P27 Kip1 Expression Research Articles

Long acting β2-agonist's activation of cyclic AMP cannot halt ongoing mitogenic stimulation in airway smooth muscle cells

Airway smooth muscle cell (ASMC) hyperplasia causes airway wall remodelling, which is resisting to therapy. Long acting β2-agonists (LABA) relax airway muscles, but their effect on remodelling is unclear. This study compared the anti-proliferative effect of LABA in human primary ASMC, in situations where LABA were applied before, together, or after platelet derived growth factor (PDGF-BB). Cells obtained from controls (n = 5), and asthma patients (n = 5) were stimulated by PDGF-BB (10 ng/ml) before or after the application of formoterol or salmeterol. Proliferation was determined by direct cell counts over three days, cell cycle control proteins p21(Waf1/Cip1), p27(Kip1), signalling proteins Erk1/2 and p38 mitogen activated protein kinase (MAPK) were detected by immuno-blotting. PDGF-BB induced proliferation was significantly stronger in asthmatic ASMC versus controls. Proliferation was prevented by 30 min pre-incubation with LABA. When LABA were applied together or after PDGF-BB, their anti-proliferative effect was no longer significant. In untreated ASMC, LABA increased the expression of p21(Waf1/Cip1) and p27(Kip1) through cAMP, and this mechanism was abolished by the presence of PDGF-BB. The data show that the anti-proliferative effect of cAMP signalling cannot overcome the mitogenic signalling cascade once it was activated. Therefore, remodelling in asthma cannot be reduced by LABA.

PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis

BackgroundAltered glucose metabolism endows tumor cells with metabolic flexibility for biosynthesis requirements. Phosphoenolpyruvate carboxykinase 1 (PCK1), a key enzyme in the gluconeogenesis pathway, is downregulated in hepatocellular carcinoma (HCC) and predicts poor prognosis. Overexpression of PCK1 has been shown to suppress liver tumor growth, but the underlying mechanism remains unclear.MethodsmRNA and protein expression patterns of PCK1, AMPK, pAMPK, and the CDK/Rb/E2F pathway were determined using qRT-PCR and western blotting. Cell proliferation ability and cell cycle were assessed by MTS assay and flow cytometric analysis. The effect of PCK1 on tumor growth was examined in xenograft implantation models.ResultsBoth gain and loss-of-function experiments demonstrated that PCK1 deficiency promotes hepatoma cell proliferation through inactivation of AMPK, suppression of p27Kip1 expression, and stimulation of the CDK/Rb/E2F pathway, thereby accelerating cell cycle transition from the G1 to S phase under glucose-starved conditions. Overexpression of PCK1 reduced cellular ATP levels and enhanced AMPK phosphorylation and p27Kip1 expression but decreased Rb phosphorylation, leading to cell cycle arrest at G1. AMPK knockdown significantly reversed G1-phase arrest and growth inhibition of PCK1-expressing SK-Hep1 cells. In addition, the AMPK activator metformin remarkably suppressed the growth of PCK1-knockout PLC/PRF/5 cells and inhibited tumor growth in an orthotropic HCC mouse model.ConclusionThis study revealed that PCK1 negatively regulates cell cycle progression and hepatoma cell proliferation via the AMPK/p27Kip1 axis and supports a potential therapeutic and protective effect of metformin on HCC.

Lysophosphatidic acid increases mesangial cell proliferation in models of diabetic nephropathy via Rac1/MAPK/KLF5 signaling

