D1 mRNA Levels Research Articles

Tetrandrine inhibits deregulated cell cycle in pancreatic cancer cells: Differential regulation of p21Cip1/Waf1, p27Kip1 and cyclin D1

Current therapies in Pancreatic Cancer (PaCa) are ineffective due to deregulated cell cycle driven by landscape mutations. In this study, we show for the first time that tetrandrine (TET) inhibits proliferation of the PaCa cells and inhibits PaCa tumor growth. TET inhibits cell cycle transition at G1/S boundary. TET increased levels of p21Cip1/Waf1 and p27Kip1, had no effect on the levels of CDK4/6 proteins and decreased the levels of cyclin D1 and pRb proteins. TET resulted in changes in mRNA levels of cyclin D1 and p21Cip1/Waf1 but had no effect on the mRNA of p27Kip1. We show, for the first time in any system, that TET treatment downregulated Skp2, E3 ligase specific for degradation of p27Kip1 during the cell cycle. Taken together our results show, that TET indirectly impairs the activities of CDK4/6 to halt deregulated cell cycle and inhibit PaCa tumor growth. These results suggest that TET may serve as a novel agent for treatment of PaCa, for which there is no effective cure to date.

Increased levels of cyclin D1 negatively impacts on acute lymphoblastic leukemia overall survival

BackgroundCyclin D1 is a protein essential for transition from G1 to S phase during cell cycle progression, which has an oncogenic potential and is highly expressed in several human malignancies. However, in view of the heterogeneity of the findings in the literature, the prognostic value of cyclin D1 expression still needs to be validated in different cohorts of adult acute lymphoblastic leukemia (ALL) patients.MethodsBone marrow samples from 13 healthy donors and 45 adult patients with acute lymphoblastic leukemia were included. Cyclin D1 gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Fisher’s exact test, Chi-squared test and Cox regression were used, as appropriate. All p values were two-sided with a significance level of 5%.ResultsCyclin D1 mRNA levels were similar between primary cells from ALL patients and healthy donors. In ALL patients, high cyclin D1 expression was associated with older age at the diagnosis, presence of BCR-ABL1, and lower white blood cell counts. Importantly, increased cyclin D1 expression was an independent factor that predicted worse overall survival in our adult ALL cohort.ConclusionIncreased levels of cyclin D1 negatively impacted on ALL survival outcome, suggesting that this gene is involved in the malignant phenotype of ALL.

Research of the inhibitory effect and the mechanisms of plumbagin on large cell lung cancer NL9980 cell line

Objective To investigate the effects of plumbagin on human NL9980 large-cell lung cancer cells and the role of interleukin-6/signal transducers and activators of transcription 3 (IL-6/STAT3) signaling pathway in the anticancer procedure of plumbagin. Methods Different concentrations of plumbagin were administrated in NL9980 cell line. Inhibitory effects of plumbagin on NL9980 cells were analyzed using methyl thiazol tetrazolium (MTT) assay. Groups of different drug doses (5.0, 7.5, 10.0 μmol/L) and blank control (without administration of plumbagin) were set with simple random sampling method. Cells were collected after 24 h of administration. Expression of IL-6 and STAT3 and the downstream genes B-cell lymphoma/leukemia-2 (bcl-2), vascular endothelial growth factor (VEGF), and Cyclin D1 were assessed by real-time quantitative polymerase chain reaction (Real-time PCR). Enzyme linked immunosorbent assay (ELISA) was used to measure secreted IL-6 levels and western blotting to measure intracellular phospho-STAT3 levels. Results Plumbagin significantly inhibited NL9980 cells via suppressing IL-6/STAT3 signaling. Expression of all genes was inhibited by plumbagin in a dose-dependent manner (F=32.170, P=0.000). As the concentration of plumbagin increased, the expression of IL-6 mRNA decreased significantly (F=173.067, P=0.000) to 0.50±0.02, 0.44±0.1, 0.34±0.04 respectively and IL-6 protein decreased to 4.71±0.02, 4.08±0.14, 2.01±0.07 significantlly (F=1 805.177, P=0.000). Besides, the expression of STAT3 mRNA (0.86±0.08, 0.72±0.03, 0.52±0.06, F=230.918, P=0.000) and protein (0.65±0.19, 0.45±0.22, 0.31±0.11, F=17.332, P=0.000) were reduced with the increase of concentration of plumbagin. Compared with the control group, expression of downstream genes bcl-2, VEGF and Cyclin D1 mRNA were decreased significantly after administration of plumbagin (F=33.698, P=0.000; F=337.743, P=0.000; F=180.136, P=0.000), and declined gradually as drug concentration increased. Moreover, bcl-2, VEGF, and Cyclin D1 mRNA levels positively correlated with levels of IL-6 and STAT3 gene expressions. Conclusion IL-6-mediated STAT3 suppression decreased bcl-2, VEGF, and Cyclin D1 expression. Thus, plumbagin may be an effective therapy for large-cell lung cancer that acts by targeting the IL-6/STAT3 signaling pathway. Key words: Plumbagin; Interleukin-6; Signal transducers and activators of transcription 3; Large cell lung cancer

