Human Epithelial Cancer Cell Line Research Articles

LncRNA NEAT1 mediates intestinal inflammation by regulating TNFRSF1B.

BackgroundInflammatory bowel disease (IBD) is a chronic nonspecific intestinal disease. Our previous work showed that long non-coding RNA (LncRNA) nuclear enriched abundant transcript 1 (NEAT1) plays an important role in IBD. In the current study, we aimed to explore the underlying mechanism by which NEAT1 participates in the development of the disease.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of NEAT1 and tumor necrosis factor superfamily member 1B (TNFRSF1B) in clinical specimens and dextran sulfate sodium (DSS) colitis mice. Inflammatory cell models were established by stimulating human normal intestinal epithelial cell line NCM460 and human colon cancer cell line HT-29 with tumor necrosis factor alpha (TNF-α). Expressions of inflammatory cytokines such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) or RT-qPCR, TNFRSF1B, NF-κB p65 and p-NF-κB p65 followed by the knockdown or overexpression of NEAT1 and TNFRSF1B were analyzed by western blotting, and the regulatory effects of NEAT1 on TNFRSF1B were detected by RNA pull-down experiments and RNA-decay assay. The translocation of NF-κB p65 to the nucleus was detected by immunofluorescence.ResultsIn patients’ specimens and DSS colitis mouse models, NEAT1 and TNFRSF1B expression were up-regulated compared with the control group. TNF-α stimulation increased NEAT1 and TNFRSF1B expression and activated NF-κB signaling pathway by increasing the translocation of NF-κB p65 to the nucleus. In the presence of TNF-α stimulation, NEAT1 knockdown reduces the expression of TNFRSF1B and the translocation of NF-κB p65, thereby relieves cell inflammation. These effects can be reversed by the overexpression of TNFRSF1B.In addition, NEAT1 is involved in inflammatory response by up-regulating the mRNA levels of TNFRSF1B, and knocking down NEAT1 can alleviate inflammation by down-regulating TNFRSF1B. Moreover, NEAT1 co-precipitates TNFRSF1B mRNA in RNA-pulldown assay, and the presence of NEAT1 stabilizes the mRNA of TNFRSF1B.ConclusionsOur results showed that LncRNA NEAT1 promotes NF-κB p65 translocation and mediates intestinal inflammation by regulating TNFRSF1B.

Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells

Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.

Isoflavones from Placolobium vietnamense, an indigenous plant of Vietnam

AbstractPlacolobium vietnamense N.D.Khoi & Yakovlev (Fabaceae) is an indigenous plant of Vietnam. Up to now, this plant has not yet been chemically and biologically studied. In this paper, six isoflavones including afrormosin (1), cladrastin (2), 8‐O‐methylretusin (3), millesianin C (4), barbigerone (5), and durallone (6) were isolated from the ethyl acetate extract of the stem bark of Placolobium vietnamense. Their structures were established by means of physical data (ESI MS, 1D and 2D NMR) and evaluated for the cytotoxic activities against liver hepatocellular carcinoma (HepG2), human lung cancer (NCI‐H460), human epithelial carcinoma (HeLa) and human breast cancer (MCF‐7) cell lines. This is the first report on phytochemical studies of this plant.

Method development for the quantitative determination of captopril from Caco-2 cell monolayers by using LC-MS/MS

Aim. Caco-2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Although compounds were used in the original Caco-2 cells monolayer assays, compounds have been replaced in most laboratories by the use of liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS/MS). Mass spectrometry not only eliminates the need for compounds, but permits the simultaneous measurement of multiple compounds. The measurement of multiple compounds per assay reduces the number of incubations that need to be carried out, thereby increasing the throughput of the experiments. Furthermore, LC-MS and LC-MS-MS add another dimension to Caco-2 assays by facilitating the investigation of the metabolism of compounds by Caco-2 cells. A simple, rapid LC-MS/MS method has been developed for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Materials and methods. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl. Results. Under these conditions, captopril was eluted at 1.42 min. A linear response function was established at 2 – 200 ng/mL. The regression equation for the analysis was y =0.0187x+0.000248 with coefficient of correction (r2) = 0.9993. According to the Caco-2 test results, captopril showed low permeability. It should be noted that the recovery value is 103.20%. The within-run coefficients of variation ranged between 0.321% and 0.541%. The within-run percentages of nominal concentrations ranged between 99.13% and 101.12%. The between-run coefficients of variation ranged between 0.314% and 0.663%. The between-run percentages of nominal concentrations ranged between 99.17% and 101.03%.The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits. Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of captopril from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of captopril from Caco-2 cell monolayers.

