CCND1 mRNA Expression Research Articles

71 - Insulin (Ins2) Is Expressed in the Brain and Its Global Deletion Leads to Learning Deficits, Anxiety and Reduced Hippocampal Cyclin D1 Expression in Female Mice

Insulin dysregulation independently underlies diabetes and Alzheimer’s Disease (AD). The former has also been shown to be a risk factor for the latter. The ancestral insulin gene (Ins2), but not the Ins1 gene, is transcribed locally within the brain in mice. We confirmed that neuronal expression of Ins2 is abundant within the hippocampus, a region with established roles in learning and memory. Public single cell RNA sequencing datasets were analyzed to determine the specific cell types where Ins2 is expressed in the mouse brain. To determine how brain-derived insulin influences hippocampal function, learning and memory, we eliminated Ins2 with germline Ins2 knockouts (Ins2-/-). Glucose homeostasis was maintained owing to the presence of wildtype Ins1 alleles. Using the Morris water maze, we found that learning and memory performance of female Ins2-/- mice was significantly impaired relative to wild-type mice, whereas the performance of male Ins2-/- and wild-type mice did not differ. During acquisition training, the swim-speed in female Ins2-/- was faster than wild-type mice, suggesting increased stress reactivity to escape from water. Indeed, anxiety-like behaviour was increased in female mice as assessed by the open-field test. RNA sequencing of isolated hippocampi revealed that female Ins2-/- mice had significantly reduced Cyclin D1 (Ccnd1) mRNA expression compared with littermate controls. This observation points to a possible defect in hippocampal neurogenesis, a physiological hallmark of impaired memory and emotionality implicated in both diabetes and AD. Together, these data suggest that Ins2 plays sex- and brain region-specific roles in neuronal function and, perhaps, adult neurogenesis.

Evaluation of miR-34a Effect on CCND1 mRNA Level and Sensitization of Breast Cancer Cell Lines to Paclitaxel.

Background:A growing body of literature has revealed the effective role of miR-34a, as a tumor suppressor and regulator of expression of multiple targets in tumorigenesis and cancer progression. This study aimed at evaluating the potential effects of miR-34a alone or in combination with paclitaxel on breast cancer cells. Methods:After miR-34a transduction by lentiviral vectors in two MCF-7 and MDA-MB-231 cell lines of breast cancer, effects of the elevated expression of miR-34a in the cell viability and the cell proliferation were determined using MTT assay in treated and untreated cells with paclitaxel. The mRNA level of the CCND1 gene was then measured in the two cell lines using the qRT-PCR assay. Finally, the influence of miR-34a and paclitaxel on apoptosis and cell cycle progression were examined by flow cytometry. Results:The CCND1 mRNA expression levels were significantly down-regulated by overexpressed lentiviral miR-34a in MCF-7 and MDA-MB-231 cells. Combined treatment by miR-34a and paclitaxel reduced the cell viability and proliferation compared to single-drug treatment. In addition, the cell cycle arrest appeared at two phases by the combination of miR-34a and paclitaxel in MDA-MB-231 cells. Conclusion:Our results suggest that miR34a, in combination with paclitaxel, has a potential for decreasing the cell viability and proliferation. Moreover, it can reduce the expression of CCND1 mRNA independent of the paclitaxel effect.

CCND1 and FGFR1 gene amplifications are associated with reduced benefit from aromatase inhibitors in metastatic breast cancer.

Endocrine therapy is a mainstay for the treatment of hormone receptor-positive breast cancer (BC); however, only a fraction of patients experience a pronounced response to antagonists of estrogen signaling. There is a need to identify predictors for efficacy of this treatment. This study included 138 patients with newly diagnosed metastatic BC, who received upfront endocrine therapy. Archival biopsy specimens were tested for CCND1 and FGFR1 gene amplification and mRNA expression by PCR-based methods. CCND1 and FGFR1 amplification was detected in 24 (17.9%) and 28 (20.9%) of 134 evaluable cases, respectively; 9 carcinomas had concurrent alterations of these two genes. Presence of amplification in at least one locus was more common in tumors of higher grade (p = 0.018) and was associated with higher Ki-67 proliferation index (p = 0.036). CCND1 gene amplification was associated with shorter progression-free survival (PFS) in patients receiving aromatase inhibitors (AI) [16.0months vs. 32.4months, HR = 3.16 (95% CI 1.26-7.93), p = 0.014]. FGFR1 status did not significantly affect PFS of AI-treated women; however, objective response to AI was observed less frequently in FGFR1-amplified BC as compared to cases with normal FGFR1 copy number [2/15 (13.3%) vs. 22/46 (47.8%), p = 0.031]. Meanwhile, CCND1/FGFR1 gene status did not influence the outcome of tamoxifen-treated patients. Presence of CCND1 and/or FGFR1 amplification is associated with worse outcomes of AI therapy in patients with metastatic BC.