Effects of dietary spermine supplementation on cell cycle, apoptosis, and amino acid transporters of the thymus and spleen in piglets.

ObjectiveThis study investigated whether spermine supplementation could regulate cell cycle, apoptosis, and amino acid transporter-related genes expression in the thymus and spleen of early weaned piglets.MethodsEighty female piglets were randomly distributed to receive adequate nutrients supplemented with spermine (0.4 mmol/kg body weight/24 h) or to be provided with restricted nourishment supplemented with normal saline for 7 h or 3, 6, or 9 d in pairs.ResultsRegardless of administration time, spermine supplementation significantly up-regulated cyclin A2 gene expression but down-regulated p21 and cyclin D3 mRNA levels in the thymus and spleen and reduced cyclin E2 gene expression in the thymus of piglets (p< 0.05). Irrespective of the treatment period, the reduced Bax and caspase-3 gene expressions and improved Bcl-2 mRNA level were observed in the thymus and spleen of spermine-administrated piglets (p<0.05). Regardless of supplementation time, spermine intake significantly enhanced the expressions of amino acid transporter-related genes (SLC1A1, SLC1A5, SLC7A1, SLC7A7, and SLC15A1) in both thymus and spleen, as well as SLC7A9 in the spleen of piglets (p<0.05). In addition, extended spermine administration also markedly promoted cell proliferation, depressed apoptosis and modulated amino acid transport (p< 0.05), and such effects were the greatest during prolonged spermine supplementation (6 d) compared to the other time periods (p<0.05).ConclusionSpermine supplementation may regulate cell cycle during the G1/S phase, suppress apoptosis and modulate amino acid transport. A period of 6 d of spermine supplementation is required to produce the optimal effects on nutritional implications.

Cytotoxic activity of the twigs of Cinnamomum cassia through the suppression of cell proliferation and the induction of apoptosis in human colorectal cancer cells

