Hsa_circ_0060927 Is a Novel Tumor Biomarker by Sponging miR-195-5p in the Malignant Transformation of OLK to OSCC

ObjectiveTo investigate the clinical significance of differentially expressed circRNAs and candidate circRNAs in the transformation of oral leukoplakia (OLK) to oral squamous cell carcinoma (OSCC).MethodsWe performed high-throughput circRNA sequencing in six cases of normal oral mucosal (NOM) tissues, six cases of OLK tissues, and six cases of OSCC tissues. Ten circRNAs with significant differential expression were verified by qRT-PCR. Enzyme tolerance assay and Sanger sequencing were performed on the screened target circRNA hsa_circ_0060927, and a qRT-PCR assay of hsa_circ_0060927 was performed in three tissues (24 cases in each group); this was followed by an ROC analysis. The ceRNA network was predicted using TargetScan and miRanda. MiR-195-5p and TRIM14 were selected as the downstream research objects of hsa_circ_0060927. The sponge mechanism of hsa_circ_0060927 was detected by AGO2 RIP. The interaction between hsa_circ_0060927 and miR-195-5p was verified by RNA pull-down assay and dual luciferase reporter gene assay. The expressions of hsa_circ_0060927, miR-195-5p, and TRIM14 were verified by normal oral epithelial primary cells and cell lines of LEUK1, SCC9, and SCC25. The hsa_circ_0060927 overexpressed plasmid and miR-195-5p mimics were constructed to transfection LEUK1 to detect the changes in cell proliferation, apoptosis, and migration.ResultsThe results of qRT-PCR validation were consistent with the sequencing results. Hsa_circ_0060927 is a true circRNA with trans-splicing sites. The expression of hsa_circ_0060927 increased in NOM, OLK, and OSCC. Overexpression of hsa_circ_0060927 enhanced the ability of cell proliferation and migration, and decreased cell apoptosis capacity. The prediction of ceRNA network suggested that hsa_circ_0060927 could regulate the target gene TRIM14 through sponging miR-195-5p. AGO2 RIP indicated that hsa_circ_0060927 had a sponge mechanism. RNA pull-down and dual luciferase reporter gene assay suggested that hsa_circ_0060927 interacted with miR-195-5p. Hsa_circ_0060927 was positively correlated with the expression of TRIM14, and could relieve the inhibition of miR-195-5p on TRIM14 to regulate cell proliferation, apoptosis, and migration of LEUK1 cells.ConclusionHsa_circ_0060927 acted as a potential key ceRNA to sponge downstream miR-195-5p and promote OLK carcinogenesis by upregulating TRIM14. Hsa_circ_0060927 was expected to be a molecular marker for the prevention and treatment of OLK carcinogenesis through the hsa_circ_0060927/miR-195-5p/TRIM14 axis.

Use of PiggyBac Transposon System Constructed Murine Breast Cancer Model For Reporter Gene Imaging And Characterization of Metastatic Tumor Cells

The piggyBac transposon system is known to non-viral integrate exogenous genes to chromosomes of mammalian cells. For reporter gene imaging, this transposon system is believed to efficiently establish xenograft tumor model with low immunogenicity. Because tumor cells usually exhibit genomic instability, it is important to investigate if piggyBac mediated transduction of reporter genes would change tumor characteristics. In this study, reporter gene imaging mediated by the piggyBac transposon system was exploited to track the growth and dissemination of 4T1 triple-negative murine breast cancer cells in vivo, followed by ex vivo analysis of the metastatic cells expressing reporter genes. We demonstrated that several cell properties, including proliferation rate, invasion and migration rate, and mammosphere formation ability of 4T1 cells were not influenced by piggyBac transposon system. Further, we isolated the liver metastatic cells, named 4T1-3R_L cells for further analysis. Compared to parental 4T1 cells, 4T1-3R_L cells exhibited several cancer stem cells (CSC) related characteristics, including significant mammosphere formation ability, resistance to doxorubicin, high tumorigenicity potential in Balb/C mice and expression of CD44 CSC marker. We also found that 4T1-3R_L cells exhibited stronger migrated and invasive abilities, by wound healing assay and in vitro invasion assay, respectively. The cell adhesive ability of 4T1-3R_L cells was also lower than that of 4T1 cells. The microarray assay showed that several epithelial-mesenchymal transition (EMT) promoting markers, including vimentin, N-cadherin, Twist1, and Snail were up-regulated, and anti-EMT marker E-cadherin was down-regulated in 4T1-3R_L cells. Current data suggest that the piggyBac transposon system is a reliable and biocompatible tool to engineer cancer cells for tacking and characterizing tumor development in vivo and in vitro.