CCND2 mRNA Expression Is Correlated With R-CHOP Treatment Efficacy and Prognosis in Patients With ABC-DLBCL.

In this study we investigated whether the expression of cyclin D2 (CCND2) mRNA in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) was correlated with the efficacy of Rituximab combined with chemotherapy (R-CHOP) treatment and patient prognosis. Tissue microarray and RNAscope in situ hybridization were used to detect CCND2 mRNA expression in 117 ABC-DLBCL tumor tissues and associations between CCND2 expression and progression-free survival was analyzed. We also downloaded data from the Gene Expression Omnibus database to analyze CCND2 expression and the efficacy of R-CHOP treatment and prognosis of patients with newly diagnosed ABC-DLBCL. The positive expression rate of CCND2 mRNA in patients with ABC-DLBCL was 41%. Progression-free survival was significantly lower in patients with positive rather than those negative CCND2 expression (P = 0.005). Further, R-CHOP treatment was significantly more effective for patients with ABC-DLBCL with high CCND2 mRNA expression than those with low expression (P = 0.039). Multivariate regression analysis suggested that high CCND2 expression was an independent prognostic risk factor for progression-free survival for patients with ABC-DLBCL who achieved complete remission after R-CHOP treatment. CCND2 expression in ABC-DLBCL tumors, detected by RNA in situ hybridization, is closely related to the curative effect of R-CHOP and patient prognosis following R-CHOP treatment, and represents a potential biomarker for treatment efficacy and prognostic evaluation in patients with ABC-DLBCL.

Induction of t(11;14) IgH enhancer/promoter-cyclin D1 gene translocation using CRISPR/Cas9.

Chromosomal translocation is a key process in the oncogenic transformation of somatic cells. Previously, artificial induction of chromosomal translocation was performed using homologous recombination-mediated loxP labeling of target regions followed by Cre-mediated recombination. Recent progress in genome editing techniques has facilitated the easier induction of artificial translocation by cutting two targeted genome sequences from different chromosomes. The present study established a system to induce t(11;14)(q13;q32), which is observed primarily in multiple myeloma (MM) and involves the repositioning of the cyclin D1 (CCND1) gene downstream of the immunoglobulin heavy chain (IgH) constant region enhancers by translocation. The placing of tandem gRNAs designed to cut both the IgH Eµ and CCND1 15-kb upstream regions in lentiCRISPRv2 enabled the induction of chromosomal translocation in 293T cells, with confirmation by translocation-specific PCR and fluorescence in situ hybridization probing with IgH and CCND1. At the translocation junctions, small deletions and the addition of DNA sequences (indels) were observed in several clones. Cloned cells with t(11;14) exhibited slower growth and lower CCND1 mRNA expression compared to the parent cells, presenting the opposite phenomena induced by t(11;14) in MM cells, indicating that the silent IgH gene juxtaposed to CCND1 may negatively affect CCND1 gene expression and cell proliferation in the non-B lymphocyte lineage. Therefore, the present study achieved the induction of silent promoter/enhancer translocation in t(11;14)(q13;q32) as a preparatory experiment to study the role of IgH constant region enhancer-driven CCND1 overexpression in oncogenic transformation processes in B lymphocytes.

SAT-353 The Synthetic Vitamin D Analog EB1089 Restricts Adrenocortical Tumor Proliferation in NCI-H295 Xenograft Model

