DNA Synthesis In Cells Research Articles

Palmitic acid-induced defects in cell cycle progression and cytokinesis in Neuro-2a cells

ABSTRACT Obesity is associated with elevated levels of free fatty acids (FFAs). Excessive saturated fatty acids (SFAs) exhibit significant deleterious cytotoxic effects in many types of cells. However, the effects of palmitic acid (PA), the most common circulating SFA, on cell cycle progression in neuronal cells have not been well-examined. The aim of this study was to examine whether PA affects the proliferation and cell cycle progression in mouse neuroblastoma Neuro-2a (N2a) cells. Our studies found that 200 µM PA significantly decreased DNA synthesis and mitotic index in N2a cells as early as 4 h following treatment. 24 h treatment with 200 µM PA significantly decreased the percentage of diploid (2 N) cells while dramatically increasing the percentage of tetraploid (4 N) cells as compared to the BSA control. Moreover, our studies found that 24 h treatment with 200 µM PA increased the percentage of binucleate cells as compared to the BSA control. Our studies also found that unsaturated fatty acids (UFAs), including linoleic acid, oleic acid, α-linolenic acid, and docosahexaenoic acid, were able to abolish PA-induced decrease of 2 N cells, increase of 4 N cells, and accumulation of binucleate cells. Taken together, these results suggest that PA may affect multiple aspects of the cell cycle progression in N2a cells, including decreased DNA synthesis, G2/M arrest, and cytokinetic failure, which could be abolished by UFAs. Abbreviations: 4-PBA, 4-Phenylbutyric Acid; ALA, α-linolenic acid; BrdU, 5-bromo-2’-deoxyuridine; DAPI, 4′,6-diamidino-2-phenylindole; ER, endoplasmic reticulum; FFA, free fatty acids; FITC, fluorescein isothiocyanate; LA, linoleic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; N2a, Neuro-2a; NAC, N-acetyl cysteine; OA, oleic acid; PA, palmitic acid; pHH3, Phosphorylation of histone H3; PI, propidium iodide; SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; UFA, unsaturated fatty acids

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

LncRNA TP73\u2010AS1 promotes nasopharyngeal carcinoma progression through targeting miR-342-3p and M2 polarization via exosomes

BackgroundNasopharyngeal carcinoma (NPC) is a deadly cancer, mainly presenting in southeast and east Asia. Long noncoding RNAs (lncRNAs) play essential roles in cancer progression. Exosomes are critical for intercellular communication. Thus, the aim of this study was to identify the functional lncRNAs in NPC and its relevant mechanisms.MethodsData from public databases were utilized to screen for functional lncRNAs in NPC. Functional and mechanical experiments were performed to determine the role of lncRNAs in NPC and its relative molecular mechanisms. Exosomes derived from NPC cells were isolated to determine their function in tumor-associated macrophages.ResultsLncRNA TP73-AS1 was increased in NPC cells and tissues and was associated with a poor prognosis. TP73-AS1 overexpression promoted proliferation, colony formation, and DNA synthesis of NPC cells while TP73-AS1 knockdown showed opposite roles. TP73-AS1 could directly bind with miR-342-3p. MiR-342-3p overexpression attenuated the effect of TP73-AS1 in NPC cells. Furthermore, TP73-AS1 was transferred by exosomes to promote M2 polarization of macrophages. Lastly, exosomal TP73-AS1 enhanced the motility and tube formation of macrophages.ConclusionsTogether, this study suggests that TP73-AS1 promotes NPC progression through targeting miR-342-3p and exosome-based communication with macrophages and that TP73-AS1 might be an emerging biomarker for NPC.

Insulin-like growth factor-1: A potential target for bronchopulmonary dysplasia treatment (Review).

