichment analysis revealed that DEARGs were primarily enriched in processes related to autophagy regulation and macroautophagy. KEGG enrichment analysis showed that DEARGs were predominantly associated with NOD-like receptor signaling and other signaling pathways. Disease enrichment analysis indicated that DEARGs were significantly linked to diseases such as malignant glioma and middle cerebral artery occlusion. Immune infiltration analysis demonstrated a higher presence of immune cells like activated memory CD4 T cells and follicular helper T cells in active UC patients than in healthy controls. CONCLUSION: Autophagy is closely related to the active phase of UC and the potential targets obtained from the analysis in this study may provide new insight into the treatment of active UC patients.

Hepatocellular carcinoma cells remodel the pro-metastatic tumour microenvironment through recruitment and activation of fibroblasts via paracrine Egfl7 signaling.

BACKGROUND: The tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear. METHODS: Primary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model. RESULTS: We showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανß3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis. CONCLUSION: Our data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανß3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients. Video Abstract.

Piezo1 promoted hepatocellular carcinoma progression and EMT through activating TGF-ß signaling by recruiting Rab5c.

BACKGROUND: Piezo1 has been revealed to play a regulatory role in vascular development and progression of variety tumors. However, whether and how the progression of hepatocellular carcinoma (HCC) regulated by Piezo1 remains elusive. This study aimed to elucidate the effect and mechanisms of Piezo1 in HCC. METHODS: The mRNA and protein expression level of Piezo1 in HCC samples and cell lines was determined by qRT-PCR, western blot and immunohistochemistry analyses. Two independent study cohorts containing 280 patients were analyzed to reveal the association between Piezo1 expression and clinicopathological characteristics. Series of in vitro and in vivo experiments were used to validate the function of Piezo1 in HCC. Gene set enrichment analysis (GSEA) was performed to explore the signaling pathway of Piezo1. Immunoprecipitation, immunofluorescence and in vitro and in vivo experiments were used to explore the molecular mechanism of Piezo1 in HCC progression. RESULTS: Our results demonstrated the Piezo1 expression was significantly upregulated in HCC tissues and cell lines, and upregulation of Piezo1 closely correlated with aggressive clinicopathological features and poor prognosis. Knockdown of Piezo1 in HCCLM3 and Hep3B cells significantly restrained proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of HCC cells in vitro, and tumor growth, metastasis, EMT in vivo. TGF-ß signaling pathway was most significant enriched pathway in GSEA. Finally, tumor promotion effect of Piezo1 was found to exerted through recruiting and combining Rab5c to activating TGF-ß signaling pathway. CONCLUSIONS: Piezo1 significantly related to poor prognosis and promotes progression of hepatocellular carcinoma via activating TGF-ß signaling, which suggesting that Piezo1 may serve as a novel prognostic predictor and the potential therapeutic target for HCC patients.

Ischemia-Reperfusion Injury and Immunosuppressants Promote Polyomavirus Replication Through Common Molecular Mechanisms.