Linc00887 suppresses tumorigenesis of cervical cancer through regulating the miR-454-3p/FRMD6-Hippo axis

BackgroundEmerging evidence suggested that long intergenic noncoding RNA (lincRNA) 00887 (NR_024480) reduced the invasion and metastasis of non-small cell lung cancer by sponging miRNAs degradation. However, the role and regulatory mechanism of linc00887 in the progression of cervical cancer remain largely unknown.MethodsIn vivo or vitro, RT-qPCR assay was used to detect the expression of linc00887 in human normal (N = 30), cervical cancer tissues (N = 30), human normal cervical epithelial cells (Ect1/E6E7) and cervical cancer cell lines (HeLa, C33A). Then, CCK-8 and Transwell assays were used to examine cell proliferation and invasion when linc00887 was overexpressed or knocked down. In addition, bioinformatics, luciferase reporter gene and pull-down assays were used to predict and validate the relationship between linc00887 and miR-454-3p. Moreover, we detected the expression of miR-454-3p in Ect1/E6E7, HeLa and C33A cells when linc00887 was overexpressed or knocked down. Cell proliferation and invasion were also measured when pcDNA-linc00887 and miR-454-3p were transfected alone or together. Next, miR-454-3p target gene was predicted and validated by bioinformatics and luciferase reporter gene assays. Gain- and loss-of-function experiments were performed in HeLa cells to evaluate the effect of miR-454-3p or linc00887 on the expression of FERM domain containing protein 6 (FRMD6) protein and several key proteins in the FRMD6-Hippo signaling pathway.ResultsLinc00887 was downregulated in cervical cancer tissues or human cervical cancer cell lines (Hela, C33A) compared with normal tissues or cell lines. Overexpression of linc00887 inhibited proliferation and invasion HeLa and C33A cells, while linc00887 knockdown had the opposite effect. Linc00887 bound with miR-454-3p, and overexpression of miR-454-3p rescued linc00887-induced inhibition proliferation and invasion of HeLa cells. MiR-454-3p targeted and suppressed the expression of FRMD6, and linc00887 suppressed tumorigenesis of cervical cancer through activating the FRMD6-Hippo signaling pathway.ConclusionsLinc00887, sponging miR-454-3p, inhibited the progression of cervical cancer by activating the FRMD6-Hippo signaling pathway.

Biological Activity of Some Pyrimidine Derivatives: Cytotoxicity and Oxidative Stress Potential in Human Lung Cancer Cell line (A549).

Compounds with pyrimidine ring in their structure have many biological activities including antimicrobial, antiviral and anticancer. Recently, studies related to their synthesis and so their applications have increased. In a previous study, a solid-phase microwave method was used to synthesized 25 new hydroxy- and methoxy-substituent 4,6-diarylpyrimidin-2 (1H) -ol and 4,6 diarylpyrimidin-2 (1H) –thiol compounds. In the present study, as a preliminary estimation of the anticancer activity, the cytotoxicity and oxidative stress induction potential of 6 derivatives that show highest antibacterial activities was evaluated in human lung epithelial cancer cell line (A549). Results of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity test indicate that pyrimidine derivatives caused concentration-dependent cell death that IC50 values were calculated between 16.7 and 41.5 µg/mL. additonally, pyrimidine derivatives induced significant changes in malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) activity; oxidative stress could be the mechanism of action of the tested pyrimidine derivatives in the cancerous cells. The results could be used to design the further in vivo and in vitro detailed studies to appreciate these pyrimidine derivatives anticancer activity, compare this activity with in-use known drugs, elucidate their mechanisms of action and estimate their safety.