Mesangial cell proliferation has been identified as a major factor contributing to glomerulosclerosis, which is a typical symptom of diabetic nephropathy (DN). Lysophosphatidic acid (LPA) levels are increased in the glomerulus of the kidney in diabetic mice. LPA is a critical regulator that induces mesangial cell proliferation; however, its effect and molecular mechanisms remain unknown. The proportion of α-SMA+/PCNA+ cells was increased in the kidney cortex of db/db mice compared with control mice. Treatment with LPA concomitantly increased the proliferation of mouse mesangial cells (SV40 MES13) and the expression of cyclin D1 and CDK4. On the other hand, the expression of p27Kip1 was decreased. The expression of Krüppel-like factor 5 (KLF5) was upregulated in the kidney cortex of db/db mice and LPA-treated SV40 MES13 cells. RNAi-mediated silencing of KLF5 reversed these effects and inhibited the proliferation of LPA-treated cells. Mitogen-activated protein kinases (MAPKs) were activated, and the expression of early growth response 1 (Egr1) was subsequently increased in LPA-treated SV40 MES13 cells and the kidney cortex of db/db mice. Moreover, LPA significantly increased the activity of the Ras-related C3 botulinum toxin substrate (Rac1) GTPase in SV40 MES13 cells, and the dominant-negative form of Rac1 partially inhibited the phosphorylation of p38 and upregulation of Egr1 and KLF5 induced by LPA. LPA-induced hyperproliferation was attenuated by the inhibition of Rac1 activity. Based on these results, the Rac1/MAPK/KLF5 signaling pathway was one of the mechanisms by which LPA induced mesangial cell proliferation in DN models.

CCR2 Chemokine Receptors Enhance Growth and Cell-Cycle Progression of Breast Cancer Cells through SRC and PKC Activation.

Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2. CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation. IMPLICATIONS: This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells.

MiR-222 promotes the progression of polycystic ovary syndrome by targeting p27 Kip1

Polycystic ovary syndrome (PCOS) is one of the most complex and common reproductive and endocrinologic disorders in the child-bearing age of women. Recently, miR-222 were reported to be associated with the etiology of PCOS. However, the function of miR-222 during the pathogenesis of PCOS remains unclear. In the present study, we aimed to investigate the role of miR-222 in PCOS. Firstly, miR-222 expression was examined by quantitative real-time PCR (qRT-PCR) in PCOS. The effects of miR-222 on proliferation, apoptosis and cell cycle in KGN cells were analyzed by CCK-8 assay and flow cytometry analysis, respectively. In addition, bioinformatics analysis was used to predict the target genes of miR-222, and dual-luciferase reporter assay was applied to verified the interaction between miR-222 and p27 Kip1 in KGN cells. Moreover, the expressions of p27 Kip1 in KGN cells treated with miR-222 mimics or miR-222 inhibitor were evaluated by qRT-PCR and western blot assays. The results showed that the expression of miR-222 was remarkably upregulated in PCOS tissues compared with corresponding normal tissues. In the gain-of-function and loss-of-function assays, we revealed that miR-222 mimics significantly promoted cell proliferation, while miR-222 inhibitor induced cell apoptosis and cell cycle arrested. Furthermore, p27 Kip1 was identified as a target gene of miR-222, and could be negatively regulated by miR-222 mimics in KGN cells. In conclusion, our findings suggested that miR-222 may promote the progression of PCOS by targeting p27 Kip1.

The long non-coding RNA Kcnq1ot1 controls maternal p57 expression in muscle cells by promoting H3K27me3 accumulation to an intragenic MyoD-binding region

BackgroundThe cell-cycle inhibitor p57kip2 plays a critical role in mammalian development by coordinating cell proliferation and differentiation in many cell types. p57kip2 expression is finely regulated by several epigenetic mechanisms, including paternal imprinting. Kcnq1ot1, a long non-coding RNA (LncRNA), whose gene maps to the p57Kip2 imprinting domain, is expressed exclusively from the paternal allele and participates in the cis-silencing of the neighboring imprinted genes through chromatin-level regulation. In light of our previous evidence of a functional interaction between myogenic factors and imprinting control elements in the regulation of the maternal p57Kip2 allele during muscle differentiation, we examined the possibility that also Kcnq1ot1 could play an imprinting-independent role in the control of p57Kip2 expression in muscle cells.ResultsWe found that Kcnq1ot1 depletion by siRNA causes the upregulation of the maternal and functional p57Kip2 allele during differentiation, suggesting a previously undisclosed role for this LncRNA. Consistently, Chromatin Oligo-affinity Precipitation assays showed that Kcnq1ot1 physically interacts not only with the paternal imprinting control region of the locus, as already known, but also with both maternal and paternal alleles of a novel p57Kip2 regulatory region, located intragenically and containing two binding sites for the muscle-specific factor MyoD. Moreover, chromatin immunoprecipitation assays after Kcnq1ot1 depletion demonstrated that the LncRNA is required for the accumulation of H3K27me3, a chromatin modification catalyzed by the histone-methyl-transferase EZH2, at the maternal p57kip2 intragenic region. Finally, upon differentiation, the binding of MyoD to this region and its physical interaction with Kcnq1ot1, analyzed by ChIP and RNA immunoprecipitation assays, correlate with the loss of EZH2 and H3K27me3 from chromatin and with p57Kip2 de-repression.ConclusionsThese findings highlight the existence of an imprinting-independent role of Kcnq1ot1, adding new insights into the biology of a still mysterious LncRNA. Moreover, they expand our knowledge about the molecular mechanisms underlying the tight and fine regulation of p57Kip2 during differentiation and, possibly, its aberrant silencing observed in several pathologic conditions.