Background: BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) causes renal allograft dysfunction and graft loss. However, the mechanism of BKPyV replication after kidney transplantation is unclear. Clinical studies have demonstrated that immunosuppressants and renal ischemia-reperfusion injury (IRI) are risk factors for BKPyV infection. Studying the pathogenic mechanism of BKPyV is limited by the inability of BKPyV to infect the animal. Mouse polyomavirus (MPyV) is a close homolog of BKPyV. We used a model of MPyV infection to investigate the core genes and underlying mechanism of IRI and immunosuppressants to promote polyomavirus replication. Materials and Methods: One-day-old male C57BL/6 mice were intraperitoneally injected with MPyV. At week 9 post-infection, all mice were randomly divided into IRI, immunosuppressant, and control groups and treated accordingly. IRI was established by clamping the left renal pedicle. Subsequently, kidney specimens were collected for detecting MPyV DNA, histopathological observation, and high-throughput RNA sequencing. Weighted gene correlation network analysis (WGCNA), protein-protein interaction network analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to screen for core genes and common signaling pathways involved in promoting MPyV replication by IRI and immunosuppressants. Results: After primary infection, MPyV established persistent infection in kidneys and subsequently was significantly increased by IRI or immunosuppressant treatment individually. In the IRI group, viral loads peaked on day 3 in the left kidney, which were significantly higher than those in the right kidney and the control group. In the immunosuppressant group, viral loads in the left kidney were significantly increased on day 3, which were significantly higher than those in the control group. Protein-protein interaction network analysis and WGCNA screened complement C3, epidermal growth factor receptor (EGFR), and FN1 as core genes. Pathway enrichment analysis based on the IRI- or immunosuppressant-related genes selected by WGCNA indicated that the NF-κB signaling pathway was the main pathway involved in promoting MPyV replication. The core genes were further confirmed using published datasets GSE47199 and GSE75693 in human polyomavirus-associated nephropathy. Conclusions: Our study demonstrated that IRI and immunosuppressants promote polyomavirus replication through common molecular mechanisms. In future studies, knockdown or specific inhibition of C3, EGFR, FN1, and NF-κB signaling pathway will further validate their critical roles in promoting polyomavirus replication.

GNA14's interaction with RACK1 inhibits hepatocellular carcinoma progression through reducing MAPK/JNK and PI3K/AKT signaling pathway.

Gαq subfamily proteins play critical roles in many biological functions including cardiovascular development, angiogenesis, and tumorigenesis of melanoma. However, the understanding of G Protein Subunit Alpha 14 (GNA14) in diseases, especially in cancers is limited. Here, we revealed that GNA14 was significantly low expression in Human hepatocellular carcinoma (HCC) samples. Low GNA14 expression was correlated with aggressive clinicopathological features. Moreover, the overall survival (OS) and disease-free survival (DFS) of high GNA14 expression HCC patients were much better than low GNA14 expression group. Lentivirus-mediated GNA14 knockdown significantly promoted the growth of liver cancer in vitro and in vivo. However, opposing results were observed when GNA14 is upregulated. Mechanistically, We identified receptor for activated C kinase 1 (RACK1) as a binding partner of GNA14 by co-immunoprecipitation and mass spectrometry (MS). Glutathione-S-transferase (GST) pull-down assay further verified the direct interaction between GNA14 and RACK1. RNA-Seq and loss- and gain-of-function assays also confirmed that GNA14 reduced the activity of both MAPK/JNK and PI3K/AKT signaling pathways through RACK1. GNA14 synergized with U73122 (PLC inhibitor) to enhance this effect. Further studies suggested that GNA14 potentially competed with protein kinase C (PKC) to bind with RACK1, consequently reducing the stability of PKC. Moreover, we also showed that GNA14'supression of p-AKT protein level depended on sufficient RACK1 expression. In conclusion, we indicated a different role of GNA14, which acted as a suppressor inhibiting liver cancer progression through MAPK/JNK and PI3K/AKT signaling pathways. Due to this, GNA14 served as a potentially valuable prognostic biomarker for liver cancer.

Programmed cell death 10 promotes metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma via PP2Ac-mediated YAP activation.

