Novel circular RNA hsa_circ_0036683 suppresses proliferation and migration by mediating the miR-4664-3p/CDK2AP2 axis in non-small cell lung cancer.

BACKGROUND: The aim of the present study was to investigate the function of novel circular RNA hsa_circ_0036683 (circ-36683) in non-small cell lung cancer (NSCLC). METHODS: RNA sequencing was used to screen out differentially expressed miRNAs. Expression levels of miR-4664-3p and circ-36683 were evaluated in lung carcinoma cells and tissues by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effects of miR-4664-3p and circ-36683 on proliferation and migration were assessed using cell counting kit-8 (CCK-8), wound healing and transwell migration assays and xenograft experiments. The targeting relationship of circ-36683/miR-4664-3p/CDK2AP2 was assessed by luciferase reporter assays, western blot, qRT-PCR and argonaute2-RNA immunoprecipitation (AGO2 RIP). Co-immunoprecipitation (Co-IP), 5-ethynyl-2'-deoxyuridine (EdU) staining and CCK-8 were used to validate the indispensable role of CDK2AP2 in suppressing cell proliferation as a result of CDK2AP1 overexpression. RESULTS: By RNA sequencing, miR-4664-3p was screened out as an abnormally elevated miRNA in NSCLC tissues. Transfection of miR-4664-3p could promote cell proliferation, migration and xenograft tumor growth. As a target of miR-4664-3p, CDK2AP2 expression was downregulated by miR-4664-3p transfection and CDK2AP2 overexpression could abolish the proliferation promotion resulting from miR-4664-3p elevation. Circ-36683, derived from back splicing of ABHD2 pre-mRNA, was attenuated in NSCLC tissue and identified as a sponge of miR-4664-3p. The functional study revealed that circ-36683 overexpression suppressed cell proliferation, migration and resulted in G0/G1 phase arrest. More importantly, the antioncogenic function of circ-36683 was largely dependent on the miR-4664-3p/CDK2AP2 axis, through which circ-36683 could upregulate the expression of p53/p21/p27 and downregulate the expression of CDK2/cyclin E1. CONCLUSION: The present study revealed the antioncogenic role of circ-36683 in suppressing cell proliferation and migration and highlighted that targeting the circ-36683/miR-4664-3p/CDK2AP2 axis is a promising strategy for the intervention of NSCLC.

Assessing the antitumor effects of metformin on ovarian clear cell carcinoma.

Developing novel therapies that outperform the existing chemotherapeutic treatments is required for treatment-resistant ovarian clear cell carcinoma. We investigated the antitumor effect of metformin on ovarian clear cell carcinoma, enhancement of the antitumor effect by its combination with chemotherapy, and its molecular regulatory mechanism. First, we evaluated the viability of ovarian clear cell carcinoma lines using the water-soluble tetrazolium-1 assay and found that metformin suppressed cell viability. Cell viability was significantly suppressed by co-treatment with cisplatin and metformin. In contrast, co-treatment with paclitaxel and metformin showed no significant difference in viability compared with the group without metformin. Western blot analysis showed increased phosphorylation of AMP-activated protein kinase in some cell lines and suppressed phosphorylation of the mammalian target of rapamycin in a particular cell line. Flow cytometry analysis revealed a significant increase in the rate of apoptosis in the metformin-treated group and rate of cell cycle arrest at the G2/M phase in a particular cell line. These results indicated that metformin may be effective against cultured ovarian clear cell carcinoma cells, particularly in combination with cisplatin.

Selinexor targeting XPO1 promotes PEG3 nuclear accumulation and suppresses cholangiocarcinoma progression.