BackgroundBecause twigs of Cinnamomum cassia (TC) have been reported to exert anti-cancer activity, the mechanistic study for TC’s anti-cancer activity is required. Thus, we elucidated the potential molecular mechanism of TC’s anti-proliferative effect and the induction of apoptosis in human colorectal cancer cells.MethodsHow water extracts form TC (TC-HW) was used in this study. Anti-cell proliferative effect of TC-HW was evaluated by MTT assay. The change of protein or mRNA level by TC-HW was evaluated by Western blot and RT-RCR, respectively. The promoter construct for ATF3, NF-κB, TOP-FLASH or FOP-FLASH was used for the investigation of the transcriptional activity for ATF3, NF-κB or Wnt. siRNA for ATF3 or p65 was used for the knockdown of ATF3 and p65.ResultsTC-HW reduced the cell viability in human colorectal cancer cells. TC-HW decreased cyclin D1 protein level through cyclin D1 degradation via GSK3β-dependent threonine-286 (T286) phosphorylation of cyclin D1, indicating that cyclin D1 degradation may contribute to TC-HW-mediated decrease of cyclin D1 protein level. TC-HW downregulated the expression of cyclin D1 mRNA level and inhibited Wnt activation through the downregulation of β-catenin and TCF4 expression, indicating that inhibition of cyclin D1 transcription may also result in TC-HW-mediated decrease of cyclin D1 protein level. In addition, TC-HW was observed to induce apoptosis through ROS-dependent DNA damage. TC-HW-induced ROS increased NF-κB and ATF3 activation, and inhibition of NF-κB and ATF3 activation attenuated TC-HW-mediated apoptosis.ConclusionsOur results suggest that TC-HW may suppress cell proliferation through the downregulation of cyclin D1 via proteasomal degradation and transcriptional inhibition, and may induce apoptosis through ROS-dependent NF-κB and ATF3 activation. These effects of TC-HW may contribute to the reduction of cell viability in human colorectal cancer cells. From these findings, TC-HW has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

RFRP-3, the mammalian ortholog of GnIH, induces cell cycle arrest at G2/M in porcine ovarian granulosa cells

RFamide-related peptide-3 (RFRP-3), the mammalian ortholog of gonadotropin-inhibitory hormone (GnIH), has been proposed as a key inhibitory regulator of mammal reproduction. Our previous studies have demonstrated that RFRP-3 inhibited the expression of proliferation-related proteins in porcine granulose cells (GCs), but the inhibitory mechanism causing this has not been discovered. Here, we aim to elucidate the underlying mechanism and determine the cell cycle regulatory sites of action of RFRP-3 on porcine GC proliferation. To this end, the viability of porcine GCs was initially estimated by cell counting kit-8 (CCK-8). We confirmed that different doses of RFRP-3 decreased the cellular viability, suggesting that RFRP-3 could inhibit the proliferation of GCs. Subsequently, we evaluated the direct effects of RFRP-3 on the expression of cell cycle regulators. Compared to the control treated cells, 10−6 and 10−8 M of RFRP-3 effectively reduced the transcription of Cyclin B1 and CDK1 mRNAs. However, treatment with RFRP-3 did not alter Cyclin A2, Cyclin D1, CDK2, or CDK4 mRNA levels. These results suggest that RFRP-3 might be inducing G2/M-phase arrest in porcine GCs. Finally, to further determine the molecular mechanism underlying RFRP-3-mediated G2/M cell cycle arrest, we observed the levels of G2/M cell cycle regulatory factors in RFRP-3-treated porcine GCs. The results showed that RFRP-3 treatment significantly increased the expression of Myt1, p-Wee1 and p-Cdc2, whereas the level of Cyclin B1 significantly decreased in porcine GCs treated with 10−6 M of RFRP-3. Taken together, our data suggest that RFRP-3 regulates the phosphorylation or expression of G2/M cell cycle regulatory factors to induce G2/M-phase arrest via inhibition Cyclin B-CDK1 complex activation in porcine GCs, which might provide an unfavorable condition for porcine GC proliferation.

Effect of thimerosal on thyroid hormones metabolism in rats.

Mercury seems to exert an inhibitory effect on deiodinases, but there are few studies using Thimerosal (TM) as the mercury source. We aimed to elucidate the effect of TM on thyroid hormones peripheral metabolism. Adult Wistar female rats received 0.25 µg or 250 µg TM/100 g BW, IM, twice a week, for a month. We evaluated serum total T3 and T4, D1 activity using 125I-rT3 as tracer, and D2 activity using 125I-T4. NADPH oxidase activity was measured by Amplex-red/HRP method and mRNA levels by real time PCR. Serum T4 was increased and T3 decreased by the greatest dose of TM. Even though D1 activity in pituitary and kidney was reduced by the highest dose of TM, hepatic D1 activity and D1 mRNA levels remained unchanged. D2 activity was also significantly decreased by the highest dose of TM in all CNS samples tested, except cerebellum, but D2 mRNA was unaltered. mRNA levels of the tested NADPH oxidases were not affected by TM and NADPH oxidase activity was either unaltered or decreased. Our results indicate that TM might directly interact with deiodinases, inhibiting their activity probably by binding to their selenium catalytic site, without changes in enzyme expression.