Bronchopulmonary dysplasia (BPD) is a common respiratory disorder among preterm infants, particularly low-birth-weight infants (LBWIs) and very-low-birth-weight infants (VLBWIs). Although BPD was first reported 50 years ago, no specific drugs or efficient measures are yet available for prevention or treatment. Insulin-like growth factor-1 (IGF-1) belongs to the insulin family. It promotes mitosis and stimulates cell proliferation and DNA synthesis, the primary factors involved in pulmonary development during the fetal and postnatal periods. Several studies have reported that IGF-1 exerts certain effects on BPD genesis and progression by regulating BPD-related biological processes. In addition, exogenous addition of IGF-1 can alleviate lung inflammation, cell apoptosis and eliminate alveolar development disorders in children with BPD. These findings suggest that IGF-1 could be a new target for treating BPD. Here, we summarize and analyze the definition, pathogenesis, and research status of BPD, as well as the pathogenesis of IGF-1 in BPD and the latest findings in related biological processes.

Forkhead box protein O1 (FoxO1) regulates lipids metabolism and cell proliferation mediated by insulin and PI3K-Akt-mTOR pathway in goose primary hepatocytes

In order to explore the role of forkhead box protein O1 (FoxO1) in the lipid metabolism and cell proliferation, goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVP-BEZ235, and then transfected with FoxO1 interference plasmid. The related parameters of lipid metabolism and cell proliferation were measured. The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration (P < 0.05); and increased the proliferative index (PI), cell DNA synthesis, protein expression of Cyclin D1 in goose primary hepatocytes (P < 0.05). Secondly, the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1 (P < 0.05); however, there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI (P > 0.05). Lastly, the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly (P < 0.05). In summary, FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes. Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.

Transient Receptor Potential Melastatin 8, a sensor of cold temperatures mediates expression of cyclin-dependent kinase inhibitor, p21/Cip1, a regulator of epidermal cell proliferation.

Transient Receptor Potential Melastatin 8 (TRPM8) is a calcium-permeable, non-selective cation channel of the transient receptor potential superfamily, required for the transduction of moderate cold temperatures. TRPM8 is also known to regulate proliferation of prostate, pancreatic, breast, and melanoma carcinoma cells. Here, we examined a key factor in the regulation of TRPM8-mediated proliferation of epidermal cells, which are directly affected by cold temperatures. Experiments involving knockdown and ectopic expression of TRPM8 in normal keratinocyte HaCaT and squamous carcinoma SAS cells suggest that TRPM8 inhibits cell proliferation by upregulating the expression of cyclin-dependent inhibitor p21/Cip1. Whereas these findings were observed in the absence of an endogenous agonists, additions of the synthetic TRPM8 agonist icilin reduced DNA synthesis in HaCaT cells but stimulated that in SAS cells by altering p21/Cip1 levels in a TRPM8-independent manner, indicating that icilin poses a risk of stimulating carcinoma cell proliferation. Unexpectedly, the TRPM8 blocker, used for the treatment of overactive bladder and bladder pain, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl] oxy}-N-(2-thienylmethyl) benzamide hydrochloride salt (AMTB) reduced DNA synthesis by upregulating p21/Cip1 expression. However, another TRPM8 blocker, N-(4-Tertiarybutylphenyl)-4-(3-chloropyridin- 2-yl) tetrahydropyrazine-1 (2H)-carbox-amide (BCTC), stimulated DNA synthesis by downregulating p21/Cip1 expression, indicating that it may pose a risk of carcinogenesis associated with dysregulated cell cycles when used to treat overactive bladder and bladder pain.

Hypomethylation-driven AKT Serine/Threonine Kinase 3 promotes testicular germ cell tumors proliferation and negatively correlates to immune infiltration