BACKGROUND: The role of selinexor, a targeted inhibitor of exportin 1 (XPO1), in the treatment of cholangiocarcinoma is not yet fully understood. This study conducted comprehensive in vitro and in vivo investigations to elucidate the effects of selinexor on cholangiocarcinoma, with a focus on its mechanistic relationship with the cellular localization of Paternally Expressed Gene 3 (PEG3). METHODS: A patient-derived xenograft (PDX) model was established using samples from a cholangiocarcinoma patient in immunodeficient mice to assess the in vivo effects of selinexor. Additionally, cholangiocarcinoma cell lines HuCC-T1 and BRE were cultured to evaluate selinexor's impact on cell proliferation, invasion, migration, cell cycle, and apoptosis. HuCC-T1 cells were also implanted in immunodeficient mice for further investigation. Immunofluorescence and Western blotting were employed to observe the expression and localization of the PEG3 protein. RESULTS: The results demonstrated that selinexor significantly inhibited tumor growth in the cholangiocarcinoma PDX model and promoted the accumulation of PEG3 protein within the nuclei of tumor cells. In vitro experiments showed that selinexor effectively suppressed cholangiocarcinoma cell proliferation, invasion, and migration, while also impeding the cell cycle and inducing apoptosis. Notably, selinexor markedly facilitated the nuclear accumulation of PEG3 protein in cholangiocarcinoma cells. However, when PEG3 expression was knocked down, the effects of selinexor on cholangiocarcinoma were significantly reversed. CONCLUSION: These findings suggest that selinexor inhibits the progression of cholangiocarcinoma by targeting XPO1 and promoting the nuclear accumulation of PEG3 protein, thereby hindering the cell cycle and inducing apoptosis.

Evidence for phosphate-dependent control of symbiont cell division in the model anemone Exaiptasia diaphana.

Reef-building corals depend on symbiosis with photosynthetic algae that reside within their cells. As important as this relationship is for maintaining healthy reefs, it is strikingly delicate. When ocean temperatures briefly exceed the average summer maximum, corals can bleach, losing their endosymbionts. Although the mechanisms governing bleaching are unknown, studies implicate uncoupling of coral and algal cell divisions at high temperatures. Still, little is known regarding the coordination of host and algal cell divisions. Control of nutrient exchange is one likely mechanism. Both nitrogen and phosphate are necessary for dividing cells, and although nitrogen enrichment is known to increase symbiont density in the host, the consequences of phosphate enrichment are poorly understood. Here, we examined the effects of phosphate depletion on symbiont growth in culture and compared the physiology of phosphate-starved symbionts in culture to symbionts that were freshly isolated from a host. We found that available phosphate is as low in freshly isolated symbionts as it is in phosphate-starved cultures. Furthermore, RNAseq revealed that phosphate-limited and freshly isolated symbionts have similar patterns of gene expression for phosphate-dependent genes, most notably upregulation of phosphatases, which is consistent with phosphate recycling. Similarly, lipid profiling revealed a substantial decrease in phospholipid abundance in both phosphate-starved cultures and freshly isolated symbionts. These findings are important because they suggest that limited access to phosphate controls algal cell divisions within a host. IMPORTANCE: The corals responsible for building tropical reefs are disappearing at an alarming rate as elevated sea temperatures cause them to bleach and lose the algal symbionts they rely on. Without these symbionts, corals are unable to harvest energy from sunlight and, therefore, struggle to thrive or even survive in the nutrient-poor waters of the tropics. To devise solutions to address the threat to coral reefs, it is necessary to understand the cellular events underpinning the bleaching process. One model for bleaching proposes that heat stress impairs algal photosynthesis and transfer of sugar to the host. Consequently, the host's demands for nitrogen decrease, increasing nitrogen availability to the symbionts, which leads to an increase in algal proliferation that overwhelms the host. Our work suggests that phosphate may play a similar role to nitrogen in this feedback loop.

Hyperglycemia-independent neonatal streptozotocin-induced retinopathy (NSIR) in rats.