The inhibitory effect of curcumin via fascin suppression through JAK/STAT3 pathway on metastasis and recurrence of ovary cancer cells

BackgroundFascin is an actin-binding protein and highly expressed in ovarian cancer cells. It is associated with metastasis of cancer and may be a useful prognostic factor. Anticancer activity of curcumin is related to its effect on several signaling mechanisms. Although there have been many reports regarding the anticancer properties of curcumin, its inhibitory effects on migration and invasion of ovarian cancer cells, particularly in the context of fascin expression, have not been reported. The purpose of this study was to investigate the effect of curcumin on fascin expression in ovarian cancer cells and to propose a possible mechanism for the anticancer activity of curcumin through reduced fascin expression.MethodsSKOV3, human epithelial ovary cancer cell line, was cultured with curcumin at various dose and duration. The fascin was quantified using cell viability test and Western blot. To determine the effect of curcumin on the upstream pathway of fascin expression, the signal transducer and activator of transcription 3 (STAT3) was analyzed by sandwich-ELISA. Attachment assay, migration assay and invasion assay were analyzed to approve the change of cellular invasiveness of ovary cancer after curcumin. To determine the morphological changes of ovarian cancer cells by curcumin, immunofluorescence was performed.ResultsMTS assays showed that cell viability was different at various concentration of curcumin, and as concentration increased, cell viability tended to decrease. Curcumin appears to suppress fascin expression, even with a minimal concentration and short exposure time. Also, curcumin may suppress fascin expression in ovarian cancer cells through STAT3 downregulation. The attachment assay, migration assay and invasion assay of the ovarian cancer cells exhibited a statistically significant decrease. Immunofluorescence revealed a change of cell shape from a typical form of uninfluenced cells to a more polygonal appearance, with a significant reduction in filopodia formation.ConclusionsCurcumin reduces fascin expression through JAK/STAT3 pathway inhibition, which interferes with the cellular interactions essential for the metastasis and recurrence of ovarian cancer cells. Higher curcumin concentrations and longer exposure times concomitantly decreased fascin expression.

Molecular Modeling, Synthesis and Biological Evaluation of N-Phenyl-4-Hydroxy-6-Methyl-2-Quinolone-3-CarboxAmides as Anticancer Agents.

The emergence of phosphatidylinositol 3-kinase (PI3Kα) in cancer development has accentuated its significance as a potential target for anticancer drug design. Twenty one derivatives of N-phenyl-4-hydroxy-6-methyl-2-quinolone-3-carboxamide were synthesized and characterized using NMR (1H and 13C) and HRMS. The derivatives displayed inhibitory activity against human epithelial colorectal adenocarcinoma (Caco-2) and human colon cancer (HCT-116) cell lines: compounds 8 (IC50 Caco-2 = 98 µM, IC50 HCT-116 = 337 µM) and 16 (IC50 Caco-2 = 13 µM, IC50 HCT-116 = 240.2 µM). Results showed that compound 16 significantly affected the gene encoding AKT, BAD, and PI3K. The induced-fit docking (IFD) studies against PI3Kα demonstrated that the scaffold accommodates the kinase domains and forms H-bonds with significant binding residues.

Synthesis, Characterization and Biological Studies of Organoselenium trans-Palladium(II) Complexes.

