Cyclin D1 mRNA Research Articles

Puerarin alleviates the progression of non-small cell lung cancer by regulating the miR-342/CCND1 axis.

Puerarin has recently been demonstrated to play anti-cancer roles in a series of human cancers, including non-small cell lung cancer (NSCLC), possibly through regulation of cancer-related microRNAs (miRNAs). The purpose of the present study was to further investigate the detailed role and underlying mechanism of puerarin on NSCLC progression. Cell viability and apoptosis were assessed using the Cell Counting kit-8 (CCK-8) assay and flow cytometry, respectively. Transwell assays were performed to determine cell migration and invasion abilities. The qRT-PCR assay was employed to detect the expression of miR-342 and cyclin D1 (CCND1) mRNA, and CCND1 protein expression was evaluated by western blotting. The targeted interaction between miR-342 and CCND1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. We found that our data demonstrated that puerarin repressed cell viability, migration, invasion, and cell cycle progression, and enhanced the apoptosis of NSCLC cells. miR-342 overexpression hindered the migration, invasion and cell cycle progression, and accelerated the apoptosis of NSCLC cells. miR-342 inhibited CCND1 expression by directly binding to the 3'-UTR of CCND1. Moreover, miR-342 overexpression-mediated anti-migration, anti-invasion, anti-cell cycle progression, and pro-apoptotic effects were abated by co-transfection of pcDNA-CCND1. More importantly, puerarin inhibited CCND1 expression by upregulating miR-342. Additionally, puerarin hampered NSCLC cell progression in vitro and tumor growth in vivo by upregulating miR-342. In conclusion, our study suggested that puerarin hampered NSCLC progression in vitro and in vivo at least partly through regulating miR-342/CCND1 axis, highlighting a novel mechanism of puerarin exerting anti-cancer property in NSCLC.

Downregulation of miR-4755-5p promotes fluoride-induced osteoblast activation via tageting Cyclin D1

BackgroundEndemic fluorosis remains a major public health issue in many countries. Fluoride can cause abnormalities in osteoblast proliferation and activation, leading to skeletal fluorosis. However, its detailed molecular mechanism remains unclear. Based on a previous study, the aim of this study is to explore the role of miRNA in osteoblast activation of skeletal fluorosis via targeting of Cyclin D1. MethodsA population study of coal-burning fluorosis and in vitro experiments were performed in this study. Urine fluoride (UF) concentrations of the participants were determined using a national standardized ion selective electrode approach. Based on our previous miRNA sequence results, bioinformatic analysis was used to predict miR-4755-5p targeting Cyclin D1. Quantitative real-time PCR (qRT-PCR) was used to verify the expression of miR-4755-5p. The expression of Cyclin D1 mRNA was detected by qRT-PCR. The expression of Cyclin D1 protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Cell viability was detected by CCK-8 method. The distribution of the cell cycle was analyzed by flow cytometry. The alkaline phosphatase (ALP) activity and bone Gla protein (BGP) content were detected by micronutrient enzymes standard method and ELISA. The target binding between miR-4755-5p and Cyclin D1 was verified using dual-luciferase reporter assay. ResultsIn the fluoride-exposed population, the results showed that with the increase in UF content, the expression of miR-4755-5p decreased gradually, while the mRNA transcription and protein expression of Cyclin D1 increased gradually. The relative miR-4755-5p expression showed a negative correlation with Cyclin D1 expression. Subsequently, in human osteoblasts treated with sodium fluoride (NaF), the results also showed that NaF caused low expression of miR-4755-5p and increased expression of Cyclin D1. Further, the results of miR-4755-5p mimic transfection confirmed that under the action of NaF, miR-4755-5p overexpression reduced Cyclin D1 protein expression within osteoblasts and further inhibited cell proliferation and activation. Simultaneously, luciferase reporter assays verified that Cyclin D1 was the miR-4755-5p direct target. ConclusionThe results demonstrate that fluoride exposure induced the downregulation of miR-4755-5p and downregulated miR-4755-5p promoted fluoride-induced osteoblast activation by increasing Cyclin D1 protein expression. This study sheds new light on biomarkers and potential treatment for endemic fluorosis.

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.

Long Noncoding RNA FGD5-AS1 Promotes Glioma Cell Proliferation, Migration and Invasion by Regulating wnt/β-Catenin Pathway.