ABSTRACT AKT Serine/Threonine Kinase 3 (AKT3) has been reported to play an important role in different tumors. However, its clinical value, biological function, and molecular mechanism in testicular germ cell tumors (TGCT) remains unclear. In the current study, we applied the Gene Set Cancer Analysis (GSCA), UCSC XENA, Gene Expression Omnibus (GEO), the Human Protein Atlas (HPA), LinkedOmics, DiseaseMeth version 2.0, TISIDB, and other databases for TGCT data mining. Then, we investigated AKT3’s mechanism of action and clinical survival significance via bioinformatics followed by in vitro experiments. We found that AKT3 was upregulated and had frequent copy number amplifications in TGCT, which were associated with poor survival outcomes of patients. On the other hand, mutations that led to AKT3 loss-of-function were correlated to a better prognosis in patients. Moreover, AKT3 silencing significantly inhibited the proliferation, DNA synthesis and colony formation of NCCIT cells (a TGCT cell line). AKT3 might participate in TGCT progression through multiple signaling pathways, such as ErbB, oxidative phosphorylation, and affecting tumor immune infiltration. Also, the upregulation of AKT3 mRNA expression might be driven by the hypomethylation of its promoter region. Overall, AKT3 is a potential TGCT oncogene and can be further used as a therapeutic target.

HORMONE-ASSOCIATED METABOLIC DISORDERS OF THE ORAL CAVITY ORGANS IN WOMEN OF REPRODUCTIVE AGE

There is emerging evidence of a possible relationship between the oral cavity and reproductive organs. Recent studies suggest these functional relations. The aim of this review was to synthesize the available evidence on this relationship. Clinical observation established that sex hormones enhance gingival inflammation in periodontal healthy women during pregnancy and that periodontal condition is associated with variation of sex hormones in blood. Estrogen regulates DNA synthesis in human gingival epithelial cells and periodontal ligament, estrogen reduces down regulation of cytokines. Estrogen and progesterone affect the periodontium via appropriate receptors (estrogen receptor and progesterone receptor). They are localized in human periodontium, demonstrating that periodontal tissues are the target tissues for these hormones. Testosterone receptors are found in the periodontal tissues. It inhibits prostaglandin secretion and reduces interleukin production. At the same time testosterone stimulates osteoblast proliferation and differentiation, also enhances matrix synthesis by fibroblast, osteoblasts, and periodontal ligament. The role of testosterone in the formation of teeth is demonstrated in the paper. In females and males, in saliva there are sex steroid hormones. The study examined the entry mode of hormones into saliva. The results suggest that lipid soluble unconjugated steroids (estriol, testosterone, progesterone) enter saliva via intracellular route; the conjugated steroids (lipid insoluble (dehydroepiandrosterone, conjugated estrogens)) enter via the ‘tight junctions’ (infiltrations through the tight junctions between the acinar cells). Recent evidence indicates that organs of the oral cavity (salivary glands, periodontal tissues, oral epithelial cells mucus) produce ghrelin-hormone which affects organs of the reproductive system directly or indirectly via hypothalamic-pituitary-gonadal axis. In all these organs, there is an appropriate receptor. In conclusion, the organs of oral cavity and organs of reproductive system are functionally linked by sex steroid hormones and ghrelin, besides that periodont can influence ovaries by neuro-reflectory link.

Combined treatment with glucagon-like peptide-1 receptor agonist exendin-4 and metformin attenuates breast cancer growth.

Cancer is a major cause of death in patients with diabetes. Incretin therapy has received much attention because of its tissue-protective effects. We have previously reported an anti-breast cancer effect of glucagon-like peptide-1 receptor agonist exendin-4 (Ex-4). An anti-cancer effect of metformin is well recognized. Therefore, we examined the effect of combined treatment with Ex-4 and metformin in breast cancer cells. In human breast cancer cell lines MCF-7, MDA-MB-231, and KPL-1, 0.1-10mM metformin significantly reduced the cell number in growth curve analysis in a dose-dependent manner. Furthermore, combined treatment with 0.1mM metformin and 10nM Ex-4 additively attenuated the growth curve progression of breast cancer cells. In a bromodeoxyuridine (BrdU) assay, Ex-4 or metformin significantly decreased breast cancer cell proliferation and further reduction of BrdU incorporation was observed by combined treatment with Ex-4 and metformin, which suggested that Ex-4 and metformin additively decreased DNA synthesis in breast cancer cells. Although apoptotic cells were not observed among Ex-4-treated breast cancer cells, apoptotic cells were clearly detected among metformin-treated breast cancer cells by apoptosis assays. Furthermore, metformin decreased BCL-2 expression in MCF-7 cells. In vivo experiments using a xenograft model showed that Ex-4 and metformin significantly decreased the breast tumor weight and Ki67-positive proliferative cancer cells, and metformin reduced the serum insulin level in mice. These data suggested that Ex-4 and metformin attenuated cell proliferation and metformin induced apoptosis in breast cancer cells. Combined treatment of Ex-4 and metformin may be an optional therapy to inhibit breast cancer progression.