Over the years, transition metal complexes have exhibited significant antimicrobial and antitumor activity. It all started with cisplatin discovery, but due to the large number of side effects it shows, there is a growing need to find a new metal-based compound with higher selectivity and activity on more tumors. Two novel trans-palladium(II) complexes with organoselenium compounds as ligands, [Pd(L1)2Cl2] (L1 = 5-(phenylselanylmethyl)-dihydrofuran-2(3H)-one) and [Pd(L2)2Cl2] (L2 = 2- methyl-5-(phenylselanylmethyl)- tetrahydrofuran) were synthesized, in the text referred to as Pd-Se1 and Pd-Se2. Also, a structurally similar trans-palladium(II) complex, [Pd(L3)2Cl2] (L3= 2,2- dimethyl-3-(phenylselanylmethyl)-tetrahydro-2H-pyran) was synthesized according to an already published work and is referred to as Pd-Se3. The interaction of synthesized complexes with DNA and bovine serum albumin was observed. Also, antimicrobial activity and in vitro testing, cell viability, and cytotoxic effects of synthesized ligands and complexes on human epithelial colorectal cancer cell line HCT-116 were studied. Molecular docking simulations were performed to understand better the binding modes of the complexes reported in this paper with DNA and BSA, as well as to comprehend their antimicrobial activity. The interactions of the synthesized complexes with DNA and bovine serum albumin were done using UV-Vis and emission spectral studies as well as docking studies. Antimicrobial activity was tested by determining the minimum inhibitory concentrations (MIC) and minimum microbicidal concentration (MMC) using the resazurin microdilution plate method. Cytotoxic activity on cancer cells was studied by MTT test. The Pd(II) complexes showed a significant binding affinity for calf thymus DNA and bovine serum albumin by UV-Vis and emission spectral studies. The intensity of antimicrobial activity varied with the complexes Pd-Se1 and Pd-Se3, showing significantly higher activity than the corresponding ligand. The most significant activity was shown on Pseudomonas aeruginosa. Under standardized laboratory conditions for in vitro testing, cell viability and cytotoxic effects of synthesized ligands and complexes were studied on human epithelial colorectal cancer cell line HCT-116, where Pd-Se2 showed some significant cytotoxic effects. The newly synthesized complexes have the potential to be further investigated as metallodrugs.

MiR-212 as potential biomarker suppresses the proliferation of gastric cancer via targeting SOX4.

BackgroundCirculating microRNAs that post‐transcriptionally regulate gene expressions have been reported as promising biomarkers in cancer monitoring. This study was to identify the potential role of circulating miR‐212 in gastric cancer and whether it could serve as a novel biomarker for gastric cancer.MethodsWe detected the serum levels of miR‐212 in 100 health people and 110 gastric cancer patients and analyzed the relationships of the serum level of miR‐212 with gastric cancer. We detected the expression of miR‐212 in human gastric mucosal epithelial cell line (GES‐1) and human gastric cancer cell lines (NCI‐N87 and SNU‐16) using qRT‐PCR. Then, we detected the role of 5‐aza‐deoxycytidine on the epigenetic regulation of miR‐212 in human gastric cancer cell lines. Furthermore, luciferase reporter assay was used to detect binding activity of miR‐212 on SOX4 mRNA, and their functions on the cell proliferation and apoptosis.ResultsThe expression of miR‐212 was higher in health people than that in gastric cancer patients, higher in gastric mucosal epithelial cell line than that in gastric cancer cells. miR‐212 can be a circulating biomarker and an independent prognostic factor of gastric cancer. Moreover, miR‐212 can directly regulate the 3′UTR of SOX4 mRNA to suppress p53 and Bax, resulting gastric cancer cells proliferation inhibition and apoptosis induction.ConclusionOur study demonstrated that miR‐212 was epigenetically downregulated in gastric cancer, and resulting low level of miR‐212 can be a potential circulating biomarker and poor prognosis predicator of gastric cancer.

Design and synthesis of some barbituric and 1,3-dimethylbarbituric acid derivatives: A non-classical scaffold for potential PARP1 inhibitors

Six series based on barbituric acid 5a-e, 10a-d; thiobarbituric acid 6a-e, 11a-d and 1,3-dimethylbarbituric acid 7a-e, 12a-d were prepared and screened for their in vitro PARP1 inhibition. They revealed promising inhibition at nanomolar level especially compounds 5c, 7b, 7d and 7e (IC50 = 30.51, 41.60, 41.53 and 36.33 nM) with higher potency than olaparib (IC50 = 43.59 nM). Moreover, compounds 5b, 5d, 7a, 12a and 12c exhibited good comparable activity (IC50 = 65.93, 58.90, 66.57, 45.40 and 50.62 nM, respectively). Furthermore, the most active compounds 5c, 7b, 7d, 7e, 12a and 12c against PARP1 in vitro were evaluated in the BRCA1 mutated triple negative breast cancer cell line MDA-MB-436 where 5c and 12c showed higher potency compared to olaparib and result in cell cycle arrest at G2/M phase. 5c and 12c showed apoptotic effects in MDA-MB-436 and potentiated the cytotoxicity of temozolomide in A549 human lung epithelial cancer cell line. Compounds 5c and 12c represent interesting starting points towards PARP1 inhibitors.