The vitamin D (VD) receptor (VDR) is underexpressed in adult and pediatric adrenocortical tumors (ACT), especially in carcinomas (ACC). VDR activation by VD represses B-catenin signaling in different types of tumors that like ACT, present aberrant B-catenin expression. EB1089 is a potent synthetic VD analog that presents lower calcemic effects and is well tolerated at therapeutic doses. Since there is no effective adjuvant target-therapy for patients with ACT, we evaluated the effects of EB1089 in adrenocortical tumor proliferation. Methods:In vitro, we treated NCI-H295 (B-catenin mutant/activated) and Y1 (normal B-catenin) ACC cells with EB1089 (10-7M) and evaluated VDR and B-catenin signaling (qPCR) and cell proliferation (MTS). In vivo, we bilaterally inoculated subcutaneously 10 female NGS mice (8 week-old) with NCI-H295 cells (2.5x106 / 100 mcL). We assessed body weight (BW) and tumor volume (TuV; d2D/2) weekly. Once the TuV reached 100 mm3, we randomized the animals for EB1089 (n=5; 0.8mcg/kg BW) or vehicle [Control group (CG) n=5; 80% propylene glycol-20% PBS] treatment (sc injection; 5 days/week during 5 weeks). At the end of the protocol, mice were sacrificed and each tumor excised, weighted and fixed for pathological evaluation (H&E). Blood samples were taken for serum calcium (colorimetry), cortisol and testosterone (RIA) assessment. Data were presented as percentage or mean ± SEM. Results: In NCI-H295 cells the treatment with EB1089 (48h) increased VDR mRNA expression (47%; p=0.01) and activated its target gene CYP24A1 (p<0.0001). B-catenin expression and signaling decreased, as shown by reduced CTNNB1 (-24%; p=0.001), MYC (-40%; p=0.0001), CCND1 (-38%; p=0.02), and DKK3 (-40%; p=0.02) mRNA expression. Cell proliferation reduced after 96h (-15%; p=0.001). None of these observations were present in Y1 cells. In vivo, EB1089 significantly restricted tumor growth starting after 2 weeks of treatment (TuV: 284 ± 52 vs. 156 ± 20 mm3; p=0.05). At the end of the experiment TuV was ~50% lower in EB1089 xenografts (683 ± 345 vs. 368 ± 211 mm3; p=0.03). TuV and tumor weight correlated positively (r=0.88; p<0.0001). Both, EB1089 and CG tumors presented highly proliferative areas (pleomorphism, mitosis and few apoptotic bodies). However, EB1089 xenografts presented large necrotic areas. BW (p=0.98) and calcium levels (9.96 ± 0.4 vs. 11.3 ± 0.3 mg/dL; p=0.06) did not differ between groups. EB1089 mice presented reduced serum testosterone (76.7 ± 11.6 vs. 39 ± 5.6 ng/dL; p=0.02) and undetectable cortisol levels (CG: 2 ± 1.2 mcg/dL). Conclusion: The activation of VDR signaling by EB1089 inhibited B-catenin activated ACT proliferation. Furthermore, in vivo, EB1089 reduced tumor steroid secretion and did not result in important VD side effects like significant hypercalcemia and weight loss. VD analogs have antitumor activity and may emerge as an adjuvant therapy for patients with ACT.

Elimination of Wnt secretion from hepatic stellate cells impairs hepatocyte proliferation after partial hepatectomy

Wnt‐dependent β‐catenin activation contributes to hepatocyte proliferation following 2/3 partial hepatectomy (PH) through regulation of Cyclin‐D1 (Ccnd1) expression, as we have previously shown using β‐catenin or Wnt coreceptors low‐density lipoprotein receptor‐related protein 5/6 conditional knockouts (KO). However, given the redundancy between the 19 mammalian Wnt proteins and various cell sources for Wnt secretion, the precise mechanisms of Wnt signaling in liver regeneration (LR) are not completely understood. Recently, using endothelial cell (EC)‐specific deletion of Wntless (Wls), a cargo receptor required for Wnt secretion, we have reported that sinusoidal ECs secrete Wnts (Wnt2 and Wnt9b) to activate β‐catenin in hepatocytes and enhance Ccnd1 expression, which is indispensable for proliferation of hepatocytes during LR after PH. Alternatively, conditional deletion of Wls in hepatocytes or cholangiocytes did not impact LR, whereas Wls deletion in macrophages only marginally affected LR post‐PH. Here, to investigate the roles for hepatic stellate cell (HSC)‐secreted Wnts in LR, we have characterized HSC‐Wls‐KO, generated by interbreeding Wls‐floxed and Lecithin Retinol Acyl Transferase (LRAT)‐driven Cre (Lrat‐cre) transgenic mice. HSC‐Wls‐KO mice showed no effect on liver weight or β‐catenin‐dependent pericentral gene expression at baseline. However, hepatocyte‐specific Cyclin‐D1 expression was abolished in baseline KO liver, whereas Cyclin‐D1 expression in non‐parenchymal cells was intact in KO liver. After PH, HSC‐Wls‐KO showed diminished protein and mRNA expression of Ccnd1, Ccnb1 and Ccna2 at 24–40 hour (h) post‐PH, but displayed slight increases at 72h, which led to a lower number of BrdU+ hepatocytes at 24 and 40h but a rebound increase by 72h. Interestingly, among several Wnts, HSC‐Wls‐KO livers at 24h post‐PH showed decrease of Wnt3 and Wnt9b mRNA compared to controls, although expression became comparable at 40–72h post‐PH.ConclusionAt baseline, HSCs secrete Wnt ligands essential for Cyclin‐D1 expression in midzonal hepatocytes but not for β‐catenin‐dependent hepatic zonation. Following PH, HSCs‐derived Wnts also contribute to Cyclin‐D1‐dependent hepatocyte proliferation during LR.Support or Funding InformationThe work was supported by NIH grants to S.P.M. (DK62277, DK100287, CA204586). S.P.M. is an Endowed Chair for Experimental Pathology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