Introduction: Chemicals, such as MNU (N-methyl-N-nitrosourea) and NaIO3 (sodium iodate), are widely used to induce retinal degeneration in rodents. Streptozotocin (STZ) is an analog of N-acetyl glucosamine in which an MNU moiety is linked to a hexose and has a special toxic effect on insulin-producing pancreatic ß-cells. It is commonly used to induce hyperglycemia to model diabetes. While intracerebroventricular injection of STZ can produce Alzheimer's disease independent of hyperglycemia, most retinal studies using STZ focus on the effects of hyperglycemia on the retina, but whether STZ has any impact on retinal cells independent of hyperglycemia is unknown. We aimed to investigate the role of cytotoxicity of STZ in rat retina. Methods: Intravitreal or subcutaneous injection of STZ was performed on newborn rats. Electroretinogram (ERG) and H&E staining investigated retinal function and morphological changes. Retinal cell types, cell death, proliferation, inflammation, and angiogenesis were studied by immunostaining. RNA sequencing was performed to examine the transcriptome changes of retinal cells after intravitreal injection of STZ. Results: Intravitreal (5 µg or 10 µg) or subcutaneous (30 mg/kg) injection of STZ at the early stage of newborn rats couldn't induce hyperglycemia but caused NSIR (Neonatal STZ-induced retinopathy), including reduced ERG amplitudes, retinal rosettes and apoptosis, cell cycle arrest, microglial activation, and delayed retinal angiogenesis. STZ did not affect the early-born retinal cell types but significantly reduced the late-born ones. Short-term and long-term hyperglycemia had no significant effects on the NSIR phenotypes. RNA sequencing revealed that STZ induces oxidative stress and activates the p53 pathway of retinal cells. Locally or systemically, STZ injection after P8 couldn't induce SINR when all retinal progenitors exit the cell cycle. Conclusion: NSIR in rats is independent of hyperglycemia but due to STZ's direct cytotoxic effects on retinal progenitor cells. NSIR is a typical reaction to STZ-induced retinal oxidative stress and DNA damage. This significant finding suggests that NSIR may be a valuable model for studying retinal progenitor DNA damage-related diseases, potentially leading to new insights and treatments.

Shatavarin-IV, a steroidal saponin from Asparagus racemosus, inhibits cell cycle progression and epithelial-to-mesenchymal transition in AGS cells under hyperglycemic conditions.

Gastric cancer (GC)-diabetes co-morbidity is nowadays growing into a rising concern. However, no separate treatment procedures have been outlined for such patients. Phytochemicals and their derivatives can therefore be used as therapeutics as they have greater effectiveness, reduced toxicity, and a reduced likelihood of developing multi-drug resistance in cancer treatments. The present study intended to assess the therapeutic efficacy of Shatavarin-IV - a major steroidal saponin from the roots of Asparagus racemosus, in human gastric adenocarcinoma cell line under hyperglycemic conditions and explore its mechanism of action in controlling GC progression. For the present study, AGS cells were incubated in high glucose-containing media and the effects of Shatavarin-IV therein have been evaluated. Cell proliferation, confocal microscopic imaging, flow-cytometric analysis for cell cycle and apoptosis, immunoblotting, zymography, reverse zymography, wound-healing, colony formation, and invasion assays were performed. Shatavarin-IV has a prominent effect on AGS cell proliferation; with IC50 of 2.463 µâ€¯M under hyperglycemic conditions. Shatavarin-IV induces cell cycle arrest at the G0/G1 phase, thereby preventing hyperglycemia-induced excessive cell proliferation that later on leads to apoptotic cell death at 36 h of incubation. Shatavarin-IV further inhibits the migratory and invasive potential of AGS cells by altering the expression patterns of different EMT markers. It also inhibits MMP-9 while promoting TIMP-1 activity and expression; thereby regulating ECM turnover. This is the first report demonstrating the therapeutic efficacy of Shatavarin-IV against AGS cells grown in hyperglycemic conditions, implicating new insights into the treatment paradigm of patients with GC-diabetes co-morbidity.

ARG2 knockdown promotes G0/G1 cell cycle arrest and mitochondrial dysfunction in adenomyosis via regulation NF-κB and Wnt/Β-catenin signaling cascades.