In Vitro Photodynamic Therapy of Mononuclear and Dinuclear Iridium(III) Bis(terpyridine) Complexes

Three mononuclear or dinuclear bis(terpyridine) (tpy) iridium(III) complexes bearing pyren-1-yl (pyr) group(s) were synthesized. Their photophysical properties in water and in vitro photodynamic therapy (PDT) effects toward the human lung epithelial cancer cell line A549 and the human epidermal skin cancer cell line A431 were investigated to evaluate the effects of dinuclear versus mononuclear complexes and the impact of the oligoether substituent at the ligand. All complexes possessed pyr-tpy ligand-associated charge transfer (1CT)/1π,π* absorption bands at 350-550 nm, with the dinuclear complex Ir3 showing the much enhanced absorptivity of this band. These complexes exhibited dual emission upon excitation at >430 nm in most cases, with the emitting states being ascribed to 1ILCT (intraligand charge transfer) and 3π,π*/3CT states, respectively. All complexes exhibited relatively weak to moderate cytotoxicity in the dark but high photocytotoxicity upon broadband visible light irradiation. Among them, the dinuclear complex Ir3 showed the highest intracellular reactive oxygen species (ROS) generation and PDT efficiency compared to its mononuclear counterpart Ir1. Introducing an oligoether substituent on one of the tpy ligands in Ir2 also improved its intracellular ROS generation and PDT efficacy compared to those induced by Ir1. Ir3 induced both mitochondrial dysfunction and lysosomal damage upon light activation toward both cell lines, whereas Ir1 and Ir2 caused both mitochondrial dysfunction and lysosomal damage in A431 cells but only lysosomal damage in A549 cells. The dominant cell death pathway induced by Ir1-Ir3 PDT is apoptosis.

Evaluation of intercellular communication between normal human prostate epithelial cells and prostate cancer cell lines in co-culture model

Evaluation of intercellular communication between normal human prostate epithelial cells and prostate cancer cell lines in co-culture model

The Gli1-Snail axis contributes to Salmonella Typhimurium-induced disruption of intercellular junctions of intestinal epithelial cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that damages gastrointestinal tissue and causes severe diarrhoea. The mechanisms by which Salmonella disrupts epithelial barrier and increases the paracellular permeability are incompletely understood. Our present study aims to determine the role of Gli1, a transcription factor activated in the sonic hedgehog (Shh) pathway, in decreasing the levels of apical junction proteins in a Salmonella-infected human colonic epithelial cancer cell line, Caco-2, and in the intestinal tissue of Salmonella-infected mice. Here, we report that S. Typhimurium increased the mRNA and protein levels of Gli1 and Snail, a downstream transcription factor that plays an important role in the epithelial-to-mesenchymal transition (EMT). S. Typhimurium also decreased the levels of E-cadherin and three tight junction proteins (ZO-1, claudin-1, and occludin). Gli1 siRNA and GANT61, a Gli1-specific inhibitor, blocked S. Typhimurium-induced Snail expression, restored the levels of E-cadherin and tight junction proteins, and prevented S. Typhimurium-increased paracellular permeability. Further study showed that Gli1 was cross-activated by the MAP and PI-3 kinase pathways. S. Typhimurium devoid of sopB, an effector of the Type 3 secretion system (T3SS) responsible for AKT activation, was unable to induce Snail expression and to decrease the expression of apical junction proteins. Our study uncovered a novel role of Gli1 in mediating the Salmonella-induced disruption of the intestinal epithelial barrier.