PurposeTo investigate the specific function of long noncoding RNA FGD5 antisense RNA 1 (lncRNA FGD5-AS1) in glioma.Materials and MethodsThe level of FGD5-AS1 was detected in clinical samples and cell lines by qRT-PCR. Small interfering RNA (siRNA) of FGD5-AS1 or scramble siRNA was transfected into U87 cell lines to examine the role of FGD5-AS1 on glioma development. The proliferation of glioma cells was tested by Cell Counting Kit-8 (CCK-8), the migration and invasion of glioma cells were tested by transwell assay without matrigel or with matrigel. Western blot was used to detect the protein expression, and XAV-939 was used to inhibit wnt/β-catenin pathway. The effect of FGD5-AS1 on tumorigenesis of glioma was confirmed by xenograft nude mice model.ResultsFGD5-AS1 was significantly increased in glioma tissues and cells. Loss of FGD5-AS1 inhibited the proliferation, migration and invasion of U87 cells. Furthermore, overexpression of FGD5-AS1 increased the mRNA and protein levels of β-catenin and cyclin D1. Blocking of wnt/β-catenin using XAV-939 reversed the promotion role of FGD3-AS1 on glioma cells’ migration and invasion. The in vivo tumor growth assay showed that FGD3-AS1 accelerated glioma tumorigenesis with activating wnt/β-catenin pathway.ConclusionOur research emphasized FGD5-AS1 acting as an oncogene by regulating wnt/β-catenin signaling pathway, thus providing some novel experimental basis for clinical treatment of glioma.

RBM5 Acts as Tumor Suppressor in Medulloblastoma through Regulating Wnt/β-Catenin Signaling

Introduction: RBM5 acts as a tumor suppressor gene in lung and breast cancers; however, its role in the pathogenesis of medulloblastoma (MB) remains unclear. We previously identified 4 RBM5 mutations in whole exome sequencing analysis of 40 MB patients. This study examined the role of RBM5 in MB progression. Methods: The expression patterns of RBM5 in tissues of 40 MB patients were analyzed using immunohistochemistry. Associations between RBM5 expression and overall survival (OS) were evaluated using Kaplan-Meier analysis. The RBM5 role in Daoy cells’ proliferation, migration, and Wnt/β-catenin signaling was analyzed after RBM5 knockdown and overexpression. Results: The expression level of RBM5 mRNA and protein was significantly lower in MB than that in adjacent normal control tissues, and low RBM5 expression was significantly associated with reduced OS (p = 0.034). RBM5 knockdown induced Daoy and ONS-76 cells proliferation, while RBM5 overexpression repressed cell proliferation and migration in vitro (all p < 0.05). β-Catenin, LEF1, and cyclin D1 mRNA levels were upregulated, while DKK1 expression was downregulated in Daoy cells following RBM5 knockdown. Conclusion: RBM5 may function as a tumor suppressor in MB by regulating Wnt/β-catenin signaling, and its reduced expression is associated with lower OS.

Methylation-associated silencing of miR-193b improves the radiotherapy sensitivity of esophageal cancer cells by targeting cyclin D1 in areas with zinc deficiency

BackgroundAlthough radiotherapy is an important treatment mode for esophageal cancer (EC), the outcome remains unsatisfactory due to radioresistance, and the key cause of radiotherapy resistance is a change in the cell cycle. Zinc deficiency (ZD) has a significant influence on the cell cycle, and this effect is a common phenomenon in areas with a high incidence of esophageal cancer. MethodsRadioresistant sub-cell lines were established by exposing esophageal cancer cells to nine rounds of X-ray irradiation at a dose of 2 Gy. The cells were treated with a range of different concentrations of zinc and overexpression of miR-193b. And proliferation, colony formation, cell cycle and apoptosis assays were then conducted. Luciferase reporter assays were performed to confirm direct interactions between miR-193b and ZRT/IRT-like protein 5 (ZIP5) and between miR-193b and Cyclin D1. Analysis of clinical and follow-up data was performed using data obtained from 75 patients from Cixian, a well-known high incidence area of esophageal cancer. All these patients are unable to tolerate surgery due to their advanced age or advanced stage, and serum specimens were obtained before the patients received therapy. ResultsThe cell cycle of radioresistant cells is blocked in G0/G1 phase (from 50% to 68%). The expression level of Cyclin D1 was decreased and miR-193b was increased in radioresistant cells (P < 0.001). ZD decreased the proportion of cells in G0/G1 phase both in EC (from 50% to 32%) and radioresistant (from 68% to 47%) cells. And the radioresistance of these two cells were decreased. ZD increased the expression of Cyclin D1 (P < 0.001) and inhibited the level of miR-193b (P < 0.001). Up-regulation of miR-193b recovered the proportion of cells in G0/G1 phase and the radioresistance, meanwhile, recovered the altered expression levels of Cyclin D1 and miR-193b of these two cells by ZD. ZD enhanced DNMT activity both in EC (32%) and radioresistant (26%). And miR-193b was hypermethylated both in EC and radioresistant cells. MiR-193b suppressed Cyclin D1 expression by targeting the 3′UTR of Cyclin D1 mRNA. The expression level of miR-193b in the serum of patients was correlated with the disease control rate (DCR) and had a good diagnostic value for distinguishing DCR of EC patients (AUC = 0.710, 95%CI: 0.580–0.839, P = 0.003). And the level of miR-193b was correlated with overall survival (OS) (HR = 0.208, 95%CI: 0.094–0.464). ConclusionsThe methylation-mediated silencing of miR-193b in EC cells due to ZD increased the expression of ZIP5, and the overexpression of ZIP5 increased the intracellular zinc levels to maintain zinc homeostasis. Meanwhile, the silencing of miR-193b increased the sensitivity of radiotherapy by promoting the expression of Cyclin D1.