Tumour metastasis is the main cause of postoperative tumour recurrence and mortality in patients with hepatocellular carcinoma (HCC), but the underlying mechanism remains unclear. Accumulating evidence has demonstrated that programmed cell death 10 (PDCD10) plays an important role in many biological processes. However, the role of PDCD10 in HCC progression is still elusive. In this study, we aimed to explore the clinical significance and molecular function of PDCD10 in HCC. PDCD10 is significantly upregulated in HCC, which also correlates with aggressive clinicopathological characteristics and predicts poor prognosis of HCC patients after liver resection. High PDCD10 expression promotes HCC cell proliferation, migration, and invasion in vitro and tumour growth, metastasis in vivo. In addition, PDCD10 could facilitate epithelial-to-mesenchymal transition (EMT) of HCC cells. In terms of the mechanism, PDCD10 directly binds to the catalytic subunit of protein phosphatase 2A (PP2Ac) and increases its enzymatic activity, leading to the interaction of YAP and dephosphorylation of the YAP protein. This interaction contributes to YAP nuclear translocation and transcriptional activation. PP2Ac is necessary for PDCD10-mediated HCC progression. Knocking down PP2Ac abolished the tumour-promoting role of PDCD10 in the migration, invasion and EMT of HCC. Moreover, a PP2Ac inhibitor (LB100) could restrict tumour growth and metastasis of HCC with high PDCD10 expression. Collectively, PDCD10 promotes EMT and the progression of HCC by interacting with PP2Ac to promote YAP activation, which provides new insight into the mechanism of cancer metastasis. PDCD10 may be a potential prognostic biomarker and therapeutic target for HCC.

Serum epidermal growth factor-like domain 7 serves as a novel diagnostic marker for early hepatocellular carcinoma.

BACKGROUND: Epidermal growth factor-like domain 7 (Egfl7), a recently identified secreted protein, was significantly increased in patients with HCC by our previous studies. However, its efficacy in the diagnosis of early HCC remains unknown. In this study, we therefore evaluate the efficacy of serum Egfl7 for early HCC diagnosis and compare it with alpha-fetoprotein (AFP). METHODS: Serum Egfl7 levels in testing cohort (1081 participants) and validation cohort (476 participants) were measured by a sandwich enzyme-linked immunoassay (ELISA). The cut-off value of Egfl7 was determined by Youden's index and the efficacies of Egfl7 and AFP in diagnosing early HCC were estimated by receiver operating characteristic (ROC). RESULTS: Serum Egfl7 was significantly elevated in patients with early HCC than all non-HCC controls in whatever Testing Cohort or Validation Cohort. In the Testing Cohort, ROC curves showed the optimum cut-off value of Egfl7 was 2610 ng/mL and Egfl7 showed a significantly higher sensitivity than AFP in discriminating early HCC from healthy individuals (77.4% vs. 65.3%, P = 0.0013) but the area under ROC (AUROC) and accuracy of Egfl7 and AFP were similar (0.860 vs. 0.868, P = 0.704; 80.2% vs. 83.8%, P = 0.184). In distinguishing patients with early HCC from patients with chronic liver disease (CLD), the AUROC, sensitivity, specificity and accuracy of Egfl7 were 0.800, 75.2, 71.7 and 73.5%, which were all significantly higher than AFP (0.675, 61.8, 62.0 and 61.9% in order). Egfl7 also showed a significant higher sensitivity and accuracy than AFP (76.6% vs. 64.0%, P = 0.0031; 79.9% vs. 66.1%, P < 0.0001) in differentiating early HCC patients from non-HCC individuals. Additionally, 70.8% of early HCC patients with negative AFP could be diagnosed by Egfl7 and the combined use of Egfl7 and AFP increased the sensitivity to 91.0%. These results were confirmed by a validation cohort. CONCLUSION: Egfl7 is a valuable serum marker in the diagnosis of early HCC and could complement the efficacy of AFP, especially in distinguishing early HCC from CLD and identifying patients with AFP-negative early HCC.

Metabolomics profiling for identification of potential biomarkers in chickens infected with avian leukosis virus subgroup J (ALV-J).