8:2 Fluorotelomer alcohol causes G1 cell cycle arrest and blocks granulocytic differentiation in HL-60 cells.

Fluorotelomer alcohols (FTOHs) are fluorinated intermediates used in manufacturing specialty polymer and surfactants, with 8:2 FTOH the homologue of largest production. FTOHs were found to pose acute toxicity, hepatotoxicity, nephrotoxicity, developmental toxicity and endocrine-disrupting risks, whereas research regarding immunotoxicity and its underlying mechanism, especially on specific immune cells is limited. Here, we investigated the immunotoxicity of 8:2 FTOH on immature immune cells in an in vitro system. We observed that exposure of HL-60 cells, a human promyelocytic leukemic cell line, to 8:2 FTOH reduced cell viability in a dose- and time-dependent manner. In addition, 8:2 FTOH exposure caused G1 cell cycle arrest in HL-60 cells, while it showed no effect on apoptosis. Exposure to 8:2 FTOH inhibited the mRNA expression of cell cycle-related genes, including CCNA1, CCNA2, CCND1, and CCNE2. Moreover, exposure to 8:2 FTOH inhibited the mRNA expression of granulocytic differentiation-related genes of CD11b, CSF3R, PU.1, and C/EPBε in HL-60 cells . Furthermore, 8:2 FTOH exhibited no effect on intracellular ROS level, while hydralazine hydrochloride (Hyd), one reactive carbonyl species (RCS) scavenger, partially blocked 8:2 FTOH-caused cytotoxicity in HL-60 cells. Overall, the results obtained in the study show that 8:2 FTOH poses immunotoxicity in immature immune cells and RCS may partially underline its mechanism.

Abstract PD2-08: Ribociclib with endocrine therapy for premenopausal patients with hormone receptor-positive, HER2-negative advanced breast cancer: Biomarker analyses from the phase III randomized MONALEESA-7 trial

Abstract Background: In the MONALEESA-7 study (NCT02278120), ribociclib (RIB) + a non-steroidal aromatase inhibitor (NSAI)/tamoxifen (TAM) + goserelin (GOS) significantly improved progression-free survival (PFS) vs placebo (PBO) + NSAI/TAM + GOS in premenopausal patients (pts) with hormone receptor-positive (HR+), HER2-negative (HER2–) advanced breast cancer (ABC; hazard ratio 0.553; 95% confidence interval [CI] 0.441–0.694; p=0.0000000983). Here we present PFS data by baseline (prior to study treatment) tumor (primary and metastatic) Ki67, total Rb, and p16 protein expression, and CCND1 (cyclin D1), CDKN2A (p16), and ESR1 (estrogen receptor) messenger RNA (mRNA) levels. Methods: Pre-/perimenopausal pts (N=672; ≤1 line of prior chemotherapy and no prior endocrine therapy for ABC) were randomized 1:1 to either RIB (600 mg/day; 3-weeks-on/1-week-off) or PBO + an NSAI (letrozole [2.5 mg/day]/anastrozole [1 mg/day]) or TAM (20 mg/day) + GOS (3.6 mg every 28 days). The primary endpoint was local PFS, while correlation with biomarker expression was an exploratory endpoint. Baseline tumor tissue was evaluated for Ki67, Rb, and p16 protein expression by immunohistochemistry and CCND1, CDKN2A, and ESR1 gene expression by NanoString® nCounter. To assess correlations between protein/gene expression and PFS, pts were classified into prespecified low vs high expression subgroups; 14% of positively stained cells was used as a cutoff for Ki67, 10th percentile was used as a cutoff for total Rb, and median expression was used as a cutoff for all other proteins/genes. Results: Based on a data cutoff of August 20, 2017, PFS hazard ratios for most biomarker subgroups favored RIB + NSAI/TAM + GOS vs PBO + NSAI/TAM + GOS (Table). RIB + NSAI/TAMPBO + NSAI/TAM Events, n/NMedian PFS, monthsEvents, n/NMedian PFS, monthsHazard ratio; 95% CIKi67Low16/5627.522/49Not reached0.523; 0.270–1.013High52/11719.471/11712.60.495; 0.341–0.719Total RbLow8/1927.54/12Not reached0.239; 0.034–1.667High63/15123.889/15212.90.483; 0.342–0.681p16Low28/85Not reached41/7916.40.570; 0.348–0.935High35/7423.847/749.10.403; 0.250–0.651CCND1Low40/8819.151/9213.60.672; 0.442–1.022High36/9723.847/8314.60.381; 0.236–0.614CDKN2ALow29/85Not reached53/9515.60.476; 0.300–0.756High47/10019.445/8012.60.485; 0.310–0.758ESR1Low42/9722.145/8312.90.571; 0.362–0.901High34/8827.553/9214.60.568; 0.359–0.899 It is difficult to draw conclusions for total Rb protein expression due to the small sample size and limited number of events. RIB treatment benefit was numerically greater in pts with high vs low CCND1 mRNA expression levels. Conclusions: Generally consistent PFS benefits with RIB + NSAI/TAM + GOS vs PBO + NSAI/TAM + GOS were observed irrespective of baseline biomarker expression in cell cycle- and proliferation-related genes. Updated results will be presented at the meeting. Citation Format: Bardia A, Colleoni M, Campos-Gomez S, Jung KH, Wheatley-Price P, Kümmel S, Srimuninnimit V, Taylor D, Im Y-H, Hughes G, Diaz-Padilla I, Miller M, Su F, Tripathy D. Ribociclib with endocrine therapy for premenopausal patients with hormone receptor-positive, HER2-negative advanced breast cancer: Biomarker analyses from the phase III randomized MONALEESA-7 trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-08.