Regulation of NSCLC Cell Proliferation By MARCH7 Via the NF-κB and Wnt/β-Catenin Signaling Pathways

The aim of the study was to explore the role of the E3 ubiquitin ligase MARCH7 in the development of non-small-cell lung cancer (NSCLC) and to explore the underlying molecular mechanism.Western blot and immunohistochemistry results showed that the expression of MARCH7 in NSCLC cancer tissues was higher than that in paracancerous tissues. Tissue microarray staining results and clinicopathological parameters of NSCLC patients revealed that MARCH7 expression was closely related to TNM stage, degree of tumor differentiation and lymph node metastasis of NSCLC patients. Furthermore, univariate and multivariate analyses and survival curve analysis showed that high expression of MARCH7 was associated with poor prognosis.In vitro, siRNA was constructed and transfected into A549 cells to inhibit the expression of MARCH7. The CCK-8 assay indicated that the growth rate of tumor cells in the interference group was reduced. The number of colonies and cells in the interference group decreased in the plate clone formation experiment. Flow cytometry showed that G0/G1 phase cells were predominantly increased after blocking endogenous MARCH7 expression, and G0/G1 phase arrest occurred in A549 cells. The reporter gene activity of the NF-κB signaling pathway and Wnt/β-catenin signaling pathway was reduced, as validated by a double luciferase reporter gene assay. Western blot analysis showed that the expression of NF-κB P50, NF-κB P65 and β-catenin was decreased, while the expression of E-cadherin was elevated.In vivo, MARCH7-overexpressing virus was constructed and transfected into A549 cells and then subcutaneously injected into nude mice. It was demonstrated that the tumor volume was significantly larger in the MARCH7 overexpression group than in the control nude mice during the same period. Elevated expression of PCNA and Ki-67 was observed in the tumor mass of the MARCH7 overexpression group, as measured by immunohistochemical analysis, accompanied by enhanced levels of NF-κB P50, NF-κB P65 and β-catenin, as detected by Western blot. These results provide a new idea for the experimental basis for the treatment of NSCLC in the future.

Circular RNA CCDC66 Regulates Osteoarthritis Progression by Targeting miR-3622b-5p

<b><i>Objective:</i></b> CircCCDC66 is involved in cancer progression, but its role in osteoarthritis (OA) remains unknown. This study was carried out to explore the biological role of circCCDC66 in OA and its underlying mechanism. <b><i>Methods:</i></b> The expression levels of miR-3622b-5p and circCCDC66 in OA cartilage tissues were detected by qRT-PCR. Cell Counting Kit-8 (CCK8) and flow cytometry were used to detect the chondrocyte viability and apoptosis. The expression of chondrocyte inflammatory factors (IL-6 and TNF-α) was measured by ELISA. The target genes of circCCDC66 and miR-3622b-5p were analyzed by bioinformatics analysis and luciferase reporter gene assay. The relationship between circCCDC66 and miR-3622b-5p was analyzed by bioinformatics analysis and luciferase reporter gene assay. <b><i>Results:</i></b> It was found that circCCDC66 expression in OA cartilage tissues was upregulated. CircCCDC66 overexpression inhibited proliferation and promoted apoptosis of chondrocytes and increased IL-6 and TNF-α levels in chondrocytes. miR-3622b-5p was predicted to be a downstream target gene of circCCDC66, and circCCDC66 overexpression inhibited miR-3622b-5p expression in chondrocytes. Moreover, miR-3622b-5p expression was downregulated in OA cartilage tissues. miR-3622b-5p overexpression increased chondrocyte proliferation, inhibited chondrocyte apoptosis, and enhanced the expression of IL-6 and TNF-α in chondrocytes. In addition, circCCDC66 overexpression enhanced SIRT3 expression in chondrocytes, while miR-3622b-5p overexpression inhibited SIRT3 expression in chondrocytes. <b><i>Conclusion:</i></b> CircCCDC66 promoted OA chondrocyte apoptosis by regulating the miR-3622b-5p/SIRT3 axis. CircCCDC66 may be a new therapeutic target of OA.

Xenobiotic metabolism and its physiological consequences in high-Antarctic Notothenioid fishes