BACKGROUND: Adenomyosis is a common gynecological disease, characterized by overgrowth of endometrial glands and stroma in the myometrium, however its exact pathophysiology still remains uncertain. Emerging evidence has demonstrated the elevated level of arginase 2 (ARG2) in endometriosis and adenomyosis. This study aimed to determine whether ARG2 involved in mitochondrial function and epithelial to mesenchymal transition (EMT) in adenomyosis and its potential underlying mechanisms. MATERIALS AND METHODS: RNA interference was used to inhibit ARG2 gene, and then Cell Counting Kit (CCK-8) assay and flow cytometery were performed to detect the cell proliferation capacity, cell cycle, and apoptosis progression, respectively. The mouse adenomyosis model was established and RT-PCR, Western blot analysis, mitochondrial membrane potential (Δψm) detection and mPTP opening evaluation were conducted. RESULTS: Silencing ARG2 effectively down-regulated its expression at the mRNA and protein levels in endometrial cells, leading to decreased enzyme activity and inhibition of cell viability. Additionally, ARG2 knockdown induced G0/G1 cell cycle arrest, promoted apoptosis, and modulated the expression of cell cycle- and apoptosis-related regulators. Notably, the interference with ARG2 induces apoptosis by mitochondrial dysfunction, ROS production, ATP depletion, decreasing the Bcl-2/Bax ratio, releasing Cytochrome c, and increasing the expression of Caspase-9/-3 and PARP. In vivo study in a mouse model of adenomyosis demonstrated also elevated levels of ARG2 and EMT markers, while siARG2 treatment reversed EMT and modulated inflammatory cytokines. Furthermore, ARG2 knockdown was found to modulate the NF-κB and Wnt/ß-catenin signaling pathways in mouse adenomyosis. CONCLUSION: Consequently, ARG2 silencing could induce apoptosis through a mitochondria-dependent pathway mediated by ROS, and G0/G1 cell cycle arrest via suppressing NF-κB and Wnt/ß-catenin signaling pathways in Ishikawa cells. These findings collectively suggest that ARG2 plays a crucial role in the pathogenesis of adenomyosis and may serve as a potential target for therapeutic intervention.

Oncolytic Virus Senecavirus A Inhibits Hepatocellular Carcinoma Proliferation and Growth by Inducing Cell Cycle Arrest and Apoptosis.

Background and Aims: Hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options and high mortality. Senecavirus A (SVA) has shown potential in selectively targeting tumors while sparing healthy tissues. This study aimed to investigate the effects of SVA on HCC cells in vitro and in vivo and to elucidate its mechanisms of action. Methods: The cell counting kit-8 assay and colony formation assay were conducted to examine cell proliferation. Flow cytometry and nuclear staining were employed to analyze cell cycle distribution and apoptosis occurrence. A subcutaneous tumor xenograft HCC mouse model was created in vivo using HepG2 cells, and Ki67 expression in the tumor tissues was assessed. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay and hematoxylin and eosin staining were employed to evaluate HCC apoptosis and the toxicity of SVA on mouse organs. Results: In vitro, SVA effectively suppressed the growth of tumor cells by inducing apoptosis and cell cycle arrest. However, it did not have a notable effect on normal hepatocytes (MIHA cells). In an in vivo setting, SVA effectively suppressed the growth of HCC in a mouse model. SVA treatment resulted in a significant decrease in Ki67 expression and an increase in apoptosis of tumor cells. No notable histopathological alterations were observed in the organs of mice during SVA administration. Conclusions: SVA inhibits the growth of HCC cells by inducing cell cycle arrest and apoptosis. It does not cause any noticeable toxicity to vital organs.

TTI-101 targets STAT3/c-Myc signaling pathway to suppress cervical cancer progression: an integrated experimental and computational analysis.

BACKGROUND: Cervical cancer (CC) is a significant global health concern, demanding the consideration of novel therapeutic strategies. The signal transducer and activator of transcription 3 (STAT3) pathway has been implicated in cancer progression and is a potential target for therapeutic intervention. This study aimed to explore the therapeutic potential of TTI-101, a small molecule STAT3 inhibitor, in CC and investigate its underlying mechanisms. METHODS: Molecular docking studies and molecular dynamics simulations were performed to explore the binding interaction between TTI-101 and STAT3 and assess the stability of the STAT3-TTI-101 complex. Cell viability assays, wound healing assays, colony formation assays, flow cytometry analysis, and gene expression analysis were conducted. In vivo xenograft models were used to assess the antitumor efficacy of TTI-101. RESULTS: The in silico analysis shows a stable binding interaction between TTI-101 and STAT3. TTI-101 treatment inhibits cell viability, clonogenic ability, and cell migration in CC cells. Furthermore, TTI-101 induces apoptosis and cell cycle arrest. Analysis of apoptosis-related markers demonstrated dysregulation of Bax, Bcl-2, and Caspase-3 upon TTI-101 treatment. Moreover, TTI-101 caused G2/M phase arrest accompanied by a decrease in CDK1 and Cyclin B1 at mRNA levels. In the xenograft model, TTI-101 significantly inhibited tumor growth without adverse effects on body weight. CONCLUSION: TTI-101 exhibited anticancer effects by targeting the STAT3/c-Myc signaling pathway, inducing cell cycle arrest, and promoting apoptosis in CC cells. These findings provide valuable insights into the development of novel therapeutic strategies for cervical cancer. Further investigation is warranted to validate the clinical application of TTI-101.