Suppressive activities of mangiferin on human epithelial ovarian cancer

BackgroundEpithelial carcinoma is a subtype of ovarian cancers, with the highest lethality among all ovarian cancer subtypes. Hitherto surgical excision combined with chemotherapy has been the most extensively employed method in clinical treatment. However, the disease relapses very frequently, calling for more effective therapies. Mangiferin, a natural xanthone glucoside, has displayed promising anti-cancer activities by in vitro studies, but its therapeutic value in epithelial ovarian cancer treatment, either by in vivo or in vitro studies, remained to be known. PurposeThis study aimed to determine the suppressive activities of mangiferin on human epithelial ovarian cancer and elucidate the underlying molecular mechanisms. Study design and methodsWe employed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the crystal violet assay to determine the half maximal inhibitory concentration (IC50) values of mangiferin with paclitaxel as a positive control and the inhibitory effects of mangiferin on the proliferation of two human epithelial ovarian cancer cell lines. Wound healing and Transwell assays were used to determine anti-metastastic activities of mangiferin. ES-2 xenograft nude mouse model was used for the in vivo experiments. Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) assays were carried out for evaluating the expression level of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). ResultsIn the present study, we demonstrated by both in vitro and in vivo assays that mangiferin suppressed the progress of epithelial ovarian cancer in a dose-dependent manner. In the animals treated with mangiferin, the tumor volume and weight were reduced significantly. Analyses of involved molecular events demonstrated that mangiferin down-regulated the expression of metastasis-associated proteins MMP2 and MMP9. ConclusionMangiferin strongly inhibited the progression of human epithelial ovarian cancer by down-regulating MMP2 and MMP9.

MCF7 Human Breast Cancer Cells Engineered to Stably Overexpress DEPTOR via CRISPR/Cas9 to Reduce Anabolic Capacity

As a potent cellular inhibitor of both mTOR complexes, DEPTOR protein has the potential to dramatically alter the anabolic potential of many different cell types and conditions. Manipulation of DEPTOR protein content means that cellular anabolism can be more aggressively regulated at a single bottleneck of key cellular signaling pathways. This approach could be particularly advantageous in a number of disease states where anabolically aggressive behavior gives rise to specific pathology. To evaluate this concept, we developed a novel subset of the immortalized MCF7 human epithelial breast cancer cell line. By utilizing CRISPR/Cas9 to facilitate a targeted insertion of a constitutively active DEPTOR gene into the human AAVS1 genomic safe harbor site, we examined direct and indirect measures of cellular anabolism to determine the efficacy of abating the anabolic potential of this type of human cancer.MCF7 cells overexpressing DEPTOR (DEP) were generated by genome integration of a constitutively active DEPTOR gene via a homologous directed repair (HDR) based template following a double stranded break (DSB) at the human AAVS1 safe harbor site by Cas9 nuclease. A second group of cells were used as a control by treating them with a vector containing a red fluorescent protein (RFP) gene in lieu of the DEPTOR gene. Both vectors contain a separate promoter for green fluorescence protein (GFP) gene, and N‐acetyltransferase. Both groups of cells were subjected to 14 days of puromycin selection, and appropriate fluorophore presence was confirmed. Rates of protein synthesis were measured using deuterium heavy water stable isotope methods, and cellular anabolic signaling was assessed via immunoblotting.Twenty‐four hour rates of protein synthesis were significantly reduced in DEP compared to RFP (23.390 ± 2.26 vs. 37.314 ± 0.21 %/day, respectively; p=0.029), representing a 37.6% reduction. DEPTOR protein content was found to be significantly higher in the DEP group compared to RFP controls (9.593 ± 3.209 vs. 1 ± 0.552, respectively; p<0.001). Total (0.978 ± 0.202 vs 1 ± 0.162; p=0.841) and phosphorylated (0.65 ± 0.445 vs 1 ± 0.406; p=0.186) p70 S6 KinaseThr389 were not found to be different between DEP and RFP. The phosphorylated to total ratio of p70 S6 Kinase was lower in DEP vs RFP (0.619 ± 0.315 vs 0.978 ± 0.255, respectively; p=0.05). Interestingly, both total 4E‐BP1 (1.340 ± 0.107 vs 1 ± 0.102, p<0.001) and Thr 37/46 phosphorylation (2.106 ± 0.742 vs 1 ± 0.559, p=0.015) were significantly higher in the DEP vs RFP groups, respectively, but the phosphorylated to total ratios were not different between groups.These results indicate that DEPTOR overexpression dramatically impacts the anabolic activity of MCF7 human cancer cells, as shown by significant reductions in protein synthesis rates, as well as phosphorylation of the downstream mTORC1 target p70 S6 Kinase. Increases in phosphorylated and total 4E‐BP1 could be the result of long‐term inhibition of mTORC1 by DEPTOR protein, possibly indicating the need for evaluation of 4E‐BP1’s unique role in regulating transitions between Cap‐Dependent and Cap‐Independent mRNA translation.Support or Funding InformationFunding provided by the Sydney & JL Huffine’s Institute and the College of Education and Human Development.