Abstract 14091: Systemic Hypoxemia Induces Cardiomyocyte Cell Cycle Re-Entry but Few Myocytes Appear to Truly Divide

Rationale: The adult mammalian heart has a very limited capacity to regenerate cardiomyocytes that die from pathological damage. Several studies suggest that those few new myocytes found after injury are derived from pre-existing cardiomyocytes. A recent study suggests that systemic hypoxemia in adult mice can induce their cardiac myocytes to reenter the cell cycle and form two daughter cells. The goal of the present experiments was to determine if hypoxemia can induce adult murine cardiomyocytes to reenter the cell cycle and divide. Hypothesis: Hypoxia induces adult cardiomyocytes to complete the cell cycle and form new cardiac myocytes. Methods and Results: EdU mini pumps were implanted in 2-month-old C57BL/6 mice. Mice were then placed in a hypoxia chamber and the oxygen was lowered by 1% every day for 14 days to reach a final concentration of 7% oxygen. The animals remained in hypoxia (7% Oxygen) for 2 weeks and then were euthanized. Hypoxic mice had an increase in heart weight and echocardiography documented an increase in cardiac pump function and LV wall thickness when compared to control mice. Analysis of isolated cardiac myocytes in C57BL/6 animals showed an increase in EdU+ cardiomyocytes in hypoxemia and DAPI fluorescence intensity was also increased in these EdU+ myocytes which may indicate DNA synthesis. In fixed heart tissue sections, hypoxic mice contained more EdU+ myocytes and had an increase in the total amount of EdU+ cells in the heart. To further evaluate cardiomyocyte proliferation, we employed a mosaic mouse model with double markers (MADM) in which myocytes entering the cell cycle express both red fluorescent protein (RFP) and green fluorescent protein (GFP) and therefore are yellow in color. When cells complete the cell cycle and divide they express only RFP or GFP. MADM mice were exposed to hypoxia at 7% Oxygen as described above. MADM mice subjected to hypoxemia had increased numbers of yellow myocytes versus controls. There was a much smaller number of hypoxic myocytes with just RFP or GFP. Conclusion: Systemic hypoxia induces cardiac myocyte cell cycle re-entry and DNA synthesis, but it seems that very few of these myocytes complete the cell cycle and divide into two daughter cells.

H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma.To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM.We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM.To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c‐Myc using 10058‐F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM.Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. DisclosuresNo relevant conflicts of interest to declare.

Intrinsic S phase checkpoint enforced by an antiproliferative oncosuppressor cytokine

The cell cycle is strictly programmed with control mechanisms that dictate order in cell cycle progression to ensure faithful DNA replication, whose deviance may lead to cancer. Checkpoint control at the G1/S, S/G2 and G2/M portals have been defined but no statutory time-programmed control for securing orderly transition through S phase has so far been identified. Here we report that in normal cells DNA synthesis is controlled by a checkpoint sited within the early part of S phase, enforced by the βGBP cytokine an antiproliferative molecule otherwise known for its oncosuppressor properties that normal cells constitutively produce for self-regulation. Suppression of active Ras and active MAPK, block of cyclin A gene expression and suppression of CDK2-cyclin A activity are events which while specific to the control of a cell cycle phase in normal cells are part of the apoptotic network in cancer cells.

Dihydroartemisinin Suppresses the Tumorigenesis and Cycle Progression of Colorectal Cancer by Targeting CDK1/CCNB1/PLK1 Signaling.