Pyridaben inhibits cell cycle progression and delays early embryonic development in zebrafish (Danio rerio).

Pyridaben is a broad-spectrum, contact-killing acaricide that can be used to control a variety of harmful food and plant mites. Pyridaben displays cardiotoxicity and liver toxicity toward fish, but the effects on fish embryonic development have not been characterized. We exposed early zebrafish embryos to 20, 30, and 40 µg/L concentrations of pyridaben. The exposure caused developmental abnormalities, including delayed embryonic shield formation, yolk sac resorption, decreases in body length, reduced pigmentation, and delays in hatching. Pyridaben caused a significant increase in the transcription level of the endoderm marker foxa2, but the transcription levels of the ectoderm development marker foxb1a and the mesoderm development marker snaila were not significantly altered. The transcription levels of the genes SOX17 in early embryos were significantly reduced. After exposure to pyridaben, catalase (CAT) activity and glutathione (GSH) content were increased, and cyclin D1, that is involved in early embryonic development, was abnormally expressed. This study shows that pyridaben causes anomalous development in zebrafish embryos by interfering with the cell cycle order of early embryonic development and inducing excessive oxidative stress. Colivelin, an agonist of the STAT3 signaling pathway, acted as a salvage drug to restore the cell cycle order during embryonic development following exposure to pyridaben. Thus, the toxic effects may be caused by pyridaben's regulation of the STAT3 signaling pathway.

Molecular insights into epoxyazadiradione induced death in triple-negative breast cancer cells: A system biology approach.

Epoxyazadiradione is an important limonoid with immense pharmacological potential. We have reported previously that epoxyazadiradione (EAD) induces apoptosis in triple negative breast cancer cells (MDA-MB 231) by modulating diverse cellular targets. Here, we identify the key genes/pathways responsible for this effect through next-generation sequencing of the transcriptome from EAD treated cells and integrated molecular data analysis using bioinformatics. In silico analysis indicated that EAD displayed favourable drug-like properties and could target multiple macromolecules relevant to TNBC. RNA sequencing revealed that EAD treatment results in the differential expression of 1838 genes in MDA-MB 231 cells, with 752 downregulated and 1086 upregulated. Gene set enrichment analysis of these genes suggested that EAD disrupts protein folding in the endoplasmic reticulum, triggering the unfolded protein response (UPR) and potentially leading to cell death. EAD also induced oxidative stress and DNA damage, downregulated pathways linked to metabolism, cell cycle progression, pro-survival signalling, cell adhesion, motility and inflammatory response. The identification of protein cluster and hub genes were also done. The validation of the identified hub genes gave an inverse correlation between their expression in EAD treated cells and TNBC patient samples. Thus, the identified hub genes could be explored as therapeutic or diagnostic markers for TNBC. Hence, EAD appears to be a promising therapeutic candidate for TNBC by targeting various hallmarks of cancer, including cell death resistance, uncontrolled proliferation and metastasis. To conclude, the identified pathways and validated targets for EAD will provide a roadmap for further in vivo studies and preclinical/clinical validation required for potential drug development.

Dynamics of Replication-Associated Protein Levels through the Cell Cycle.