MicroRNA-186 targets SKP2 to induce p27Kip1-mediated pituitary tumor cell cycle deregulation and modulate cell proliferation.

Pituitary tumors are usually benign but can occasionally exhibit hormonal and proliferative behaviors. Dysregulation of the G1/S restriction point largely contributes to the over-proliferation of pituitary tumor cells. F-box protein S-phase kinase-interacting protein-2 (SKP2) reportedly targets and inhibits the expression of p27Kip1, a well-known negative regulator of G1 cell cycle progression. In this study, SKP2 expression was found to be upregulated while p27Kip1 expression was determined to be downregulated in rat and human pituitary tumor cells. Furthermore, SKP2 knockdown induced upregulation of p27Kip1 and cell growth inhibition in rat and human pituitary tumor cells, while SKP2overexpression elicited opposite effects on p27Kip1 expression and cell growth. The expression of microRNA-186 (miR-186) was reported to be reduced in pituitary tumors. Online tools predicted SKP2 to be a direct downstream target of miR-186, which was further confirmed by luciferase reporter gene assays. Moreover, miR-186 could modulate the cell proliferation and p27Kip1-mediated cell cycle alternation of rat and human pituitary tumor cells through SKP2. As further confirmation of these findings, miR-186 and p27Kip1 expression were downregulated, while SKP2 expression was upregulated in human pituitary tumor tissue samples; thus, SKP2 expression negatively correlated with miR-186 and p27Kip1 expression. In contrast, miR-186 expression positively associated with p27Kip1 expression. Taken together, we discovered a novel mechanism by which miR-186/SKP2 axis modulates pituitary tumor cell proliferation through p27Kip1-mediated cell cycle alternation.

Gestational diabetes alters the expression of genes involved in Sertoli cells maturation in testis tissue from adult rat offspring

Introduction: Previous Studies indicate that both type 1 and 2 diabetes mellitus have adverse effects on the male reproductive system. Furthermore, it has recently shown that induced gestational diabetes significantly reduces the Sertoli cells number in the rat offspring. This study was done to evaluate the effect of gestational diabetes mellitus (GDM) on the expression of genes involved in Sertoli cell proliferation and maturation in the adult rat offspring. Material and Methods: To test this hypothesis, 12 Wistar rat dams were randomly allocated to control and diabetic groups. The diabetic group received 40 mg/kg/body weight of streptozotocin on day 0 of gestation. After delivery, six offspring of each group at the age of 12 weeks scarified and testis tissue harvested. After RNA extraction, the expression of p27kip1, A-kinase anchoring protein 9, CX43, and aromatase genes in both groups was evaluated by quantitative real-time polymerase chain reaction. Results: Our data showed that the expression of all examined genes which are important in Sertoli cells maturity and function were lower in GDM offspring. p27kip1 and aromatase were significantly downregulated in GDM offspring by 57% and 41%, respectively (P * 0.05*). Discussion and Conclusion: In summary, we provide evidence that GDM may adverse effects on the male reproductive system in the offspring by alterations in the expression of genes responsible for Sertoli cell proliferation and differentiation.

Resistance to ERK1/2 pathway inhibitors; sweet spots, fitness deficits and drug addiction.