The effect of different types of exercise training on diet-induced obesity in rats, cross-talk between cell cycle proteins and apoptosis in testis.

Obesity is associated with germ cell apoptosis, spermatogenesis arrest, and testicular endocrine suppression. The aim of the present study was to investigate the crosstalk between germ cell apoptosis and cell cycle machinery in sedentary and obese rats after moderate-intensity continuous (MICT), high-intensity continuous (HICT) and High-intensity interval (HIIT) exercise trainings. Male Wistar rats (n= 30) were randomly divided into 5 groups; the control, sedentary high-fat diet (HFD)-received (HFD-sole), MICT, HICT and HIIT-induced HFD-received groups. The serum levels of LDL-C, HDL-C, triglyceride, and testosterone, mRNA and protein levels of Cyclin D1, Cdk4, p21, apoptotic cell number/mm2 of testicular tissue and testicular DNA fragmentation ratio were investigated. The obese animals in HFD-sole group represented a significant (p<0.05) reduction in serum HDL-C and testosterone levels, Cyclin D1, Cdk4 expressions, and exhibited a remarkable (p<0.05) increment in LDL-C, triglyceride, p21 expression, apoptotic cell number and DNA fragmentation ratio versus control animals. However, the animals in MICT, HICT, HIIT groups exhibited a significant (p<0.05) increment in serum HDL-C and testosterone, Cyclin D1 and Cdk4 expressions and showed a significant (p<0.05) reduction in serum LDL-C and triglyceride, p21 expression, apoptotic cell number and DNA fragmentation versus the HFD-sole group. In conclusion, a crosslink between cell cycle machinery and apoptosis of germ cells was revealed in the testicles of HFD-sole animals, and MICT, HICT and HIIT could ameliorate the obesity-induced impairments, respectively. This effect may be attributed to the effect of exercise training protocols on maintaining Cyclin D1 and Cdk4 and suppressing p21 expression levels in the testicles.

P0870THE ACTIVATION OF KYNURENIC SYSTEM IN BONE TISSUE AS A NEW REGULATOR OF OSTEOBLASTOGENESIS IN RATS WITH EXPERIMENTAL CHRONIC KIDNEY DISEASE DURING LP533401 THERAPY

Abstract Background and Aims Abnormalities in bone metabolism represent the most complex disorder accompanying a chronic kidney disease (CKD) development. Serotonin – one of the tryptophan metabolites, has received intensive attention due to its potential role in bone metabolism. The synthesis of peripheral serotonin is initiated by the tryptophan hydroxylase-1 (TPH-1) in the gut. LP533401 is a small-molecule inhibitor of TPH-1. Previously, we found that inhibition of TPH-1 by LP533401 may improve bone mineralization in rats with experimentally induced CKD (Pawlak et al, Bone, 2018). The aim of this study was to establish whether the inhibition of serotonin synthesis by LP533401 may affect kynurenine pathway activity in bone tissue of nephrectomized rats, and to determine the potential consequence of this process in relation to osteoblastogenesis. Method Nephrectomized rats were randomized into: untreated (CKD), treated with vehicle (VEH), treated with LP533401 at a dose of 30 (LP 30) and 100 mg/kg (LP 100) daily for 8 weeks. The shame-operated animals served as controls (CON). Tryptophan (TRP) and kynurenine (KYN) concentrations in trabecular and cortical homogenates of femoral bone were determined using high-performance liquid chromatography (HPLC). The expression of tryptophan 2,3-dioxygenase (TDO) as well as genes associated with osteoblastogenesis: activating transcription factor 4 (ATF4), forkhead box protein O1 (FOXO1), Cyclin D1, osteocalcin (Bglap) and sclerostin (Sost) were assessed using the QRT-PCR method. Results We demonstrated a presence of TDO-dependent, paracrine kynurenic system in bone homogenates of rats with CKD. The significant decrease in TDO mRNA level was observed in the bone of all nephrectomized groups in comparison with CON (all p&amp;lt;0.0001). In LP 100 group, the TDO expression was still lower than in CON, but was significantly higher than in VEH and LP 30-treated groups (both p&amp;lt;0.0001). TDO expression was positively associated with KYN concentration and KYN/TRP ratio (R = 0.426, p = 0.016 and R = 0.559, p = 0.001; respectively) in trabecular bone region. ATF4 gene expression in bone was similar in LP 30 and CON, whereas it was significantly higher in other studied groups compared to healthy animals, and the positive relationship existed between ATF4 and TDO expression (R = 0.372, p = 0.042) as well as between ATF4 gene expression and KYN/TRP ratio both in trabecular and cortical bone tissue (R = 0.485, p = 0.006 and R = 0.421, p = 0.023; respectively) of LP-treated rats. In contrast, cyclin D1 mRNA level was the lowest in LP 100 group compared to other studied groups, and the inverse relationship was between this gene and TDO expression (R = -0.622, p = 0.0002). Bglap mRNA level was higher in CKD and VEH group compared to CON (p&amp;lt;0.05 and p&amp;lt;0.0001; respectively). CKD rats treated with LP 30 had the lowest Bglap mRNA levels among all analyzed uremic groups (all p&amp;lt;0.0001), and the positive relationship was between this gene expression and both TDO expression and KYN/TRP ratio (R = 0.468, p = 0.011 and R = 0.474, p = 0.008; respectively) in trabecular bone of uremic rats treated with LP. Similarly, Sost gene expression was significantly reduced in LP 30 group in comparison with other studied groups (all p&amp;lt;0.0001), and it was positively correlated with TDO expression and KYN/TRP ratio (R = 0.540, p = 0.002 and R = 0.409, p = 0.022; respectively) in trabecular bone tissue. Conclusion This study for the first time demonstrated the presence of a TDO-dependent, paracrine kynurenic system in the bone of rats with CKD. The modulation of this system during LP533401 treatment was closely associated with the expression of genes participating in osteoblastogenesis, particularly with osteoblast maturation markers. These results represent the next step in studying the role of tryptophan metabolites in renal osteodystrophy.