Circular RNA La-related RNA-binding protein 4 correlates with reduced tumor stage, as well as better prognosis, and promotes chemosensitivity to doxorubicin in breast cancer.

ObjectiveWe aimed to evaluate the correlation of circular RNA La‐related RNA‐binding protein 4 (circ‐LARP4) with tumor characteristics and prognosis, and its effect on chemosensitivity in breast cancer.MethodsCirc‐LARP4 from tumor and adjacent tissues of 283 female breast cancer patients underwent resection was detected by reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). Tumor features, disease‐free survival (DFS), and overall survival (OS) were recorded. In vitro, circ‐LARP4 in human normal mammary epithelial cells (HMEC) and breast cancer cell lines was detected by RT‐qPCR. MCF‐7 and MDA‐MB‐231 cells were transfected with circ‐LARP4 overexpression plasmid (as OE‐Circ group) and control overexpression plasmid (as OE‐Control group). Relative cell viability under different concentrations of doxorubicin was measured.ResultsCirc‐LARP4 was decreased in tumor tissues than adjacent tissues (P < .001). Tumor circ‐LARP4 negatively correlated with tumor size (P = .001), T stage (P = .009), N stage (P = .006), and TNM stage (P < .001), whereas positively correlated with DFS (P = .004) and OS (P < .001). In vitro, circ‐LARP4 was decreased MCF‐7, BT474, MDA‐MB‐231, and MDA‐MB‐468 cell lines than HMEC (all P < .001). Relatively cell viability of MCF‐7 cells (at 20 nmol/L [P < .05], 40 nmol/L [P < .01], 80 nmol/L [P < .05] of doxorubicin) and MDA‐MB‐231 cells (at 120 nmol/L [P < .05], 240 nmol/L [P < .05] of doxorubicin) was decreased in OE‐Circ group than OE‐Control group. IC50 value of doxorubicin was decreased in OE‐Circ group than OE‐Control group in MCF‐7 and MDA‐MB‐231 cell lines (both P < .01).ConclusionCirc‐LARP4 was a potential prognostic biomarker, which might improve the management of breast cancer.

LncRNA MIAT Regulates Cell Growth, Migration, and Invasion Through Sponging miR-150-5p in Ovarian Cancer.

Background: MIAT (myocardial infarction-associated transcript) regulates cell proliferation, apoptosis, and metastasis in several cancers. In this study, the authors aimed to explore the role of MIAT in ovarian cancer. Materials and Methods: The expression of MIAT in ovarian cancer subtypes, normal human ovarian surface epithelial and ovarian cancer cell lines was measured by qualitative real-time polymerase chain reaction (qRT-PCR). OVCAR3 and SKOV3 cells were transfected with MIAT overexpression plasmid or siMIAT. The cell growth ability was then evaluated by CCK-8 and colony formation assays. The cell migration and invasion rate were separately measured by wound-healing and transwell assays. The levels of epithelial-mesenchymal transition (EMT)-associated markers were evaluated by Western blotting. MIAT sponging miR-150-5p was predicted by starBase and confirmed by dual-luciferase reporter assays. The expression of miR-150-5p in OVCAR3 and SKOV3 cells with MIAT overexpression or knockdown, and in ovarian cancer subtypes was also measured by qRT-PCR. Further analyses confirmed the role of MIAT sponging miR-150-5p in ovarian cancer cells. Results: MIAT was highly expressed in mesenchymal subtype ovarian cancer tissues and ovarian cancer cells. In OVCAR3 and SKOV3 cells, overexpression of MIAT promoted, and knockdown of MIAT suppressed the cell growth, migration, invasion, and EMT. miR-150-5p was sponged and regulated by MIAT. miR-150-5p was downregulated in mesenchymal subtype ovarian cancer. Suppression of cell migration, invasion, and EMT caused by miR-150-5p overexpression was rescued by MIAT overexpression. Conclusions: MIAT acts as an oncogene in ovarian cancer cells through sponging miR-150-5p. MIAT or miR-150-5p expression might be a potential prognostic biomarker for ovarian cancer patients. MIAT and miR-150-5p are potential therapeutic targets in treatment of ovarian cancer.