The measurement of dynamic changes in protein level and localization throughout the cell cycle is of major relevance to studies of cellular processes tightly coordinated with the cycle, such as replication, transcription, DNA repair, and checkpoint control. Currently available methods include biochemical assays of cells in bulk following synchronization, which determine protein levels with poor temporal and no spatial resolution. Taking advantage of genetic engineering and live-cell microscopy, we performed time-lapse imaging of cells expressing fluorescently tagged proteins under the control of their endogenous regulatory elements in order to follow their levels throughout the cell cycle. We effectively discern between cell cycle phases and S subphases based on fluorescence intensity and distribution of co-expressed proliferating cell nuclear antigen (PCNA)-mCherry. This allowed us to precisely determine and compare the levels and distribution of multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition complex subunit 1 (ORC1, and Claspin, with high spatiotemporal resolution in HeLa Kyoto cells. Combining these data with available mass spectrometry-based measurements of protein concentrations reveals the changes in the concentration of these proteins throughout the cell cycle. Our approach provides a practical basis for a detailed interrogation of protein dynamics in the context of the cell cycle.

LncRNA PTENP1/miR-21/PTEN Axis Modulates EMT and Drug Resistance in Cancer: Dynamic Boolean Modeling for Cell Fates in DNA Damage Response.

It is well established that microRNA-21 (miR-21) targets phosphatase and tensin homolog (PTEN), facilitating epithelial-to-mesenchymal transition (EMT) and drug resistance in cancer. Recent evidence indicates that PTEN activates its pseudogene-derived long non-coding RNA, PTENP1, which in turn inhibits miR-21. However, the dynamics of PTEN, miR-21, and PTENP1 in the DNA damage response (DDR) remain unclear. Thus, we propose a dynamic Boolean network model by integrating the published literature from various cancers. Our model shows good agreement with the experimental findings from breast cancer, hepatocellular carcinoma (HCC), and oral squamous cell carcinoma (OSCC), elucidating how DDR activation transitions from the intra-S phase to the G2 checkpoint, leading to a cascade of cellular responses such as cell cycle arrest, senescence, autophagy, apoptosis, drug resistance, and EMT. Model validation underscores the roles of PTENP1, miR-21, and PTEN in modulating EMT and drug resistance. Furthermore, our analysis reveals nine novel feedback loops, eight positive and one negative, mediated by PTEN and implicated in DDR cell fate determination, including pathways related to drug resistance and EMT. Our work presents a comprehensive framework for investigating cellular responses following DDR, underscoring the therapeutic potential of targeting PTEN, miR-21, and PTENP1 in cancer treatment.

Effect of Ferulic Acid Loaded in Nanoparticle on Tissue Transglutaminase Expression Levels in Human Glioblastoma Cell Line.

Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.

Case Report: Molecular Analyses of Cell-Cycle-Related Genes in Cortical Brain Tissue of a Patient with Rasmussen Encephalitis.

Rasmussen's encephalitis (RE) stands as a rare neurological disorder marked by progressive cerebral hemiatrophy and epilepsy resistant to medical treatment. Despite extensive study, the primary cause of RE remains elusive, while its histopathological features encompass cortical inflammation, neuronal degeneration, and gliosis. The underlying molecular mechanisms driving disease progression remain largely unexplored. In this case study, we present a patient with RE who underwent hemispherotomy and has remained seizure-free for over six months, experiencing gradual motor improvement. Furthermore, we conducted molecular analysis on the excised brain tissue, unveiling a decrease in the expression of cell-cycle-associated genes coupled with elevated levels of BDNF and TNF-α proteins. These findings suggest the potential involvement of cell cycle regulators in the progression of RE.

Human induced pluripotent stem cells for live cell cycle monitoring and endogenous gene activation.

The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.

Degradation of STIM1 through FAM134B-mediated ER-phagy is potentially involved in cell proliferation.