SUN-126 Distinct Molecular Phenotypes of Non-Diseased Breast Adipose Tissue of Pre-Menopausal Obese and Non-Obese Women May Underlie Differing Breast Cancer Risks

Obesity is a major risk factor for many chronic diseases including postmenopausal breast cancer. Paradoxically, breast cancer susceptibility is inversely linked to obesity in pre-menopausal women. Adipose tissues are active endocrine organs that play major roles in tumor development and progression; however, fat depots at different anatomical sites are biologically and functionally distinct and their singular influence on breast epithelial biology remains unclear. To study the early events by which breast adiposity may provide a microenvironment predisposing normal breast epithelial cells to tumorigenesis, we collected breast tissue from pre-menopausal (n=10/group) non-obese (NO, BMI=27.6±0.8) and obese (O, BMI=44.5±2.8) women of comparable ages (NO: 36.1± 3.3; O: 40.0±2.0) with no breast cancer and undergoing elective breast reduction surgery. Breast adipose tissue and corresponding glandular cells were analyzed histologically and evaluated for expression of genes (adipokines, cytokines, steroid hormone signaling) by QPCR and proteins (proliferation, apoptosis, inflammation) by IHC. Adipocyte size distributions from NO and O breasts did not differ (P=0.9). However, adipose mRNA levels for pro-inflammatory cytokines (IL-6, IL-8, CSF-1, MCP-1) and adipokines (LEP, CFD) were higher for O than NO (P<0.05). AdipoQ, ER-α, and ER-β transcript levels were lower for O than NO (P<0.05), while those for CYP19 and PTGS2 showed reverse trends (O>NO, P<0.05). In the corresponding glandular cells, NO had higher mRNA levels for IL-6, IL-8, ER-α, and ER-β than O (P<0.05). Immunostaining with anti-Ki67 antibodies indicated that O glandular cells were 3-fold less proliferative than those for NO, consistent with their lower Cyclin D1 mRNA levels (P<0.05). Galectin-1, a pro-fibrotic protein, showed predominant myo- vs. luminal epithelial localization, with staining intensities for O tending to be higher (P=0.07) than for NO. Perilipin immunostaining was specific for adipocytes and did not differ for O and NO. A non-targeted approach using a Human Cytokine Array (R&D Systems) was employed to further evaluate the inflammation status of O vs. NO adipose. The analyses confirmed the higher expression of IL-8, Leptin and CFD (by QPCR) in O vs. NO and identified C-reactive protein, EMMPRIN, Trefoil Factor-3, Cystatin-3 and Macrophage Migration Inhibitory Factor-1 as greater in O than NO (~2-fold). Our findings demonstrate marked differences in gene and protein expression patterns of O and NO breast adipose tissue, which were accompanied by a suppression of proliferation of O relative to NO breast epithelium. We speculate that early exposure of the breast epithelium to a highly inflammatory environment fueled by breast adiposity may promote a senescent state that confers protection from pre-menopausal breast cancer.

Long Non-Coding RNA TTN-AS1 Serves as a Competing Endogenous RNA of miR-195 to Facilitate Clear Cell Renal Cell Carcinoma Progression.