MiR-485-5p inhibits the progression of breast cancer cells by negatively regulating MUC1.

To investigate the mechanism of miR-485-5p inhibiting breast cancer cells by targeting MUC1. Differentially expressed genes (DEGs) in breast cancer tissues were analyzed using breast cancer tissue microarrays (TMA) in the GEO database. Differential expression of MUC1 in breast cancer tissue samples was detected by TCGA database. qRT-PCR was used to detect the expression of MUC1 and miR-485-5p in human normal breast epithelial cell lines and human breast cancer cell lines. Bioinformatics was applied to analyze targeted binding site of miR-485-5p and MUC1 and their targeted relationship was identified by dual luciferase assay. The proliferation ability of breast cancer cells was detected by CCK-8 assay. Cell apoptosis was detected by flow cytometry. The ability of cell migration was measured by scratch healing test. Transwell assay was used to detect the invasion ability of cells. The protein expression levels of MUC1 and EMT-related molecules (E-cadherin, N-cadherin and Vimentin) were detected by Western blot. MUC1 was highly expressed in breast cancer tissue samples and breast cancer cell lines, while miR-485-5p was lowly expressed. Overexpression of miR-485-5p inhibits cell viability and invasion and migration of breast cancer cell line MCF-7 and promotes apoptosis. The same results were obtained by silencing the expression of MUC1. MiR-485-5p targets to bind to the 3'-UTR region of MUC1 and negatively regulates the expression of MUC1. Overexpressing MUC1 while overexpressing miR-485-5p reversed the inhibitory effect of miR-485-5p on breast cancer and inhibited EMT. MiR-485-5p can down-regulate the expression of MUC1, thus inhibit the proliferation, invasion and migration of breast cancer cells and promote cell apoptosis.

MiR-92b inhibits proliferation and invasion of lung cancer by targeting EZH2.

To verify that miR-92b inhibits proliferation and invasion of lung cancer by targeting EZH2. The expression levels of miR-92b and EZH2 in human bronchial epithelial cell line BEAS-2B and human lung cancer cell line (A549, NCI-H23, NCI-H358, NCI-H1975, PC-9) were detected, and miR-92b mimic, sh-EZH2 expression vector, and plasmid blank vector (blank group) were constructed. Blank group, miR-92b mimic, miR-92b mimic+sh-EZH2 group (combined group) were set up, MTT and transwell were used to detect the proliferation and invasion ability of A549 and NCI-H23 cells, and fluorescein report verified the regulatory relationship of miR-92b to EZH2. The expression level of miR-92b in A549, NCI-H23, NCI-H358, NCI-H1975, and PC-9 cells was lower than that in BEAS-2B cells (p<0.05). The expression level of EZH2 was higher than that of BEAS-2B cells (p<0.05). A549 and NCI-H23 cells were selected for transfection. After that, the expression level of miR-92 in miR-92b mimic, combined group A549 and NCI-H23 cells was higher than that in blank group (p<0.05), and miR-92b mimic had no difference with joint group (p>0.05). The expression level of EZH2 in cells of miR-92b mimic, blank group A549, and NCI-H23 was lower than that of combined group (p<0.05), and miR-92b mimic was lower than that of blank group (p<0.05). After the overexpression of miR-92b, pmirGLO-EZH2-3'UT Wt luciferase activity decreased significantly (p<0.05) but had no effect on pmirGLO-EZH2-3'UTR Mut Luciferase activity (p>0.05). Cell proliferation ability and invasion ability of A549 cells and NCI-H23 cells in miR-92b mimic group were lower than those in blank group (p<0.05), while those in combined group were higher than those in miR-92b mimic group (p<0.05). MiR-92b inhibits proliferation and invasion of lung cancer cells through targeted inhibition of EZH2, which is a potential target for future treatment of lung cancer.