Dihydroartemisinin (DHA), a well-known antimalarial drug, has been widely investigated for its antitumor effects in multiple malignancies. However, its effects and regulatory mechanisms in colorectal cancer (CRC) are still unproved. In this study, in vitro experiments including CCK8, EdU, Transwell, and flow cytometry analyses and an in vivo tumorigenesis model were conducted to assess the effects of DHA on the bio-behaviors of CRC cells. Additionally, RNA-seq combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses was used to obtain the targets of DHA, and these were verified by molecular docking, qRT-PCR, and Western blotting. As a result, we found that DHA significantly suppressed the proliferation, DNA synthesis, and invasive capabilities and induced cell apoptosis and cell cycle arrest in HCT116, DLD1, and RKO cells in vitro and in vivo. Further analyses indicated that the targets of DHA were predominantly enriched in cell cycle-associated pathways, including CDK1, CCNB1, and PLK1; and DHA could bind with the CDK1/CCNB1 complex and inhibit the activation of CDK1/CCNB1/PLK1 signaling. Moreover, cucurbitacin E, a specific inhibitor of the CDK1/CCNB1 axis, enhanced the inhibitory effects of DHA on DNA synthesis and colony formation in HCT116 and DLD1 cells. In short, DHA could suppress the tumorigenesis and cycle progression of CRC cells by targeting CDK1/CCNB1/PLK1 signaling.

Phytoestrogens on the Proliferation of Breast Cancer Cell MCF-7

A cell is an organic whole containing biological macromolecules such as nucleic acid and protein. Human cells contain about 15,000 proteins, which play an important role in the development of cell life. This article aims to study the effect of phytoestrogens on the proliferation of breast cancer cells MCF-7. The estrogen-dependent MCF-7 cells described in this article are grown on average DMEM (containing calf serum 10) and connected to single cell culture. Five days before adding the test product, the cells were washed with PBS and converted into red-free phenol. High-sugar DMEM containing 5 parts of bovine embryo serum was cultured and treated with activated charcoal glucan glycosides. In the experiment, four dose groups of solvent control, estrogen control, anti-estrogens and two test substances were used for control, and the proliferation of MCF-7 cells was analyzed by integration method and flow cytometry. The experimental results in this article show that compared with the solvent control group, GS can significantly inhibit MCF-7 cell proliferation and cell DNA synthesis, and G/M can block the cell cycle and produce a similar inhibitory effect. 96mol/L zein treatment for 24 hours can significantly promote MCF-7 cell proliferation and cell DNA composition, promote cell cycle, and increase cell separation and proliferation index.

Biological Effects of DMBA and Lagenaria siceraria:2: Modulatory Role of Lagenaria siceraria on The Ileum of Females Swiss Albino Mice Induced by The Possible carcinogenic effect of DMBA

Background: Cancer is constituted as the most famous fatal disease among human-being. DMBA is a common carcinogen and immunosuppressant and experimentally used for the induction of mutations via the creation of DNA adducts. Aim: Evaluation of the therapeutic effect of Lagenaria siceraria on 9,10 dimethyl-1, 2-benzanthracene (DMBA)-induced changes. Materials and Methods: A total number of thirty adult female albino rats within average age 3 months classified into 3 groups as follow: Group1 is designated as control. Group 2 was subcutaneously injected with a single dose of DMBA (10 mg/ 100 g b. wt) and after 90 days, animals have received a daily injection of distilled water for 30 days. Group 3 was subcutaneously mice were injected with a single dose of DMBA and after 90 days, mice were received morning and night daily injection of Lagenaria siceraria (100 mg/ 100 g b.w) for 30 days. After scarifying the mice, ileum samples were taken and passed the histological examination, also Histochemical stains were done. Results: From the cell biological, histochemical, and histopathological points of view, the carcinogen DMBA has a highly stimulatory effect on the cellular activities, DNA synthesis, RNA synthesis, and cell proliferative in the normal epithelial cells lining villi of the ileum of female mice, while Lagenaria siceraria has a highly inhibitory effect on the volume of the nuclei, DNA synthesist, and the cell proliferation of the DMBA-induced cells lining villi of the ileum of female mice. Conclusion: Finally, it could be said that the carcinogen DMBA has a proliferative and/or possibly a carcinogenic effect on the ileum of female mice. Also, It could be concluded that Lagenaria siceraria could be offered an antiproliferative and/or possibly an anti-carcinogenic effect against DMBA.