IntroductionClear cell renal cell carcinoma (ccRCC) is an aggressive human malignancy. Long non-coding RNAs (lncRNAs) are critical regulators in many malignant tumors, including ccRCC. The aim of this study is to investigate the expression, functions and molecular mechanisms of lncRNA TTN-AS1 in ccRCC.MethodsA total of 145 paired cancer and normal tissues were collected from patients with ccRCC. The expression levels of TTN-AS1 and miR-195 in the tissues or cells were measured by RT-qPCR analysis. The expression levels of cyclin D1 protein were measured by Western blot analysis. Cell proliferation and cell cycle distribution were detected by MTT assay and flow cytometer analysis, respectively. The binding relationship between miR-195 and TTN-AS1 or cyclin D1 mRNA was validated by dual-luciferase reporter assay.ResultsOur results revealed that TTN-AS1 expression levels in human ccRCC tissues and cell lines were markedly increased. High expression of TTN-AS1 was closely associated with adverse clinicopathological characteristics of ccRCC patients. Gain- and loss-of-function experiments showed that TTN-AS1 overexpression promoted the proliferation and cell cycle transition of ccRCC cells, while the malignant traits were obviously suppressed after TTN-AS1 knockdown. Mechanistically, miR-195 was found to bind with and to be negatively regulated by TTN-AS1 in ccRCC cells. Further, we showed that cyclin D1 is a direct target of miR-195 in ccRCC, and rescue assays verified that restoration of miR-195 expression partially blocked the oncogenic effects of TTN-AS1 in ccRCC cells.ConclusionOur study provides a novel mechanism of TTN-AS1/miR-195/cyclin D1 regulatory axis in ccRCC, which may become a breakthrough for ccRCC therapy in the future.

Effects of Wnt/β-Catenin Signal Pathway Regulated by miR-342-5p Targeting CBX2 on Proliferation, Metastasis and Invasion of Ovarian Cancer Cells.

ObjectiveThis study aimed to investigate the effect of Wnt/β-catenin signal pathway mediated by miR-342-5p targeting CBX2 gene on the proliferation, metastasis, invasion and apoptosis of ovarian cancer cells, and to explore its related regulatory mechanism.MethodsHuman normal ovarian epithelial cell line IOSE80, human ovarian cancer cell line SKOV3 and OVCAR3 were the subjects. Software were used to predict the binding site of miR-342-5p targeting CBX2 gene. The proliferation rate of ovarian cancer cells was detected by MTT method; the cell viability of each group was observed by colony formation test; the apoptosis of cells in each group was detected by flow cytometry; the invasive ability of cells was determined by transwell test, and the migration ability of cells was detected by scratch test. The mRNA expression levels of miR-342-5p, CBX2, Wnt1, β-catenin, C-myc and Cyclin D1 were measured by qRT-PCR. Also, Western blot was used to determine the protein expression levels of CBX2, Wnt1, β-catenin, C-myc and Cyclin D1.ResultsCBX2 was identified as the target gene of miR-342-5p. MTT test results showed that miR-342-5p could significantly inhibit the proliferation of SKOV3 and OVCAR3 cells, colony formation assay results indicated that the viability of SKOV3 and OVCAR3 cells transfected with miR-342-5p decreased significantly, and flow cytometry results suggested that miR-342-5p could promote the apoptosis of SKOV3 and OVCAR3 cells. Also, the results of transwell showed that miR-342-5p could significantly inhibit the invasive ability of SKOV3 and OVCAR3 cells, and the results of scratch assay suggested that miR-342-5p could significantly inhibit the migration of SKOV3 and OVCAR3 cells. Moreover, qRT-PCR and Western blot results indicated that the mRNA and protein expression levels of CBX2, Wnt1, β-catenin, C-myc and Cyclin D1 decreased in SKOV3 and OVCAR3 cells transfected with miR-342-5p, while the mRNA expression levels of miR-342-5p increased significantly (P<0.05).ConclusionMiR-342-5p targeted gene is CBX2, which can significantly reduce the proliferation, invasion, migration and viability of ovarian cancer cell lines SKOV3 and OVCAR3, and promote their apoptosis. The mechanism may be related to the mediation of Wnt/β-catenin signal pathway and down-regulation of the related genes expression.

TUG1 weakens the sensitivity of acute myeloid leukemia cells to cytarabine by regulating miR-655-3p/CCND1 axis.