Knockdown of MALAT1 inhibits osteosarcoma progression via regulating the miR‑34a/cyclin D1 axis.

Long non-coding (lnc)RNAs have been demonstrated to be involved in the development of various types of cancers, such as osteosarcoma (OS). Long non-coding (lnc) RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) expression was reported to be highly expressed in OS and promoted the development of this disease; however, the underlying molecular mechanism by which MALAT1 promotes the progression of OS requires further investigation. In the present study, the expression of MALAT1 and miR-34a was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The abundance of cyclin D1 (CCND1) was detected by RT-qPCR and western blotting. Cell viability, migration and invasion were examined by MTT and Transwell assays. The interaction between miR-34a and MALAT1 or CCND1 was probed by a dual luciferase reporter assay and RNA immunoprecipitation. Xenograft tumor assay was performed to verify the roles of MALAT1 and miR-34a in tumor growth in vivo. The results demonstrated that MALAT1 and CCND1 mRNA expression levels were upregulated and miR-34a was downregulated in OS tissues and cells. Additionally, MALAT1 expression was correlated with tumor size, clinical stage and distant metastasis in patients with OS. In addition, MALAT1 promoted OS cell viability, invasion and migration, while MALAT1 silencing exhibited opposing effects. Moreover, MALAT1 functioned as a ceRNA to suppress miR-34a expression and in turn upregulate CCND1 in OS cells. Rescue experiments further demonstrated that MALAT1 knockdown partially reversed anti-miR-34a-mediated promotion on OS cell viability, migration and invasion; overexpression of CCND1 partially reversed the effects of MALAT1 silencing on OS progression. Furthermore, in vivo experiments also revealed that MALAT1 promoted OS tumor growth via miR-34a inhibition and upregulating the expression of CCND1. In conclusion, the present study suggested that MALAT1 exerted its oncogenic function in OS by regulating the miR-34a/CCND1 axis in OS, which may provide novel insight into the diagnosis and therapy for OS.

Quercetin inhibits proliferation of endometriosis regulating cyclin D1 and its target microRNAs in vitro and in vivo