MEK1/2 inhibitors are clinically approved for the treatment of BRAF-mutant melanoma, where they are used in combination with BRAF inhibitors, and are undergoing evaluation in other malignancies. Acquired resistance to MEK1/2 inhibitors, including selumetinib (AZD6244/ARRY-142866), can arise through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. We have found that BRAFV600E amplification and selumetinib resistance are fully reversible following drug withdrawal. This is because resistant cells with BRAFV600E amplification become addicted to selumetinib to maintain a precise level of ERK1/2 signalling (2%-3% of total ERK1/2 active), that is optimal for cell proliferation and survival. Selumetinib withdrawal drives ERK1/2 activation outside of this critical “sweet spot” (~20%-30% of ERK1/2 active) resulting in a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death with features of autophagy; these terminal responses select against cells with amplified BRAFV600E. ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of selumetinib resistance. Growth of selumetinib-resistant cells with BRAFV600E amplification as tumour xenografts also requires the presence of selumetinib to “clamp” ERK1/2 activity within the sweet spot. Thus, BRAFV600E amplification confers a selective disadvantage or “fitness deficit” during drug withdrawal, providing a rationale for intermittent dosing to forestall resistance. Remarkably, selumetinib resistance driven by KRASG13D amplification/upregulation is not reversible. In these cells ERK1/2 reactivation does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to traditional chemotherapy agents. Our results reveal that the emergence of drug-addicted, MEKi-resistant cells, and the opportunity this may afford for intermittent dosing schedules (“drug holidays”), may be determined by the nature of the amplified driving oncogene (BRAFV600E vs. KRASG13D), further exemplifying the difficulties of targeting KRAS mutant tumour cells.

The administration of peroxisome proliferator-activated receptors α/γ agonist TZD18 inhibits cell growth and induces apoptosis in human gastric cancer cell lines.

This study is to investigate the effects of a novel peroxisome proliferator-activated receptor (PPAR) α/γ dual agonist TZD18 on cell growth, apoptosis, caspase activity, mitochondrial membrane potential, cytochrome c release, and apoptotic-related protein expression in MKN-45 cells. 3-(4, 5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay against various human cancer cell lines was performed to investigate the whether TZD18 could in reduce the proliferation rates of cancer cells. The percentages of apoptotic cells and mitochondrial membrane potential level were determined by flow cytometry. The subcellular localization of cytochrome c was examined by immunofluorescence microscopy. Western blotting assay was performed to reveal the expression of apoptosis-related proteins. The results showed that the administration of TZD18 could inhibit the growth of MKN-45 cells in a dose- and time-dependent manner. In addition, the apoptotic ratio increased sharply along with a significant increase of caspase activities, mitochondrial membrane potential, and cytochrome c release following TZD18 exposure. The expression of Bax and p27kip1 increased significantly, whereas the expression level of Bcl-2 protein was downregulated. These results indicated that the administration of PPAR α/γ agonist TZD18 may inhibit cell growth by inducing the apoptotic process in MKN-45 cells.

Herpesvirus of Turkeys (Meleagridis Herpesvirus 1) Encodes a Functional MicroRNA-221 Homolog with High Sequence Conservation

Herpesviruses account for most of the known virus-encoded miRNAs. Herpesvirus of turkey (HVT), a non-pathogenic avian herpesvirus used as an avian vaccine and viral vector, encodes 28 mature miRNAs. This included HVT-miR-H14-3p that showed almost identical sequence to gga-miR-221, suggesting that it is pirated from the avian host. Although the functional homolog between the two miRNAs has been proposed based on the sequence similarity, the direct experimental evidence is still lacking. In this report, we provide the evidence for the first time that HVT-miR-H14-3p is indeed a gga-miR-221 homolog through modulating the expression of p27Kip1, a known target of miR-221 by binding to its 3’UTR. We also created an HVT-miR-H14-3p deletion virus and show that this miRNA is not essential for in vitro replication.