There are currently no vaccines or effective drugs to prevent the disorders caused by avian leukosis virus subgroup J (ALV-J). Hence, it is critical to identify potential biomarkers in ALV-J-infected chickens to prevent ALV-J-induced disorders. We hypothesized that ALV-J infection alters metabolic profile in chickens. In the present study, a nontargeted metabolomics approach based on liquid chromatography coupled with mass spectrometry (LC-MS) was used to find differential metabolites in plasma samples from ALV-J-infected chickens and healthy controls. The parametric statistical test (Student's t-test) and fold change analysis were used for univariate analysis. Multivariate statistical analyses included principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The levels of methyl bromide, pyraclonil, hexaflumuron, lythidathion, 3-phosphoglycerol-glutathione, bis-4-nitrophenyl phosphate, 4-ketocyclophosphamide, oxidized photinus luciferin, phenyl sulfate, and aryl sulfate significantly decreased, whereas the levels of 2-methylthiobenzothiazole, irinotecan, methadone, 3-o-ethyl-l-ascorbic acid, and o-acetylneuraminic acid markedly increased in ALV-J-infected chickens as compared to those in healthy controls. These data provide metabolic evidence and potential biomarkers for ALV-J-induced alterations in plasma metabolism.

ATE1 Inhibits Liver Cancer Progression through RGS5-Mediated Suppression of Wnt/ß-Catenin Signaling.

Arginyltransferase (ATE1) plays critical roles in many biological functions including cardiovascular development, angiogenesis, adipogenesis, muscle contraction, and metastasis of cancer. However, the role of ATE1 in hepatocellular carcinoma (HCC) remains unknown. In this study, we find that ATE1 plays an essential role in growth and malignancy of liver cancer. ATE1 expression is significantly reduced in human HCC samples compared with normal liver tissue. In addition, low ATE1 expression is correlated with aggressive clinicopathologic features and is an independent poor prognostic factor for overall survival and disease-free survival of patients with HCC. Lentivirus-mediated ATE1 knockdown significantly promoted liver cancer growth, migration, and disease progression in vitro and in vivo. Opposing results were observed when ATE1 was upregulated. Mechanistically, ATE1 accelerated the degradation of ß-catenin and inhibited Wnt signaling by regulating turnover of Regulator of G Protein Signaling 5 (RGS5). Loss- and gain-of-function assays confirmed that RGS5 was a key effector of ATE1-mediated regulation of Wnt signaling. Further studies indicated that RGS5 might be involved in regulating the activity of GSK3-ß, a crucial component of the cytoplasmic destruction complex. Treatment with a GSK inhibitor (CHIR99021) cooperated with ablation of ATE1 or RGS5 overexpression to promote Wnt/ß-catenin signaling, but overexpression of ATE1 or RGS5 knockdown did not reverse the effect of GSK inhibitor. IMPLICATIONS: ATE1 inhibits liver cancer progression by suppressing Wnt/ß-catenin signaling and can serve as a potentially valuable prognostic biomarker for HCC.

NAD(P)HX epimerase downregulation promotes tumor progression through ROS/HIF-1α signaling in hepatocellular carcinoma.

Reactive oxygen species (ROS) derived from aberrant tumor metabolism could contribute to tumor invasion and metastasis. NAD(P)HX Epimerase (NAXE), an epimerase that allows the repair of damaged forms of antioxidant NADPH, is a potential cellular ROS scavenger and its role in tumor development is still elusive. Here, we found that NAXE is significantly downregulated in hepatocellular carcinoma (HCC) tissues and cell lines. NAXE downregulation is associated with poor clinicopathological characteristics and is an independent risk factor for overall and disease-free survival of HCC patients after liver resection. In addition, low NAXE expression could identify worse prognosis of HCC patients before vascular invasion or in early stages of disease. In particularly, low NAXE expression in HCC is markedly associated with microvascular invasion (MVI) and its combination with MVI predicts poorer prognosis of HCC patients after liver resection. Furthermore, in vitro and in vivo experiments both showed that knockdown of NAXE expression in HCC cells promoted migration, invasion, and metastasis by inducing epithelial-mesenchymal transition (EMT), whereas NAXE overexpression causes the opposite effects. Mechanistically, low NAXE expression reduced NADPH levels and further caused ROS level increase and hypoxia-inducible factor-1α (HIF-1α) activation, thereby promoting invasion and metastasis of HCC by facilitating EMT. What is more, the tumor-promoting effect of NAXE knockdown in HCC xenograft can be abolished by giving mice N-acetyl-l-cysteine (NAC) in drinking water. Taken together, our findings uncovered a tumor suppressor role for NAXE in HCC by scavenging excessive ROS and inhibiting tumor-promoting signaling pathways, suggesting a new strategy for HCC therapy by targeting redox signaling.