Cell cycle-coupled changes in the level of reactive oxygen species support the proliferation of human pluripotent stem cells.

The study of proliferation regulation in human pluripotent stem cells is crucial to gain insights into understanding the physiology of these cells. However, redox regulation of the pluripotent cell cycle remains largely unexplored. Here, using human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs), we demonstrate that the level of reactive oxygen species (ROS) in pluripotent cells oscillates in accordance with the cell cycle progression with the peak occurring at transition from S to G2 /M phase of the cycle. A decrease of this level by antioxidants leads to hindered S-phase initiation and progression but does not affect the early-G1 -phase or mitosis. Cells exposed to antioxidants in the early-G1 -phase accumulate the phosphorylated retinoblastoma protein and overcome the restriction point but are unable to accumulate the main regulators of the S phase-CYCLIN A and GEMININ. Based on the previous findings that CYCLIN A stability is affected by redox homeostasis disturbances in somatic cells, we compared the responses to antioxidant treatments in hESCs and in their differentiated fibroblast-like progeny cells (difESCs). In difESCs, similar to hESCs, a decrease in ROS level results in the disruption of S-phase initiation accompanied by a deficiency of the CYCLIN A level. Moreover, in antioxidant-treated cells, we revealed the accumulation of DNA breaks, which was accompanied by activation of the apoptosis program in pluripotent cells. Thus, we conclude that maintaining the physiological ROS level is essential for promotion of proliferation and accurate DNA synthesis in pluripotent cells and their differentiated descendants.

MiR-3929 Inhibits Proliferation and Promotes Apoptosis by Downregulating Cripto-1 Expression in Cervical Cancer Cells

Cripto-1 is highly expressed in many cancers, and downregulating its expression may become a promising approach for cancer treatment. However, the regulation of Cripto-1 expression is not well characterized. In this study, we focused on the post-transcriptional regulation of Cripto-1 expression and analyzed the potential miRNAs that bind to the 3′UTR of Cripto-1 mRNA. miR-3929 was found to be able to bind to the 3′UTR and downregulate the expression of Cripto-1 in cervical cancer cells. Then, we analyzed the effect of miR-3929 on the biological behavior of cervical cancer cells, finding that miR-3929 could reduce cell viability, DNA synthesis, and Ki67 expression and induce cell cycle arrest in the G2/M phase; overexpression of Cripto-1 reversed the inhibitory effect of miR-3929 on proliferation. Moreover, DAPI staining and flow cytometry revealed that miR-3929-induced cell apoptosis is dependent on the mitochondrial pathway; the overexpression of Cripto-1 reversed the proapoptotic effect of miR-3929. Finally, the in vivo results showed that miR-3929 significantly inhibits the growth of HeLa xenograft tumors in nude mice. Therefore, our findings suggest that miR-3929 inhibits the proliferation and induces the apoptosis of cervical cancer cells by downregulating Cripto-1 via specifically targeting the 3′UTR of its mRNA.

Long non-coding RNA CCAT2 drives the growth of laryngeal squamous cell carcinoma via regulating YAP activity.

Emerging evidence suggests that long non-coding RNA (lncRNA) is closely associated with numerous human diseases, including cancer. However, the functional relevance of lncRNA in human laryngeal squamous cell carcinoma (LSCC) is largely unknown. In the current study, we described CCAT2, a previously unappreciated oncogenic lncRNA in LSCC. CCAT2 was significantly upregulated in human LSCC tissue and serum samples, associated with larger tumor volume, higher clinical stage, and poorer differentiation status. Lentivirus-mediated CCAT2 knockdown notably repressed the cell viability, colony formation, and DNA synthesis rate of LSCC. Screening of transcription factors revealed that YAP/TEAD activity was affected by CCAT2 in LSCC cells. Further, CCAT2 directly binds to YAP protein and blocks the phosphorylation of YAP induced by LATS1, resulting in the nuclear translocation of YAP and the activation of YAP oncogenic targets, such as CTGF, CYR61 and AMOTL2. Importantly, we also confirmed the regulation of CCAT2 on YAP activity in vivo based on nude mice model. Altogether, we identified a novel lncRNA that controls YAP nucleocytoplasmic shuttling and promotes LSCC cell proliferation. Given the importance of YAP in tumorigenesis and progression, our results provide insights to intervene LSCC by targeting the CCAT2/YAP axis.