Long non-coding RNA taurine upregulated gene 1 (lncRNA TUG1) has been demonstrated to promote malignant phenotypes and Adriamycin resistance in acute myeloid leukemia (AML) cells. However, the function and mechanism of TUG1 in cytarabine (Ara-C) sensitivity in AML remain unclear. Levels of TUG1, microRNA (miR)-655-3p or cyclin D1 (CCND1) mRNA were examined using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation activity and apoptosis were analyzed using cell counting kit-8 (CCK-8) or flow cytometry, respectively. Western blot was utilized to detect the protein levels of Ki-67, B-cell lymphoma-2-associated X protein (Bax), and CCND1. The interaction between miR-655-3p and TUG1 or CCND1 was confirmed by Dual-Luciferase reporter and pull-down assay. TUG1 and CCND1 were higher expressed, while miR-655-3p was lower expressed in AML cells compared with that in normal cells. Higher expression levels of TUG1 or CCND1, and lower expression levels of miR-655-3p both notably reversed Ara-C-induced proliferation inhibition and apoptosis promotion in AML cells. TUG1 was a sponge of miR-655-3p, and TUG1 knockdown enhanced the sensitivity of AML cells to Ara-C by regulating miR-655-3p. MiR-655-3p directly targeted CCND1, and CCND1 overexpression attenuated miR-655-3p restoration-mediated reinforcement of Ara-C sensitivity in AML cells. Besides that, TUG1 up-regulated CCND1 expression via miR-655-3p. TUG1 weakened the sensitivity of AML cells to Ara-C by up-regulating CCND1 via miR-655-3p, suggesting a new insight into the chemotherapy of AML.

Ochratoxin A causes cell cycle arrest in G1 and G1/S phases through p53 in HK-2 cells

Ochratoxin A (OTA) is a toxic metabolite produced by Aspergillus and Penicillium fungus. OTA found in the human and animal tissues can contaminate many foods that we daily consume in our lives. It accumulates especially in kidney. Although OTA is known to cause cell cycle arrest, the molecular mechanisms underlying this effect have not been fully understood, yet. We aimed to investigate the molecular details of OTA induced inhibitory response in G1 – G1/S phase of cell cycle and also the regulatory role of p53 in OTA mediated cell cycle arrest in human proximal tubule epithelial cells, HK-2. For this purpose, Cyclin E1 and Cyclin D1 mRNA expressions and Cyclin D1, Cdk4 and Cdk2 protein expressions were evaluated in HK-2 cells transfected with either 50 nM control siRNA or p53 siRNA for 72 h in the absence or presence of OTA using RT-PCR and Western blot analyses, respectively. Our findings showed that mRNA expressions of Cyclin D1 and Cyclin E1 and protein expressions of Cyclin D1, Cdk4 and Cdk2 were inhibited in HK-2 cells treated with two different doses of OTA, 10 μM and 25 μM, for 24 h. However, the downregulation of p53 led to enhance OTA-mediated increase in mRNA expressions of Cyclin D1 and Cyclin E1 and protein expressions of Cyclin D1, Cdk4 and Cdk2 compared to control siRNA transfected HK-2 cells. Our findings strongly suggest that the cell cycle arresting effect of OTA also performs via a p53 mediated mechanism besides other possible mechanisms.

FTO Demethylates Cyclin D1 mRNA and Controls Cell-Cycle Progression

N6-Methyladenosine (m6A) modification is the major chemical modification in mRNA that controls fundamental biological processes, including cell proliferation. Herein, we demonstrate that fat mass and obesity-associated (FTO) demethylates m6A modification of cyclin D1, the key regulator for G1 phase progression and controls cell proliferation invitro and invivo. FTO depletion upregulates cyclin D1 m6A modification, which in turn accelerates the degradation of cyclin D1 mRNA, leading to the impairment of G1 progression. m6A modification of cyclin D1 oscillates in a cell-cycle-dependent manner; m6A levels are suppressed during the G1 phase and enhanced during other phases. Low m6A levels during G1 are associated with the nuclear translocation of FTO from the cytosol. Furthermore, nucleocytoplasmic shuttling of FTO is regulated by casein kinase II-mediated phosphorylation of FTO. Our results highlight the role of m6A in regulating cyclin D1 mRNA stability and add another layer of complexity to cell-cycle regulation.

The Autism-Related Protein SETD5 Controls Neural Cell Proliferation through Epigenetic Regulation of rDNA Expression.

SummaryHaploinsufficiency of SETD5 is implicated in syndromic autism spectrum disorder (ASD), but the molecular mechanism underlying the pathological role of this protein has remained unclear. We have now shown that Setd5+/– mice manifest ASD-related behavioral phenotypes and that the expression of ribosomal protein genes and rDNA is disturbed in the brain of these mice. SETD5 recruited the HDAC3 complex to the rDNA promoter, resulting in removal of the histone mark H4K16ac and its reader protein TIP5, a repressor of rDNA expression. Depletion of SETD5 attenuated rDNA expression, translational activity, and neural cell proliferation, whereas ablation of TIP5 in SETD5-deficient cells rescued these effects. Translation of cyclin D1 mRNA was specifically down-regulated in SETD5-insufficient cells. Our results thus suggest that SETD5 positively regulates rDNA expression via an HDAC3-mediated epigenetic mechanism and that such regulation is essential for translation of cyclin D1 mRNA and neural cell proliferation.