Avian leukosis virus subgroup J infection alters viral composition in the chicken gut.

Chicken is one of the economically important poultry species. Avian leucosis virus subgroup J (ALV-J) has emerged as a serious cause of mortality and suboptimal performance of domestic chickens. Changes in virome may contribute to pathogenesis. Thus, it is important to investigate the effects of ALV-J infection on the composition of the virome in chicken. In the study metagenomic sequencing was used to characterize the virome of feces collected from the AVL-J infected chickens and the controls. Our results indicated that the chicken gut virome contained a diverse range of viruses that can be found in mammal, reptile, fish, and frogs. Furthermore, at the order, family and genus levels, AVL-J infection significantly altered the chicken gut virome composition. The predominant order was Herpesvirales, accounting for more than 96% of the chicken gut virome. Furthermore, the relative abundance of Caudovirales in the controls was higher than that in the AVL-J-infected chickens. At the family level, the relative abundance of Herpesviridae, Myoviridae, Alloherpesviridae, and Genomoviridae was significantly altered in the AVL-J-infected chickens compared with that in the controls. Additionally, the relative abundance of 15 genera showed a significant difference between the AVL-J-infected chickens and controls. These results will increase our understanding of the viral diversity and changes in the virome of chicken gut, with implications in chicken health.

EB2 promotes hepatocellular carcinoma proliferation and metastasis via MAPK/ERK pathway by modulating microtubule dynamics.

Metastasis is the main cause of poor postoperative survival of hepatocellular carcinoma (HCC) patients. Cytoskeleton rearrangement is a key event in cancer metastasis. However, the significance of microtubule (MT), one of the core components of cytoskeleton, in this process is only beginning to be revealed. Here, we find that the MT dynamics regulator end-binding protein 2 (EB2) is highly expressed in HCC and predicts poor prognosis of HCC patients. Functional studies show that EB2 overexpression promotes HCC proliferation, invasion and metastasis in vitro and in vivo, while EB2 knockdown has opposite results. Mechanistically, EB2 mediates MTs destabilization, increases Src (Src proto-oncogene non-receptor tyrosine kinase) activity, and thus facilitates extracellular signal-regulated kinase (ERK) signaling activation, which could in turn promote EB2 expression in HCC, eventually resulting in enhanced HCC proliferation, invasion and metastasis. Furthermore, U0126, a specific ERK inhibitor, could effectively inhibit EB2-mediated HCC proliferation and metastasis in vitro and in vivo. In conclusion, EB2 coordinates MT cytoskeleton and intracellular signal transduction, forming an EB2-MT-ERK positive feedback loop, to facilitate HCC proliferation, invasion and metastasis. EB2 could serve as a promising prognostic biomarker and potential therapeutic target for HCC; HCC patients with high EB2 expression may benefit from treatment with ERK inhibitors.

STMN2 mediates nuclear translocation of Smad2/3 and enhances TGFß signaling by destabilizing microtubules to promote epithelial-mesenchymal transition in hepatocellular carcinoma.