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a major dietary flavonol found in diverse fruits and vegetables such as onions, cauliflower, apple skin, lettuce and chili peppers. In recent studies, quercetin is reported as a functional compound and shows a wide range of biological effects such as antioxidant, anti-inflammatory and antiangiogenic properties in obesity, diabetes, cardiovascular diseases and various cancers. However, to date, the therapeutic effect of quercetin on the progression of endometriosis, which is a common gynecological disease in reproductive-aged women and brings chronic pelvic pain and infertility, has not been examined in depth. Results of this study demonstrated that quercetin inhibited the proliferation and induced the cell cycle arrest in VK2/E6E7 and End1/E6E7 cells. Furthermore, it induced cell apoptosis with DNA fragmentation, loss of mitochondrial membrane potential and reactive oxygen species production. The effects accompanied down-regulation of ERK1/2, P38 MAPK and AKT signaling molecules. Additionally, the administration of quercetin indicated antiproliferative and anti-inflammatory effects on endometriosis autoimplanted mouse models. The mRNA expression of Ccnd1 significantly decreased in response to quercetin intraperitoneal injection when compared to that in vehicle-treated mice. The knockdown of CCND1 mRNA attenuated the proliferation with sub-G0/G1 cell cycle arrest and increased the apoptosis of VK2/E6E7 and End1/E6E7 cells. Furthermore, the treatment of quercetin induced miR-503-5p, miR-1283, miR-3714 and miR-6867-5p related to CCND1 in both cell lines and also stimulated miR-503-5p and miR-546 expression in the mouse model. Hence, quercetin may potentially act as a natural therapeutic to reduce and treat human endometriosis.

RUNX2 Promotes Malignant Progression in Glioma.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. However, therapeutic strategies against malignant gliomas have not been completely established. Runt-related transcription factor 2 (Runx2) is an essential gene for skeletal development but its regulatory role in the malignant progression of glioma remains unclear. Here we investigated expression levels of RUNX2 in glioma tissues and its regulatory effects on aberrant growth of glioma cells. RUNX2 mRNA levels were higher in GBM tissues than that of normal brains or low-grade gliomas. RUNX2 protein was detected in five out of seven human GBM cell lines and its level was positively correlated with proliferative capacity. Stable transduction of dominant-negative Runx2 in rat-derived C6 glioma cells not only inhibited the promoter activity containing Runx2 response element, but also decreased mRNA expression levels of Runx2 target genes, such as Mmp13 and Spp1, as well as the proliferative capacity. Furthermore, transient introduction of Runx2-targeted siRNAs into C6 glioma cells significantly decreased mRNA expression levels of Mmp13 and Spp1 and the proliferative capacity. Furthermore, Runx2 knockdown suppressed both Ccnd1 mRNA expression and activation of the Ccnd1 promoter by forskolin, a PKA-activating reagent, in C6 glioma cells. Our results demonstrate that cross-talk between cAMP/PKA signaling and RUNX2 promotes a malignant phenotype of glioma cells.

Selection of internal references for qRT-PCR assays of human hepatocellular carcinoma cell lines.

Selecting internal references is important for normalizing the loading quantity of samples in quantitative reverse-transcription PCR (qRT-PCR). In the present study, a systematic evaluation of reference genes among nine hepatocellular carcinoma (HCC) cell lines was conducted. After screening the microarray assay data of ten HCC cell lines, 19 candidate reference genes were preselected and then evaluated by qRT-PCR, together with ACTB, GAPDH, HPRT1 and TUBB. The expression evenness of these candidate genes was evaluated using RefFinder. The stabilities of the reference genes were further evaluated under different experimental perturbations in Huh-7 and MHCC-97L, and the applicability of the reference genes was assessed by measuring the mRNA expression of CCND1, CCND3, CDK4 and CDK6 under sorafenib treatment in Huh-7. Results showed that TFG and SFRS4 are among the most reliable reference genes, and ACTB ranks third and acts quite well as a classical choice, whereas GAPDH, HPRT1 and TUBB are not proper reference genes in qRT-PCR assays among the HCC cell lines. SFRS4, YWHAB, SFRS4 and CNPY3 are the most stable reference genes of the MHCC-97L under the perturbations of chemotherapy, oxidative stress, starvation and hypoxia respectively, whereas YWHAB is the most stable one of Huh-7 under all perturbations. GAPDH is recommended as a reference gene under chemotherapy perturbations. YWHAB and UBE2B, TMED2 and TSFM, and GAPDH and TSFM are the two best reference genes under oxidative stress, starvation and hypoxia perturbations respectively. TSFM is stable in both cell lines across all the perturbations.

Role of MicroRNA-93 I in Pathogenesis of Left Ventricular Remodeling via Targeting Cyclin-D1.