The affinity of transthyretin for T3 or T4 does not determine which form of the hormone accumulates in the choroid plexus

Normal development of the brain is dependent on the required amounts of thyroid hormones (THs) reaching specific regions of the brain during each stage of ontogeny. Many proteins are involved with regulation of TH bioavailability in the brain: the TH distributor protein transthyretin (TTR), TH transmembrane transporters (e.g. MCT8, MCT10, LAT1, OATP1C1) and deiodinases (D1, D2 and D3) which either activate or inactivate THs. Previous studies revealed that in mammals, T4, but not T3, accumulated in the choroid plexus and then entered the cerebrospinal fluid. In all mammalian species studied so far, TTR binds T4 with higher affinity than T3, whereas TTR in non-mammalian vertebrates binds T3 with higher affinity than T4. We investigated if the form of TH preferentially bound by TTR influenced the form of the TH that accumulated in the choroid plexus and consequently other areas of the brain. We measured the mRNA levels corresponding to TTR, MCT8, MCT10, LAT1, OATP1C1, D1, D2 and D3 in the brains of chickens at 11days post-hatching. TTR, D3 and OATP1C1 expression were found to be highly concentrated in the choroid plexus. D1, MCT8 and MCT10 mRNA levels were slightly greater in the choroid plexus than in other areas of the brain while D2 mRNA levels were lower. LAT1 mRNA was evenly expressed throughout the brain. Therefore, the choroid plexus appears to be a structure which exhibits sophisticated control of TH levels within the brain. We also measured the uptake of intravenously injected 125I-T3 and 125I-T4 into brains of chickens of the same age. 125I-T4 but not 125I-T3 accumulated in the choroid plexus and optic lobes. Therefore, the form of TH preferentially bound by TTR does not determine the form of TH that accumulates in the choroid plexus and other areas of the brain. As for mammals, T3 present in the avian brain therefore seems mainly produced locally by conversion of T4 into T3 by D2.

Hypoxia stimulates neural stem cell proliferation by increasing HIF‑1α expression and activating Wnt/β-catenin signaling.

Evidence indicates that after brain injury, neurogenesis is enhanced in regions such as hippocampus, striatum, and cortex. To study the role of hypoxia-inducible factor-1 (HIF‑1α) and Wnt signaling in cerebral ischemia/hypoxia-induced proliferation of neural stem cells (NSCs), we investigated the proliferation of NSCs, expression of HIF‑1α, and activation of Wnt signaling under conditions of pathologic hypoxia in vitro. NSCs were isolated from 30-day-old Sprague-Dawley rats and subjected to 0.3% oxygen in a microaerophilic incubation system. Cell proliferation was evaluated by measuring the diameter of neurospheres and by bromodeoxyuridine incorporation assays. Real-time quantitative PCR and Western blotting were used to detect mRNA and protein levels of HIF-1α, β-catenin, and cyclin D1 in the NSCs. The results showed that hypoxia increased NSC proliferation and the levels of HIF-1α, β‑catenin, and cyclin D1 (p &lt; 0.05). Blockade of the Wnt signaling pathway decreased hypoxia-induced NSC proliferation, whereas activation of this pathway increased hypoxia-induced NSC proliferation (p &lt; 0.05). Knockdown of HIF-1α with HIF-1α siRNA decreased β‑catenin nuclear translocation and cyclin D1 expression, and inhibited proliferation of NSCs (p &lt; 0.05). These findings indicate that pathologic hypoxia stimulates NSC proliferation by increasing expression of HIF-1α and activating the Wnt/β-catenin signaling pathway. The data suggest that Wnt/β-catenin signaling may play a key role in NSC proliferation under conditions of pathologic hypoxia.