Metastasis remains the major obstacle of improving the survival of patients with hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is critical to cancer metastasis. Successful induction of EMT requires dramatic cytoskeleton rearrangement. However, the significance of microtubule (MT), one of the core components of cell cytoskeleton, in this process remains largely unknown. Here we revealed that STMN2, an important MT dynamics regulator, is barely expressed in normal live tissues but markedly up-regulated in HCCs, especially in those with early recurrence. High STMN2 expression correlates with aggressive clinicopathological features and predicts poor prognosis of HCC patients. STMN2 overexpression in HCC cells promotes EMT, invasion and metastasis in vitro and in vivo, whereas STMN2 knockdown has opposite results. Mechanistically, STMN2 modulates MTs disassembly, disrupts MT-Smad complex, and facilitates release from MT network, phosphorylation and nuclear translocation of Smad2/3 even independent of TGFß stimulation, thereby enhancing TGFß signaling. Collectively, STMN2 orchestrates MT disassembly to facilitate EMT via TGF-ß signaling, providing a novel insight into the mechanisms underlying cancer metastasis. STMN2 is a promising prognostic biomarker and potential therapeutic target for HCC.

HEG1 indicates poor prognosis and promotes hepatocellular carcinoma invasion, metastasis, and EMT by activating Wnt/ß-catenin signaling.

Heart development protein with EGF-like domains 1 (HEG1) plays critical roles in embryo development and angiogenesis, which are closely related to tumor progression. However, the role of HEG1 in hepatocellular carcinoma (HCC) remains unknown. In the present study, we explored the clinical significance, biological function and regulatory mechanisms of HEG1 in HCC and found that HEG1 is significantly up-regulated in HCC cell lines and primary tumor samples. Additionally, high HEG1 expression is correlated with aggressive clinicopathological features. Patients with high HEG1 expression had shorter overall survival (OS) and disease-free survival (DFS) than those with low HEG1 expression, which indicated that HEG1 is an independent factor for poor prognosis. Lentivirus-mediated HEG1 overexpression significantly promotes HCC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro and promotes intrahepatic metastasis, lung metastasis and EMT in vivo Opposing results are observed when HEG1 is silenced. Mechanistically, HEG1 promotes ß-catenin expression and maintains its stability, leading to intracellular ß-catenin accumulation, ß-catenin nuclear translocation and Wnt signaling activation. Loss- and gain-of-function assays further confirmed that ß-catenin is essential for HEG1-mediated promotion of HCC invasion, metastasis and EMT. In conclusion, HEG1 indicates poor prognosis; plays important roles in HCC invasion, metastasis and EMT by activating Wnt/ß-catenin signaling; and can serve as a potentially valuable prognostic biomarker and therapeutic target for HCC.

Analyses of BMAL1 and PER2 Oscillations in a Model of Breast Cancer Progression Reveal Changes With Malignancy.

From an epidemiological standpoint, disruptions to circadian rhythms have been shown to contribute to the development of various disease pathologies, including breast cancer. However, it is unclear how altered circadian rhythms are related to malignant transformations at the molecular level. In this article, a series of isogenic breast cancer cells representing disease progression was used to investigate the expression patterns of core circadian clock proteins BMAL1 and PER2. Our model is indicative of 4 stages of breast cancer and includes the following cells: MCF10A (non-malignant), MCF10AT.Cl2 (pre-malignant), MCF10Ca1h (well-differentiated, malignant), and MCF10Ca1a (poorly differentiated, malignant). While studies of circadian rhythms in cancer typically use low-resolution reverse transcription polymerase chain reaction assays, we also employed luciferase reporters BMAL1:Luc and PER2:Luc in real-time luminometry experiments. We found that across all 4 cancer stages, PER2 showed relatively stable oscillations compared with BMAL1. Period estimation using both wavelet-based and damped-sine-fitting methods showed that the periods are distributed over a wide circadian range and there is no clear progression in mean period as cancer severity progresses. Additionally, we used the K-nearest neighbors algorithm to classify the recordings according to cancer line, and found that cancer stages were largely differentiated from one another. Taken together, our data support that there are circadian discrepancies between normal and malignant cells, but it is difficult and insufficient to singularly use period evaluations to differentiate them. Future studies should employ other progressive disease models to determine whether these findings are representative across cancer types or are specific to this series.

Giant surfactant-stabilized N2-foam for enhanced oil recovery after water flooding.