BackgroundThe objective of this study was to identify the pathway responsible for ventricular remodeling.Material/MethodsWe collected remodeling myocardium tissue (n=18) and control myocardium tissue (n=22), and detected the expression of 4 miRNAs in these 2 groups using real-time PCR. We then searched the miRNA database online to find the candidate genes of miR-93. Real-time PCR and Western blot analysis were used to confirm the regulatory relationship.ResultsWe found that only miR-93 was decreased in remodeling myocardium tissue, and validated CCND1 to be the direct target gene of miR-93, with the “seed sequence” located within the 3′-UTR of the target gene via luciferase reporter assay system. Furthermore, we established the negative regulatory relationship between miR-93 and CCND1 by determining the relative luciferase activity of cells transfected with wild-type or mutant 3′-UTR of CCND1. We also found that The CCND1 protein and mRNA expression level of HL-1 cells treated with 50 nM miR-93 mimics were apparently lower than the scramble control, and those of the cells treated with 100 nM miR-93 mimics and CCND1 siRNA (100 nM) were even lower than those in the 50 nM treatment group. Meanwhile, cells transfected with miR-93 mimics (50 nM) showed evidently downregulated viability when compared with the scramble controls, while cells transfected with (100 nM) and CCND1 siRNA (100nM) showed even lower viability.ConclusionsWe showed that CCND1 is a direct target of miR-93, and the dysregulation of the miR-93/CCND1 signaling pathway is responsible for the development of ventricular remodeling.

OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese.

An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1), which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05). The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05). The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05), whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05). The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05). Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese.

Bromodomain and Extraterminal Protein Inhibitor JQ1 Suppresses Thyroid Tumor Growth in a Mouse Model.

New therapeutic approaches are needed for patients with thyroid cancer refractory to radioiodine treatment. An inhibitor of bromodomain and extraterminal domain (BET) proteins, JQ1, shows potent antitumor effects in hematological cancers and solid tumors. To evaluate whether JQ1 is effective against thyroid cancer, we examined antitumor efficacy of JQ1 using the ThrbPV/PVKrasG12D mouse, a model of anaplastic thyroid cancer. We treated ThrbPV/PVKrasG12D mice with vehicle or JQ1 at a dose of 50 mg/kg body weight/day starting at the age of 8 weeks for a 10-week period and monitored thyroid tumor progression. JQ1 markedly inhibited thyroid tumor growth and prolonged survival of these mice. Global differential gene expression analysis showed that JQ1 suppressed the cMyc (hereafter referred to as Myc) transcription program by inhibiting mRNA expression of Myc, ccnd1, and other related genes. JQ1-suppressed Myc expression was accompanied by chromatin remodeling as evidenced by increased expression of histones and hexamethylene bis-acetamide inducible 1, a suppressor of RNA polymerase II transcription elongation. Analyses showed that JQ1 decreased MYC abundance in thyroid tumors and attenuated the cyclin D1-CDK4-Rb-E2F3 signaling to decrease tumor growth. Further analysis indicated that JQ1 inhibited the recruitment of BDR4 to the promoter complex of the Myc and Ccnd1 genes in rat thyroid follicular PCCL3 cells, resulting in decreased MYC expression at the mRNA and protein levels to inhibit tumor cell proliferation. These preclinical findings suggest that BET inhibitors may be an effective agent to reduce thyroid tumor burden for the treatment of refractory thyroid cancer. Clin Cancer Res; 23(2); 430-40. ©2016 AACR.

Effect of Human Umbilical Cord-derived Mesenchymal Stem Cells on Proliferation and Differentiation of Leukemia Cells

To investigate the effect of human umbilical cord-derived mesenchymal stem cells(HUC-MSC) on the proliferation and differentiation of NB4 treated with all-trans retinoid acid (ATRA) and its underlining mechanisms . Human umbilical cord mesenchymal stem cells were isolated from umbilical cord of newborns. Co-culture system was established by HUC-MSC and NB4 in vitro. The experiment was divided into 4 groups: NB4 group (NB4 cells alone) , NM group (NB4 cells co-cultured with HUC-MSC) , NA group (NB4 cells treated with ATRA) , NMA group (NB4 cells co-cultured with HUC-MSC and treated with ATRA) . NB4 cells were counted by a microscopy, NB4 proliferation was monitored by CCK-8 assay, NB4 differentiation was assessed by Wright ' s staining and nitroblue tetrazolium reduction test. IL-6 levels in the culture supernatant of different groups were tested by ELISA kit. Quantitative PCR was used to detect the transcription level of CDKN1A, CCND1 and Survivin. NB4 and HUC-MSC in the co-culturing systems were in good condition with a slight repression of NB4 proliferation by HUC-MSC. HUC-MSC could collaborate with ATRA to induce significant NB4 differentiation. Consistent with this finding, IL-6 expression levels of co-cultured groups were remarkably higher than that in any other groups or the group of HUC-MSC alone. The quantitative PCR analysis showed that the levels of CDKN1A and CCND1 mRNA expression were increased or decreased respectively in the co-cultured groups. HUC-MSC co-culture can reduce proliferation but promote the differentiation of NB4 cells, suggesting that this effect may be closely related with the secretion of IL-6 which can affect the expression of some factors in vitro.