MiR-92b targets p57kip2 to modulate the resistance of hepatocellular carcinoma (HCC) to ionizing radiation (IR) -based radiotherapy

Hepatocellular carcinoma (HCC) is one of the most common digestive system malignant tumors. Due to the resistance to radiotherapy, the prognosis in patients with HCC is poor. Based on previous studies and online tools prediction, we hypothesized that miR-92b, which was reported to promote HCC cell proliferation, might bind to p57kip2, a well-known tumor suppressor, to modulate the radioresistance of HCC to ionizing radiation (IR) -based radiotherapy. In the present study, a higher miR-92b expression in HCC tissues and cell lines was observed; a high miR-92b expression was correlated with poorer prognosis in patients with HCC. The overexpression of miR-92b enhanced the radioresistance of HCC to IR treatment by promoting cancer cell proliferation, attenuating cell apoptosis and remove IR-induced cell cycle at G2/M phase. Through directly binding to the 3′-UTR of p57kip2, miR-92b negatively regulated the protein levels of p57kip2; miR-92b inhibition enhanced the cell effect of IR on HCC cells, which could be attenuated by the p57kip2 knockdown, in other words, miR-92b modulated the radioresistance of HCC to IR-based radiotherapy through p57kip2. Taken together, miR-92b inhibits p57kip2 expression in HCC tissues and cell lines, thus enhancing the radioresistance of HCC to IR-based radiotherapy; targeting miR-92b to rescue p57kip2 expression in HCC might help sensitive HCC cells to IR-based radiotherapy.

P27Kip1 down-regulation as achieved by two clinically feasible means did not induce proliferation of supporting cells in the rat neonatal cochlea in vivo

In mammals, the cochlear sensory epithelium becomes quiescent early during development. After the first postnatal week, there is no cell replacement or proliferation, and severe damage leads to permanent deafness. Supporting cells’ trans-differentiation has been suggested as a way to regenerate cochlear hair cells after damage. However, they are also needed for proper functionality. Cdkn1b (p27Kip1) participates in the cochlear terminal mitosis state achieved during development. Its expression is maintained in adult supporting cells and its postnatal deletion has induced cochlear proliferation in vitro and in vivo. Therefore, its manipulation has been proposed as a feasible way to induce proliferation of supporting cells after birth. Nevertheless, the literature is scarce regarding feasible methods to directly decrease p27Kip1 in the clinical domain. The effects of p27Kip1 knockdown using viral vectors are not completely elucidated and no pharmacological approaches to decrease p27Kip1 in the cochlea have been tested in vivo before. This study explores the ability of p27Kip1 messenger knockdown and pharmacological transcriptional inhibition to induce proliferation of supporting cells in the P0 neonatal rat cochlea in vivo. Respectively, lentiviral vectors transducing shRNA against p27Kip1 were administered into the scala media or Alsterpaullone 2-Cyanoethyl into the round window niche. Cell markers and gene expression were assessed through immunostaining and qRT-PCR. Despite both methods significantly decreasing p27Kip1 expression in vivo, signs of toxicity in the organ of Corti were not found; however, relevant proliferation was not found either. Finally, cochlear damage was added to increase the response in vitro, achieving only a mild to moderate proliferation induction. We conclude that our approaches were not able to stimulate the recall of supporting cell proliferation despite significantly decreased p27Kip1 levels in vivo. Considering the evaluation of the cochlea at a very responsive stage, we propose that the level of isolated modification of p27Kip1 expression in living mammals achievable through these approaches is insufficient to induce proliferation of supporting cells. Future proliferation induction experiments in the cochlea should study other methods and genes.

Effects of paclitaxel intervention on pulmonary vascular remodeling in rats with pulmonary hypertension.