Autophagy is classified as non-selective or selective depending on the types of degrading substrates. Endoplasmic reticulum (ER)-phagy is a form of selective autophagy for transporting the ER-resident proteins to autolysosomes. FAM134B, a member of the family with sequence similarity 134, is a well-known ER-phagy receptor. Dysfunction of FAM134B results in several diseases including viral infection, inflammation, neurodegenerative disorder and cancer, indicating that FAM134B has crucial roles in various kinds of intracellular functions. However, how FAM134B-mediated ER-phagy regulates intracellular functions is not well understood. In this study, we found that FAM134B knockdown in mammalian cells accelerated cell proliferation. FAM134B knockdown increased the protein amount of STIM1, an ER Ca2+ sensor protein mediating the store-operated Ca2+ entry (SOCE) involved in G1 to S phase transition. FAM134B bound to STIM1 through its C-terminal cytosolic region. FAM134B knockdown reduced transport of STIM1 from the ER to autolysosomes. Finally, FAM134B knockdown accelerated G1 to S phase transition. These results suggest that FAM134B is involved in cell proliferation possibly through degradation of STIM1 via ER-phagy.

Risk assessment of 2ß,3ß-19α-trihydroxyursolic acid from Rubus imperialis (Rosaceae) in HepG2/C3A cells via genotoxicity, metabolism, and cell growth.

Rubus imperialis (Rosaceae) is a Brazilian medicinal plant that already exhibited therapeutical perspectives. However, previous studies revealed cellular and/or genetic toxicity of extracts from aerial parts of this plant, as well as other species of the Rubus genus. Being 2ß,3ß-19α-trihydroxyursolic acid (2B) one of the major compounds of this plant, with proven pharmacological effect, it is important to investigate the biosafety of this isolated compound. Therefore, in the present study, (2B) was tested by several cytogenotoxic endpoints up to 20 µg/ml in human hepatoma HepG2/C3A cells. The test compound did not produce any decreased cell viability, DNA damage, chromosomal mutations, cell cycle changes, or apoptotic effects in the tested cells. Additionally, RT-qPCR analysis revealed the downregulation of CYP3A4 (metabolism), M-TOR (cell death), and CDKN1A (cell cycle) genes. Under the experimental conditions used, the 2B compound did not show cytogenotoxic activity after a single exposure to HepG2/C3A human cells.

Lupiwighteone as an Antitumor Agent Reverses Multidrug Resistance in K562/ADR Cells by Regulating Cellular Prion Protein-Oct4 Axis.

INTRODUCTION: One of the many reasons for cancer treatment failure and recurrence is acquired Multidrug Resistance (MDR). Overcoming cancer drug resistance has been the focus of researchers' studies. Cellular prion protein (PrPC) is a glycophosphatidylinositol-anchored cell-surface glycoprotein that has been implicated in tumor behavior, including proliferation, apoptosis, invasion, metastasis, and chemoresistance. >Method: Lupiwighteone (Lup), a natural isoflavone found in the root of Glycyrrhiza glabra, has anticancer activity against prostate cancer cells, neuroblastoma cells, and human breast cancer cells. However, its pharmacological effects and mechanisms in drug-resistant cancer cells have not been reported. In this study, we used an adriamycin- resistant leukemia K562 cell model, and for the first time, we investigated the reversal effect of Lup on its MDR and the potential mechanism. RESULT: The results indicated that Lup could induce apoptosis through the mitochondrial pathway while upregulating the expression of related apoptotic proteins, such as Bax, Cyto C, Caspase-3, and PARP1. Autophagy is commonly recognized as a protective mechanism that mediates MDR during treatment. We found that Lup induced cellular autophagy while upregulating the expression of related autophagy proteins such as Beclin 1 and LC3 II. CONCLUSION: In addition, when Lup was combined with adriamycin, Lup decreased the IC50 of K562/ADR cells; moreover, Lup can downregulate the expression of drug-resistant proteins, suggesting that Lup can reverse drug resistance. Further studies have shown that Lup can downregulate the expression of PrPC-PI3K-Akt axis proteins and PrPC-Oct4 axis proteins. This study demonstrated that Lup has the potential to inhibit the proliferation of K562/ADR cells by targeting PrPC, and further study of the signaling pathway associated with PrPC may provide the experimental basis for the treatment of drug-resistant leukemia.

MANCR lncRNA Modulates Cell-Cycle Progression and Metastasis by Cis-Regulation of Nuclear Rho-GEF.

A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing in vitro, and reduced metastatic lung colonization in xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1A, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in cis, by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by cis-regulation of gene expression.