A novel giant surfactant, APOSS-PS50, possessing good surface activity, and viscosifying and reinforcing ability as a foam stabilizer, was synthesized successfully to enhance the physical properties of foaming solutions and foam. APOSS-PS50 was widely distributed at the foam gas-liquid interface and adjacent liquid layers through diffusion and adsorption, obviously decreasing the surface tension and improving the foamability and stability of the foam. Furthermore, the aggregation of APOSS-PS50 in the foam films resulted in the formation of a self-assembled nano-sized network through supramolecular interactions (such as hydrogen bonding, π-π stacking, and van der Waals attraction), thus increasing the foam viscoelasticity, including its interfacial viscoelastic modulus and apparent viscosity. Meanwhile, from the sandpack flooding experiments, compared with HPAM/AOS (HPAM: partially hydrolyzed acrylamide and AOS: alpha olefin sulfonate), the differential pressure and final oil recovery after APOSS-PS50/AOS foam flooding increased by 23.5% and 23.2%, up to 2.68 MPa and 81.7%, respectively. In general, APOSS-PS50 significantly promoted the plugging, profile control and oil displacement performance of foam.

Inhibition of MALAT1 sensitizes liver cancer cells to 5-flurouracil by regulating apoptosis through IKKα/NF-κB pathway.

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in tumor cell growth process. However, its role and molecular mechanism in liver cancer is still not fully understood. In this study, we found that MALAT1 was significantly expressed in liver cancer cell lines. And knockdown of MALAT1 suppressed proliferation, migration and invasion of HepG2 cells, accompanied with decrease of Rho-associated coiled-coil-forming protein kinase 1 (ROCK1), α-smooth muscle actin (α-SMA), N-cadherin, Vimentin and TWIST. Significantly, MALAT1 deletion sensitized HepG2 cells to 5-FU-induced cell cycle arrest in G1 phase, as evidenced by the significant reduction in Cyclin D1 and CDK4 and increase in p53, p21 and p27 protein levels. In addition, MALAT1 knockdown triggered 5-FU induced apoptosis in HepG2 cells by inducing intrinsic apoptosis-related signals, including Cyto-c, Apaf-1, cleaved Caspase-9/-7/-3 and poly (ADP-ribose) polymerase (PARP). Furthermore, phosphorylated nuclear factor-κB (p-NF-κB) was also down-regulated by MALAT1 silence. Importantly, suppression of IKKα/NF-κB significantly elevated apoptosis and reduced liver cancer cell viability in MALAT1-knockdown cells with 5-FU incubation. The nude mice transplantation model also confirmed the promoted sensitivity of MALAT1-silenced HepG2 cells to 5-FU by blocking tumor cell proliferation and inducing apoptosis. Therefore, our data supplied a potential mechanism by which knockdown of MALAT1 might play an important role in augmenting sensitivity of HepG2 cells to 5-FU in therapeutic approaches, demonstrating suppressing of MALAT1 may serve as a combination with chemotherapeutic agents in liver cancer treatment.

Downregulation of castor zinc finger 1 predicts poor prognosis and facilitates hepatocellular carcinoma progression via MAPK/ERK signaling.

BACKGROUND: Castor zinc finger 1 (CASZ1) plays critical roles in various biological processes and pathologic conditions, including cancer. However, the prognostic importance and biologic functions of CASZ1 in hepatocellular carcinoma (HCC) are still unclear. METHODS: qRT-PCR, western blot and immunohistochemistry analyses were used to determine CASZ1 expression in HCC samples and cell lines. The clinical significance of CASZ1 was assessed in two independent study cohorts containing 232 patients with HCC. A series of in vitro and in vivo experiments were performed to explore the role and molecular mechanism of CASZ1 in HCC progression. RESULTS: Here we report that CASZ1 expression was downregulated in HCC tissues and cell lines. Low CASZ1 expression was closely correlated with aggressive clinicopathological features, poor clinical outcomes and early recurrence of HCC patients. Moreover, overexpression of CASZ1 in HCCLM3 cells significantly inhibited cell proliferation, migration, invasion in vitro and tumor growth and metastasis in vivo, whereas silencing CASZ1 significantly enhanced the above abilities of PLC/PRF/5 cells. Further mechanism study indicated that these phenotypic changes were mediated by MAPK/ERK signaling pathway and involved altered expression of MMP2, MMP9 and cyclinD1. Finally, we proved that CASZ1 exerted its tumor-suppressive effect by directly interacting with RAF1 and reducing the protein stability of RAF1. CONCLUSIONS: Our study for the first time demonstrated that CASZ1 is a tumor suppressor in HCC, which may serve as a novel prognostic predictor and therapeutic target for HCC patients.