The aim of the present study was to investigate the effects of paclitaxel (PTX), at a non-cytotoxic concentration, on pulmonary vascular remodeling (PVR) in rats with pulmonary hypertension (PAH), and to explore the mechanisms underlying the PTX-mediated reversal of PVR in PAH. A total of 36 rats were divided into control group (n=12), model group (n=12) receiving a subcutaneous injection of monocrotaline (60 mg/kg) in the back on day 7 following left pneumonectomy and PTX group (n=12) with PTX (2 mg/kg) injection via the caudal vein 3 weeks following establishing the model. The degree of PVR among all groups, as well as the expression levels of Ki67, p27Kip1 and cyclin B1, were compared. The mean pulmonary artery pressure, right ventricular hypertrophy index [right ventricle/(left ventricle + septum) ratio] and the thickness of the pulmonary arterial tunica media in the model group were 58.34±2.01 mmHg, 0.64±0.046 and 65.3±3.3%, respectively, which were significantly higher when compared with 23.30±1.14 mmHg, 0.32±0.028 and 16.2±1.3% in the control group, respectively (P<0.01). The mean pulmonary artery pressure, right ventricular hypertrophy index and thickness of the pulmonary arterial tunica media in the PTX group were 42.35±1.53 mmHg, 0.44±0.029 and 40.5±2.6%, respectively, which were significantly lower when compared with the model group (P<0.01). Compared with the control group, the expression levels of Ki67 and cyclin B1 in the model group were significantly increased (P<0.01), while p27Kip1 expression was significantly reduced (P<0.01). Following PTX intervention, the expression levels of Ki67 and cyclin B1 were significantly reduced when compared with the model group (P<0.01), while p27Kip1 expression was significantly increased (P<0.01). The results of the present study suggest that PTX, administered at a non-cytotoxic concentration, may reduce PAH in rats, and prevent the effects of PVR in PAH. These effects of PTX may be associated with increased expression of p27Kip1 and decreased expression of cyclin B1.

Knockdown of nucleophosmin 1 suppresses proliferation of triple-negative breast cancer cells through activating CDH1/Skp2/p27kip1 pathway.

BackgroundNPM1 is a multifunctional phosphoprotein that commutes between the cytoplasm and nucleus in cell cycle process, which appears to be actively involved in tumorigenesis. Herein, we sought to investigate the possible role and prognostic value of NPM1 in triple-negative breast cancer (TNBC).MethodsAn array of public databases, including bc-GenExMiner v4.0, GOBO, GEPIA, UAL-CAN, ONCOMINE database and Kaplan-Meier plotter, were used to investigate the expression feature and potential function of NPM1 in TNBC. Immunohistochemistry, immunofluorescence, proliferation and colony formation, flow cytometry and western-blotting assays were used to analyze and verify the function and relevant mechanism of NPM1 in TNBC tissues and cells.ResultsAccording to analysis from bc-GenExMiner, the expression level of NPM1 was significantly higher in basal-like subtypes than luminal-A, HER-2 or normal-like subtypes of breast cancer (P<0.0001). GOBO database analysis indicated that the expression of NPM1 in basal-A or basal-B was significantly higher than luminal-like breast cancer cells. Immunohistochemistry assay in 52 TNBC tissue samples showed that positive expression of Ki-67 was 93.5% in the high-NPM1-expression group and 66.7% in the low-NPM1-expression group, respectively (P=0.032). Proliferation and colony formation assays demonstrated that inhibition of NPM1 suppressed cell growth by approximately 2-fold and reduced the number of colonies by 3-4-fold in MDA-MB-231 and BT549 cells. Moreover, inhibition of NPM1 in MDA-MB-231 and BT549 cells increased the percentage of cells at G0/G1 phase and decreased the percentage of cells at both S and G2/M phase, as compared with control counterparts. Western-blotting results showed that down-regulation of NPM1 could elevate CDH1 and p27kip1 expression, while decrease Skp2 expression both in MDA-MB-231 and BT549 cells. In addition, high mRNA expression of NPM1 correlated with shorter RFS (HR=1.64, P=0.00013) and OS (HR=2.45, P=0.00034) in patients with TNBC.ConclusionsNPM1 is significantly high expressed basal-like/triple-negative breast cancer and is correlated with shorter RFS and OS in this subset of patients. Knockdown of NPM1 impairs the proliferative capacity of TNBC cells via activation of the CDH1/Skp2/p27kip1 pathway. Targeting NPM1 is a potential therapeutic strategy against TNBC.