Impact of blue 465nm LED on suppression of cancer growth.

767 Background: Recent studies demonstrate the efficacy of irradiation from light emitting diodes (LED) for wound healing and anti-inflammation and anti-cancertherapies. In this study, the blue LED (465nm) suppressed the tumor growth in various cancers. Methods: &lt; Experiment 1, Effect of blue LED on colon cancer &gt; Human colon cancer cells (HT29 or HCT116) were seeded onto laboratory dishes that were put on LED irradiation equipment with a blue LED (465 nm). Irradiation at 15 or 30 mW was performed 10 min/day, each day for 5 days. Cell counting kit8 was then used to measure cell viability. Apoptosis and expression of several mRNAs (caspase, MAPK and autophagy pathway) in HT29 cultures irradiated with 465 nm LED were evaluated via AnnexinV/PI and RT-PCR, respectively. &lt; Experiment 2, Effect of blue LED on pancreas cancer &gt; Human colon cancer cells (SUIT2) were examined by same way and additionally cell cycle was checked. &lt; Experiment 3, Effect of LED on CSCs &gt; Cancer sphere were seeded onto laboratory dishes that were then put on LED irradiation equipment with a 465 nm-LED. Stemness gene and cell surface markers of CSCs expressions were analyzed by RT-PCR. Results: &lt; Experiment 1 &gt; Viability of HT29 and HCT116 cells was lower in blue LED irradiated cultures than in control cultures. Moreover, the expression of FAS, caspase3, capase8, and JUK were significantly higher in 465 nm-LED irradiated cultures than in control cultures, and expression of ERK1/2 and LC3 was lower in blue LED-irradiated cells. &lt; Experiment 2 &gt; Viability of SUIT2 cells was lower in blue LED irradiated cultures than in control cultures. And the CCND1 mRNA expression was significantly lower in blue LED group (p &lt; 0.05). The cells of phase G2/M was decreased. &lt; Experiment 3 &gt; Cell viability of cancer sphere were significantly decreased by LED irradiation(p &lt; 0.05). And, stemness gene and cell surface markers of CSCs expressions were significantly decreased by LED irradiation (p &lt; 0.05) Conclusions: Blue LED irradiation inhibited tumor growth in various cancer cell lines.

Reduced BCL2 and CCND1 mRNA expression in human cervical cancer HeLa cells treated with a combination of everolimus and paclitaxel

Aim of the studyCervical cancer is the second most common malignancy in women worldwide. Everolimus displays direct effects on growth and proliferation of cancer cells via inhibition of mammalian target of rapamycin (mTOR) protein, which is known to be associated with drug resistance. In this study, we aimed to investigate the effects of everolimus, gemcitabine, and paclitaxel in terms of cell viability and mRNA expression levels of GRP78, CCND1, CASP2, and BCL2 genes.Material and methodsHeLa cells were treated with different doses of everolimus, gemcitabine, and paclitaxel. Cell viability was assessed using MTT assay, and obtained dose response curves were used for the calculations of inhibitory concentration (IC) values. At the end of the treatment times with selected doses, RNA isolation and cDNA synthesis were performed. Finally, GRP78, CCND1, CASP2, and BCL2 genes mRNA expression levels were analysed using quantitative PCR.ResultsThe IC50 value of everolimus was 0.9 µM for 24-hour treatment. Moreover, the IC50 value of gemcitabine and paclitaxel was found to be around 18.1 µM and 7.08 µM, respectively. Everolimus, gemcitabine, and paclitaxel treatments alone did not change the GRP78, CCND1, BCL2 and CASP2 mRNA expression levels significantly. However, combined treatment of everolimus and paclitaxel significantly reduced BCL2 and CCND1 mRNA expression (p < 0.05). In contrast, this combination did not change GRP78 and CASP2 mRNA expression levels (p > 0.05).ConclusionsDown-regulation of CCND1 and BCL2 expression may be an important mechanism by which everolimus increases the therapeutic window of paclitaxel in cervical cancers.

Post-transcriptional Modifications Contribute to the Upregulation of Cyclin D2 in Multiple Myeloma.

Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3'UTR in CCND2 overexpression in multiple myeloma. Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3'UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3' rapid amplification of cDNA ends (RACE)-PCR. We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3'RACE experiments revealed that changes in CCND2 3'UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3'UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. Overall, these results highlight the impact of CCND2 3'UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.