MiR-874 inhibits metastasis and epithelial-mesenchymal transition in hepatocellular carcinoma by targeting SOX12.

MicroRNA-874 (miR-874), a novel cancer-associated microRNA (miRNA), has been reported to play a suppressive role in multiple malignancies. However, its function in cell migration and invasion in hepatocellular carcinoma (HCC) and the mechanisms responsible remain unclear. Here, we found miR-874 to be significantly downregulated in HCC tissues and cell lines. Moreover, this decreased expression strongly correlated with clinical stage and lymph node metastasis. Accordingly, ectopic expression of miR-874 in HCC cells markedly inhibited their migration, invasion, and epithelial-mesenchymal transition (EMT). Concerning the underlying mechanism, SRY (sex-determining region Y) -box 12 (SOX12) was identified as a direct target of miR-874, and its expression was found to be inversely correlated with that of this miRNA in HCC cells. Importantly, SOX12 knockdown had an inhibitory effect on HCC cells similar to that caused by miR-874 overexpression, whereas SOX12 overexpression in these cells negated the suppressive effects of miR-874 on migration, invasion, and EMT. Overall, these findings demonstrate that miR-874 inhibits metastasis and EMT in HCC by targeting SOX12, suggesting that this miRNA may constitute a promising therapeutic target for this disease.

miRNA-487a Promotes Proliferation and Metastasis in Hepatocellular Carcinoma.

Purpose: Hepatocellular carcinoma (HCC) harbors highly metastatic properties, accounting for postoperative recurrence and metastasis. However, the mechanisms for metastasis and recurrence remain incompletely clear. This study aimed to investigate the role of hsa-miR-487a (miR-487a) in promoting the proliferation and metastasis of HCC and to elucidate the underlying molecular mechanisms.Experimental Design: 198 HCC samples were analyzed for association between miR-487a expression and patient clinicopathological features and prognosis. The roles of miR-487a in proliferation and metastasis were validated both in vivo and in vitro The upstream regulator and downstream targets of miR-487a were determined using a dual luciferase reporter assay, chromatin immunoprecipitation and immunohistochemistry.Results: Our results demonstrate that upregulated miR-487a correlates with a poor prognosis for HCC patients. miR-487a enhances proliferation and metastasis of HCC cells by directly binding to sprouty-related EVH1 domain containing 2 (SPRED2) or phosphoinositide-3-Kinase regulatory subunit 1 (PIK3R1). Interestingly, miR-487a mainly promotes metastasis via SPRED2 induced mitogen activated protein kinase signaling and promotes proliferation via PIK3R1 mediated AKT signaling. Transcription of miR-487a was found to be activated by up-regulated heat shock factor 1, which we previously demonstrated to be an important metastasis-associated transcription factor in a previous study. Phosphorodiamidate morpholino oligomers effectively silenced miR-487a and inhibited HCC tumor progression in mouse models.Conclusions: Our findings show that miR-487a, mediated by heat shock factor 1, promotes proliferation and metastasis of HCC by PIK3R1 and SPRED2 binding, respectively. Our study provides a rationale for developing miR-487a as a potential prognostic marker or a potential therapeutic target against HCC. Clin Cancer Res; 23(10); 2593-604. ©2016 AACR.