Dexamethasone Accelerates the Transition of Human BFU-E to CFU-E and Enhances CFU-E Proliferation through Cell Cycle Regulation

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is frequently managed with glucocorticoids to increase red cell mass. Despite sustained effective clinical use for many decades in DBA and other anemias, we still do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. In order to improve the clinical management of patients with DBA, we studied the mechanisms of action of dexamethasone (Dex) on erythroid progenitors.To study the effects of Dex on erythroid progenitor biology, we employed a serum-free erythroid culture system to differentiate primary human CD34+ cells isolated from the peripheral blood of adults or DBA patients as well as cord blood samples. We found that Dex increases the total proliferation of CD34+ cells from adult peripheral blood but not in cord blood over 14 days in culture. Dex treatment also led to an acceleration of the BFU-E to CFU-E transition in peripheral blood with a minimal effect in cord blood. In methylcellulose colony forming assays, peripheral blood derived CFU-E treated with Dex for 24hrs produced more colonies or larger colonies than the untreated controls while a lesser effect was seen in cord blood derived CFU-E. Both assays revealed that Dex treatment enhanced self-renewal of peripheral blood derived CFU-E.We also noted that BFU-E and CFU-E differentially express the glucocorticoid receptor (GR) isoforms, GRα and GRβ. BFU-E expressed comparable levels of GRα and the dominant-negative GRβ, whereas only the GRα transcript was identified in CFU-E. We observed similar expression patterns for GR between peripheral and cord blood derived progenitors. Importantly, following Dex treatment, GRα was shown to translocate to the nucleus of erythroid progenitors derived from peripheral blood derived CD34+ cells but not in the cord blood derived progenitors.When we examined the cell cycle progression of sorted human erythroid progenitors, CFU-E and BFU-E, we observed a G0/G1 arrest of peripheral blood CFU-E treated with Dex compared to untreated CFU-E. In marked contrast, no such change was noted for either Dex treated BFU-E from peripheral blood or for CFU-E or BFU-E from cord blood. In addition, we found an increase in the expression of cell cycle regulators p57Kip2 and p27Kip1 in Dex treated peripheral blood CFU-E but no such increased expression was seen in cord blood CFU-E.When peripheral blood derived erythroid progenitors were treated with olomoucine (Olo), a small molecule CDK inhibitor, we observed an acceleration of the BFU-E to CFU-E transition similar to that induced by Dex. Upon co-treatment with Dex and Olo, there was a synergistic effect in the accelerated formation of CFU-E population from BFU-Es. To validate this finding of the dependence of CFU-E on cell cycle regulators, we transduced peripheral blood derived CD34+ cells with a lentiviral shRNA construct to knockdown p57 expression. Following successful knockdown of the expression levels of p57, the numbers of CFU-E indeed decreased implying a role for cell cycle in defective transition of BFU-E to CFU-E.Erythroid progenitors derived from cultures of primary CD34+ cells from DBA patients were also studied. Importantly, we noted increased baseline expression of p57Kip2 and p27Kip1 in erythroid progenitors from steroid resistant DBA patients and in contrast to controls, their expression did not increase following Dex treatment.Our findings provide novel mechanistic insights into the regulation of human erythropoiesis by Dex. We have shown that alterations in cell cycle regulation of erythroid progenitors plays a key role in the effects of glucocorticoids on red blood cells. Furthermore, this work has direct relevance for the development of improved therapeutic treatment strategies for patients with bone marrow failure syndromes such as DBA. DisclosuresNo relevant conflicts of interest to declare.

MiR-377-5p inhibits lung cancer cell proliferation, invasion, and cell cycle progression by targeting AKT1 signaling.

Lung carcinoma is the most common type of malignant tumors globally, and its molecular mechanisms remained unclear. With the aim to investigate the effects of microRNA (miR)-377-5p on the cell development, invasion, metastasis, and cycle of lung carcinoma, this study was performed. We evaluated miR-377-5p expression levels in lung cancer tissues and cell models. Cell viability, proliferation, migration, invasion abilities, and cell cycle distribution were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, crystal violet, transwell, and flow cytometry assay. Furthermore, expression levels of protein kinase B α subunit (AKT1) and proteins related to cell cycle and epithelial-mesenchymal transition (EMT) were assessed using Western blot analysis and quantitative real-time polymerase chain reaction. These results suggested that miR-377-5p was downregulated in vivo and in cell models, and miR-377-5p overexpression inhibited cell viability, proliferation, migration, invasion, and induced cell-cycle arrest. In addition, as a target of miR-377-5p, AKT1 alleviated the decreases of cell viability, proliferation, migration, invasion, the S-phase cells, the expression of cyclin D1, fibronectin, and vimentin, as well as the increases of the G0/G1-phase cells, the expression of Foxo1, p27 kip1 , p21 Cip1 and E-cadherin when miR-377-5p overexpressed. In conclusion, miR-377-5p inhibited cell development and regulated cell cycle distribution and EMT by targeting AKT1, which provided a theoretical basis for further study of lung carcinoma therapeutics.