Determination of the roles of GREM1 gene in granulosa cell proliferation and steroidogenesis of hen ovarian prehierarchical follicles

Gremlin genes are known members of the DAN family of bone morphogenetic protein (BMP) antagonists, but their functions and regulatory mechanisms in ovarian follicular development of chicken remain unknown. The current study was designed to investigate the mRNA expression patterns of gremlin1 gene (GREM1) and its protein location in the follicles sampled, and to explore the biological effect of GREM1 on the prehierarchical follicular development. This work revealed that chicken GREM1 mRNA exhibits a constant expression level across all the prehierarchical follicles (PFs) from 1-4 mm to 7–8 mm in diameter, and the preovulatory follicles (from F6 to F1) by using RT-qPCR (P > 0.05). The GREM1 protein is predominantly expressed in the oocytes and granulosa cells (GCs) of the PFs by immunohistochemistry. Furthermore, our data demonstrated that siRNA-mediated knockdown of GREM1 in the GCs resulted in a significant reduction in cell proliferation (P < 0.001); conversely, overexpression of GREM1 in the GCs led to a remarkable increase in cell proliferation (P < 0.001). Interestingly, the expression levels of proliferating cell nuclear antigen (PCNA) and cyclin D2 (CCND2) mRNA and proteins were notably increased when GREM1 expression was upregulated in the GCs (P < 0.01), however, the expression levels of CYP11A1 and StAR were markedly downregulated (P < 0.01). The current results showed that GREM1 gene plays a stimulatory role in GC proliferation during growth and development of the prehierarchical follicles in vitro but an inhibitory role in GC differentiation and steroidogenesis of the hen ovary follicles.

Effect of Silencing FMOD on Proliferation and Migration of H322 Cells and Its Mechanism

To investigate the regulation of fibromodulin (FMOD) on proliferation, adhesion and migration of non-small cell lung cancer cell line H322, and discuss its action mechanism. H322 cells were randomly divided into control group, small interfering RNA (siRNA) silencing FMOD ( FMOD siRNA) group and control siRNA (Con siRNA) group. FMOD siRNA and Con siRNA were transfected into H322 cells. The cell viability of each group was detected by CCK-8 method. The adhesion ability of cells was detected by fluorescein diacetate (FDA) fluorescent staining. The cell migration ability was detected by Transwell method. Real time-PCR was used to detect the mRNA expressions of Cyclin D1, intercellular adhesion molecule -1 （ICAM-1）, E-cadherin, FMOD, transforming growth factor-β (TGF-β), Smad2, Smad3, Smad4 and Smad7 in cells. The protein expressions of Cyclin D1, ICAM-1, E-cadherin, FMOD, TGF-β1, Smad2, Smad3, Smad4 and Smad7 were detected by Western blot. Compared with the Con siRNA group, the cell viability, cell adhesion and migration ability of the FMOD siRNA group were decreased, and the difference was statistically significant ( P<0.01). There was no significant difference between the control group and the Con siRNA group. Real time-PCR and Western blot results showed that the mRNA and protein expression levels of Cyclin D1, ICAM-1, TGF-β1, Smad2, Smad3 and Smad4 were decreased in FMOD siRNA group, compared with Con siRNA group, while the mRNA and protein expression levels of E-cadherin and Smad7 are elevated. Silencing of the FMOD gene significantly reduces the proliferation, adhesion and migration of H322 cells, which may be conducted by inhibiting the TGF-β/Smad signaling pathway.

Novel complementary antitumour effects of celastrol and metformin by targeting IκBκB, apoptosis and NLRP3 inflammasome activation in diethylnitrosamine-induced murine hepatocarcinogenesis.

One promising strategy for minimizing chemotherapeutic resistance in hepatocellular carcinoma (HCC) is the use of effective chemosensitizers. We studied the complementary multi-targeted molecular mechanisms of metformin and celastrol in mice with diethylnitrosamine-induced HCC to investigate whether metformin could augment the sensitivity of HCC tissue to the effect of celastrol. Simultaneous administration of celastrol (2mg/kg) and metformin (200mg/kg) improved liver function, enhanced the histological picture and prolonged survival. Additionally, combination therapy exerted anti-inflammatory activity, as indicated by the decreased levels of TNF-α and IL-6. This protective role could be attributed to inhibition of inflammasome activation. Herein, our data revealed downregulated NLRP3 gene expression, suppressed caspase-1 activity and reduced levels of the active forms of IL-1β and IL-18. Under this condition, pyroptotic activity was suppressed. In contrast, in the celastrol and celastrol + metformin groups, the apoptotic potential was amplified, as revealed by the increase in the caspase-9 and caspase-3 levels and Bax:BCL-2 ratio. In addition to their repressive effect on the gene expression of NFκBp65, TNFR and TLR4, metformin and celastrol inhibited phosphorylation-induced activation of IκBκB and NFκBp65 and decreased IκBα degradation. Combination therapy with metformin and celastrol repressed markers of angiogenesis, metastasis and tumour proliferation, as revealed by the decreased hepatic levels of VEGF, MMP-2/9 and cyclin D1 mRNA, respectively. In conclusion, by inhibiting NLRP3 inflammasome and its prerequisite NFκB signalling, simultaneous administration of metformin and celastrol appears to have additive benefits in the treatment of HCC compared to cela monotherapy. This effect warrants further clinical investigation.