Prognostic Value of E2F Transcription Factor Expression in Pancreatic Adenocarcinoma.

BackgroundPancreatic adenocarcinoma (PAAD) is one of the deadliest types of cancer. In the early stages, patients often have atypical symptoms, making diagnosis difficult. The prognosis of diagnosed patients is very poor and treating PAAD is challenging. Therefore, determining reliable risk factors related to PAAD development is critical for improving patient prognosis. E2F family transcription factors (TFs) are essential regulators of DNA synthesis and cell cycle progression in eukaryotic cells, and they have been identified as prognostic biomarkers associated with multiple cancer types. However, further research is necessary to establish the prognostic relevance of these TFs in PAAD patients.Material/MethodsWe assessed PAAD patient transcriptional and outcome data using the TIMER, ONCOMINE, STRING, GEPIA, cBioPortal, Kaplan-Meier Plotter, GSCALite, and starBase databases.ResultsPAAD tumor tissues exhibited increased expression of E2F1/3/5/7/8 relative to that in normal tissues, while the expression of E2F2/3/6/8 was associated with a more advanced tumor stage. Survival analyses indicated that PAAD patients expressing higher levels of E2F1/2/3/7/8 exhibited shorter overall survival (OS) and disease-free survival (DFS) than patients expressing lower levels of these TFs. In addition, E2F4 and E2F6 overexpression was associated with poorer DFS and OS, respectively. We also found that the expression of E2Fs was significantly correlated with immune infiltrates, including CD8+ T cells, CD4+ T cells, B cells, dendritic cells, neutrophils, and macrophages.ConclusionsOur study may provide new insights into the optimal choice of immunotherapy and promising novel targets for therapeutic intervention in PAAD patients.

Long Noncoding RNA OR7E156P/miR-143/HIF1A Axis Modulates the Malignant Behaviors of Glioma Cell and Tumor Growth in Mice.

Gliomas are characterized by high incidence, recurrence and mortality all of which are significant challenges to efficacious clinical treatment. The hypoxic microenvironment in the inner core and intermediate layer of the tumor mass of gliomas is a critical contributor to glioma pathogenesis. In this study, we identified an upregulated lncRNA, OR7E156P, in glioma was identified. The silencing of OR7E156P inhibited cell invasion and DNA synthesis in vitro and tumor growth in vivo. OR7E156P was intricately linked to the HIF1A pathway. Hypoxia could induce OR7E156P expression, whereas OR7E156P silencing decreased HIF1A protein levels under hypoxic conditions. Hypoxia promoted glioma cell invasion and DNA synthesis, and HUVEC tube formation, whereas OR7E156P silencing partially reversed the cellular effects of hypoxia. HIF1A overexpression promoted, whereas OR7E156P silencing inhibited tumor growth; the inhibitory effects of OR7E156P silencing on tumor growth were partially reversed by HIF1A overexpression. miR-143 directly targeted OR7E156P and HIF1A, respectively. miR-143 inhibition increased HIF1A protein levels, promoted glioma cell invasion and DNA synthesis. Moreover, they enhanced HUVEC tube formation, whereas OR7E156P silencing partially reversed the cellular effects of miR-143 inhibition. HIF1A targeted the promoter region of miR-143 and inhibited miR-143 expression. Altogether a regulatory axis consisting of OR7E156P, miR-143, and HIF1A, was identified which is deregulated in glioma, and the process of the OR7E156P/miR-143/HIF1A axis modulating glioma cell invasion through ZEB1 and HUVEC tube formation through VEGF was demonstrated.