The Antarctic ecosystem is progressively exposed to anthropogenic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). So far, it is largely unknown if PAHs leave a mark in the physiology of high-Antarctic fish. We approached this issue via two avenues: first, we examined the functional response of the aryl hydrocarbon receptor (Ahr), which is a molecular initiating event of many toxic effects of PAHs in biota. Chionodraco hamatus and Trematomus loennbergii served as representatives for high-Antarctic Notothenioids, and Atlantic cod, Gadus morhua as non-polar reference species. We sequenced and cloned the Ahr ligand binding domain (LBD) of the Notothenioids and deployed a GAL4-based luciferase reporter gene assay expressing the Ahr LBD. Benzo[a]pyrene (BaP), beta-naphthoflavone and chrysene were used as ligands for the reporter gene assay. Second, we investigated the energetic costs of Ahr activation in isolated liver cells of the Notothenioids during acute, non-cytotoxic BaP exposure. In the reporter assay, the Ahr LBD of Atlantic cod and the Antarctic Notothenioids were activated by the ligands tested herein. In the in vitro assays with isolated liver cells of high-Antarctic Notothenioids, BaP exposure had no effect on overall respiration, but caused shifts in the respiration dedicated to protein synthesis. Thus, our study demonstrated that high-Antarctic fish possess a functional Ahr that can be ligand-activated in a concentration-dependent manner by environmental contaminants. This is associated with altered cost for cellular protein synthesis. Future studies have to show if the toxicant-induced activation of the Ahr pathway may lead to altered organism performance of Antarctic fish.

Oxymatrine inhibits proliferation and migration of breast cancer cells by inhibiting miRNA-188 and upregulating its target gene, PTEN

Purpose: To explore the potential biological functions of oxymatrine on breast cancer (BCa) cells and the underlying molecular mechanism.Methods: Relative levels of microRNA-188 (miRNA-188) and PTEN (gene of phosphate and tension homology deleted on chromosome ten) in BCa cells, MDA-MB-231 and TB549, were determined. The influence of oxymatrine treatment, miRNA-188 and PTEN on proliferative and migratory abilities in BCa cells were assessed by 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell counting kit-8 (CCK-8) and Transwell assay, respectively. The binding relationship between miRNA-188 and PTEN was evaluated by dual-luciferase reporter gene assay.Results: Oxymatrine downregulated miRNA-188 and upregulated PTEN in BCa cells. Proliferative and migratory activities in BCa were inhibited by treatment of oxymatrine (p < 0.05). Dual-luciferase reporter gene assay results indicated that PTEN was the target gene of miRNA-188. Furthermore, rescue experiments demonstrated that the regulatory loop, oxymatrine/miRNA-188/PTEN, was involved in the regulation of the migration and proliferation of BCa.Conclusion: Oxymatrine treatment inhibits BCa progression by downregulating miRNA-188, leading to the upregulation of PTEN. The results of the current study may provide new insight into the diagnosis and treatment of BCa.

306 Involvement of toll-like receptor 4 in PCSK9-mediated biological effects in cell culture models

Aims The link between pro-protein convertase subtilisin/kexin 9 (PCSK9) and inflammatory-related disease was supported in several experimental and human studies, which show how the protein could acts independent its canonical mechanism, by activation of inflammatory, apoptotic and immune pathways. Among these, TLR4/NFKB signalling pathway was found to be one of the main pathways mediating the PCSK9-induced increase of pro-inflammatory and pro-thrombotic molecules, such as TNF-alpha, IL-6, IL-1, MCP-1, and TF. To investigate the direct involvement of hTLR4 in recognition of exogenous human recombinant (hr) PCSK9. Methods Experiments were carried out in three cell line: (i) HEK293-hTLR4-GFP cell lines obtained by stable co-transfection of HEK293 cells with the pUNO1-hTLR4-GFP, a plasmid expressing the hTLR4 gene fused to a GFP gene; (ii) HEK293-hTLR4+ cells obtained by co-transfection of hTLR4 receptor and its accessory proteins MD-2 and CD14, and an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene; (iii) HEK293-Null2 cells, a cell line lacking TLR4 expression, used as negative control. NFkB activity were measured by the SEAP reporter gene assay using a fluorescence detection method, while localization of hTLR4 and exogenous hrPCSK9 by confocal microscopy. Results hrPCSK9 (1 µg/mL) activates NFkB in HEK293-hTLR4+ cells [SEAP activity-OD 620 nm: from a baseline of 0.18 ± 0.06 to 0.68 ± 0.05 (N = 8), P &lt; 0.001] to an extent comparable to Lipopolysaccharide (LPS), the specific agonist of TLR4. Colocalization of hrPCSK9 and TLR4s was clearly documented by quantitative confocal microscopy in HEK293-hTLR4-GFP cell line, that comprises the analysis of more than 80 fields in ∼2.105 cells/well and showing a percentage of co-localization of ∼4% in membrane spots (P &lt; 0.01). Conclusions Our data support the results reported in previous studies that attribute to PCSK9 a pathophysiological role in development of chronic inflammation and related-diseases such as atherothrombosis. A behaviour that extends far beyond LDLR degradation, via a mechanism that might be mediated at least in part by recognition of PCSK9 by the TLRs and later activation of NFKB intracellular pathway. Future studies will be important to better investigate the specific binding site engaged in the PCSK9-TLR interaction and to discriminate the intracellular transduction pathways involved in this response, in order to provide a theoretical basis for the development of innovative therapeutic strategies.