Macrophage migration inhibitory factor regulates integrin-β1 and cyclin D1 expression via ERK pathway in podocytes

AimsMacrophage migration inhibitory factor (MIF) is found to increase in proliferative glomerulonephritis. MIF binds to the MIF receptor (CD74) that activates MAP kinase (ERK and p38). Integrins and cyclinD1 regulate cell proliferation, differentiation and adhesion. This study evaluates whether MIF can regulate integrin-β1/cyclin D1 expression and cell adhesion of podocytes. Main methodsExpression of integrin-β1 mRNA/protein and cyclin D1 mRNA under stimulation of MIF was evaluated by real-time PCR and Western blotting. MIF receptor (CD74) and MAP kinase under MIF treatment were examined to determine which pathway regulated integrin-β1 and cyclin D1 expression. Cell adhesion was evaluated under MIF treatment and/or anti-integrin-β1 antibody by cell adhesion assay. Key findingsProtein levels of integrin-β1 were up-regulated under MIF treatment in a dosage-dependent manner. CD74 protein levels were not changed after MIF treatment. Integrin-β1 and cyclin D1 mRNA levels were up-regulated after MIF 100 ng/ml treatment. ERK inhibitor U0126 reduced MIF-induced the increase in integrin-β1 mRNA and protein expression following MIF stimulation. However, p38 inhibitor SB 203580 did not inhibit MIF-induced increase in integrin-β1 mRNA and protein expression following MIF stimulation. MIF-induced increase in cyclin D1 mRNA level also was inhibited only by U0126 following MIF stimulation. Podocyte adhesion was increased after MIF treatment, but, anti-integrin-β1 antibody decreased MIF-enhanced podocyte adhesion. SignificanceMIF increases integrin-β1 and cyclin D1 expression through the ERK pathway in podocytes, and the up-regulated expression of integrin-β1 increases podocyte adhesion. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis.

Identification of a novel microRNA-141-3p/Forkhead box C1/β-catenin axis associated with rheumatoid arthritis synovial fibroblast function in vivo and in vitro.

Rationale: Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis in which synovial fibroblasts (SFs) play key roles in cartilage and bone destruction through tumor-like proliferation, migration, invasion and inflammation. This study aimed to research forkhead box protein C1 (FoxC1) and microRNA (miR)-141-3p, which modulate pathological changes in the synovial membrane, to find possible strategies for treating RA.Methods: FoxC1, β-catenin and miR-141-3p gene expression in synovial tissues and SFs was quantified by real-time PCR; FoxC1 and β-catenin protein levels were evaluated by immunohistochemistry, immunofluorescence, and Western blotting. We transiently transfected human SFs with FoxC1 and β-catenin overexpression and silencing vectors and assessed proliferation, migration, invasion and inflammation by cell function and enzyme-linked immunosorbent assays. We also assessed downstream signaling activation using immunofluorescence, real-time PCR and Western blotting. Double luciferase, coimmunoprecipitation and chromatin immunoprecipitation assays were used to verify miR-141-3p, FoxC1 and β-catenin gene and protein combinations. Finally, the therapeutic effects of FoxC1 silencing and miR-141-3p overexpression were evaluated in type II collagen-induced arthritis (CIA) rats.Results: We found that FoxC1 expression was significantly upregulated in synovium and SFs in both RA patients and rats with collagen-induced arthritis (CIA). FoxC1 overexpression increased β-catenin messenger RNA (mRNA) and protein levels and upregulated cyclin D1, c-Myc, fibronectin and matrix metalloproteinase 3 (MMP3) mRNA and protein expression in RA SFs (RASFs). In contrast, FoxC1 knockdown reduced β-catenin mRNA and protein levels as well as cyclin D1, c-Myc, and fibronectin mRNA and protein levels in RASFs. Furthermore, altering FoxC1 expression did not significantly change GSK3β and pGSK3β levels. FoxC1 overexpression promoted proliferation, migration, invasion and proinflammatory cytokine (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)) production and reduced anti-inflammatory cytokine (IL-10) levels in RASFs. FoxC1 bound to the β-catenin promoter, and β-catenin mediated the FoxC1-induced pathological changes. We also observed downregulated microRNA (miR)-141-3p expression in SFs from both RA patients and CIA rats and further found that miR-141-3p bound to the FoxC1 3′UTR and suppressed FoxC1 expression. Intra-ankle miR-141-3p agomir or FoxC1-specific siRNA injection hindered CIA development in rats.Conclusions: FoxC1 and miR-141-3p participate in RA pathogenesis by mediating inflammation and SF proliferation, migration, and invasion and thus could be novel targets for RA therapy as a nonimmunosuppressive approach.