Canonical BMP signaling in tubular cells mediates recovery after acute kidney injury

Bone morphogenetic protein (BMP) signaling has been shown to modulate the development of renal fibrosis in animal models of kidney injury, but the downstream mediators are incompletely understood. In wild-type mice, canonical BMP signaling mediated by SMAD1/5/8 transcription factors was constitutively active in healthy renal tubules, transiently down-regulated after ischemia reperfusion injury (IRI), and reactivated during successful tubular regeneration. We then induced IRI in mice with a tubular-specific BMP receptor 1A (BMPR1A) deletion. These mice failed to reactivate SMAD1/5/8 signaling in the post-ischemic phase and developed renal fibrosis after injury. Using unbiased genomic analyses, we identified three genes encoding inhibitor of DNA-binding (ID) proteins (Id1, Id2, and Id4) as key targets of BMPR1A-SMAD1/5/8 signaling. BMPR1A-deficient mice failed to re-induce these targets following IRI. Instead, BMPR1A-deficiency resulted in activation of pro-fibrotic signaling proteins that are normally repressed by ID proteins, namely, p38 mitogen-activated protein kinase and cell cycle inhibitor p27. These data indicate that the post-ischemic activation of canonical BMP signaling acts endogenously to repress pro-fibrotic signaling in tubular cells and may help to prevent the progression of acute kidney injury to chronic kidney disease.

RelB plays an oncogenic role and conveys chemo-resistance to DLD-1 colon cancer cells

BackgroundNuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemo-sensitivity. Recent studies suggest that RelB, the key subunit of the alternative NF-κB pathway, plays a critical role in the progression of diverse human malignancies. However, the significance of RelB in colorectal cancer (CRC) remains unclear. Here, we systematically explored the functions of the alternative NF-κB subunit RelB in colon cancer cells and its underlying mechanism.MethodsStably transfected RelB-shRNA DLD-1 cells were established using Lipofectamine 2000. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Cell growth was monitored by an x-Celligence system. Cell proliferation was analyzed by a CCK-8 and a Brdu proliferation assay. Response to 5-FU was assessed by an x-Celligence system. Cell apoptosis and cell cycle was detected using flow cytometry analyses. Cell migration and invasion abilities were detected by an x-Celligence system, Transwell inserts, and wound-healing assays. RelB expression and its clinical significance were analyzed using the CRC tissue microarray. The expression of NF-κB signaling subunits, AKT/mTOR signaling molecules, cell cycle related proteins, MMP2, MMP9, and Integrin β-1 were measured by Western blotting analyses.ResultsThe RelB-silencing inhibited cell growth of DLD-1 cells. The RelB-silencing exerted the anti-proliferative by downregulation of AKT/mTOR signaling. The RelB-silencing caused G0–G1 cell cycle arrested likely due to decreasing the expression of Cyclin D1 and CDK4, concomitant with increased expression of p27Kip1. The RelB-silencing enhanced cytotoxic effect of 5-FU and induced cell accumulation in S-phase. The RelB-silencing impaired the migration and invasion potential of DLD-1 cells, which was related to downregulation of MMP2, MMP9, and Integrin β-1. Importantly, the RelB expression was correlated with depth of tumor invasion, lymph node metastasis, metastasis stage, and pTNM stage. High-RelB expression was significantly correlated with poor overall survival in CRC patients.ConclusionOur studies here provided evidence that RelB plays an oncogenic role and conveys chemo-resistance to 5-FU. RelB can be considered as an independent indicator of prognosis in CRC.

A functional genetic variant in GAS5 lncRNA (rs145204276) modulates p27Kip1 expression and confers risk for gastric cancer

Gastric cancer is the fifth most common cancer type worldwide [1]. Established risk factors are environmental factors (alcohol, H pylori), atrophic gastritis, intestinal metaplasia and genetic fact...