Abstract 4489: Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis

Abstract AT-Rich Interaction Domain-containing protein 5a (ARID5A; also known as Modulator Recognition Factor 1(MRF1) ) was first cloned in our laboratory by screening a cDNA library of Tera-2 mRNA with an oligonucleotide probe from the major immediate early gene of human cytomegalo virus. It has been reported that ARID5A forms a complex with androgen receptor (AR) in yeast two hybrid assays and is a cosuppressor of AR in transient transfection assays. In this study, we report that the knockdown of ARID5a expression inhibits proliferation of LNCaP cells on the contrary to the expectation from the transient transfection assay. We found that proliferation of cells stimulated by the treatment with dihydro-testosterone (DHT) was suppressed when ARID5a expression was down-regulated by siRNA technology. Androgen stimulates cell proliferation through activation of mammalian target of rapamycin complex 1 (mTORC1). However, the suppression of ARID5a does not affect androgen-activated mTOR activity. Furthermore, the expression of androgen-inducible and androgen-suppressive genes were not affected by suppression of ARID5a. We concluded that ARID5a is not a cosuppressor of AR. Meanwhile, flow cytometer analysis showed that DHT-stimulated cell cycle was arrested at G1 phase after the knockdown of ARID5a expression. Oil Red O staining assays showed that the inhibition of ARID5a expression led to decreased DHT-induced lipid accumulation. Our data further proved that the knockdown of ARID5a expression inhibited the protein expression of DHT-induced sterol regulatory element binding transcription factor 1 (SREBP-1). In addition, Western blot analysis revealed that the down-regulation of ARID5a decreased the expression of cell survival / proliferation and cell cycle regulation proteins, such as hypoxia inducible factor 1 alpha (HIF1a), cyclin D1 and cyclin D3. Real time PCR data revealed that the knockdown of ARID5a expression did not affect the steady level of HIF1a, cyclin D1 and cyclin D3 mRNA. These suggested that knockdown of ARID5a expression affected protein expression at translational and/or post-translational level. Our data suggested that knockdown of ARID5a expression increased the phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) which inhibited translation initiation. S35 labeling assay confirmed that the knockdown of ARID5a expression suppressed the global protein synthesis. We showed that one of the four eIF2a kinases, general control non-derepressible 2 (GCN2) was activated by the suppression of ARID5a expression. We are currently studying on the mechanisms by which ARID5A regulates GCN2 kinase. Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Knockdown ARID5A suppresses proliferation of LNCaP prostate cancer cell through the inhibition of global protein synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4489. doi:10.1158/1538-7445.AM2017-4489

Abstract 3137: Breast cancer anti-estrogen resistance 4 (BCAR4) is a novel oncogene in lung cancer

Abstract BCAR4 was identified as tamoxifen resistance related gene in breast cancer due to its function to activate estrogen-independent cell growth. Instead of estrogen receptor, ERBB2 and ERBB3 genes were activated by BCAR4 in tamoxifen resistant cells. Recently, the function of BCAR4 as a long non-coding RNA (lncRNA) was reported to regulate a non-canonical Hedgehog/GLI2 pathway in breast cancer. However, the association of BCAR4 and lung cancer has not been identified. Here, we describe the oncogenic effect of BCAR4 in human lung cancer. Expression pattern of BCAR4 was examined in lung cancer cell lines, primary tumor tissues, and adjacent normal tissues. BCAR4 was highly expressed in 71% of lung cancer cell lines and especially in 43% of cancer tissues of lung adenocarcinoma patients who did not harbor the activational mutations of EGFR nor KRAS. Exogenous expression of BCAR4 protein promoted cell growth of non-tumorigenic bronchial epithelial cell line, BEAS-2B, and lung cancer cell line, NCI-H1299. We also confirmed the oncogenic feature of BCAR4 by colony forming assay using stably expressing cells. These effects were notably reduced after knockdown of BCAR4 by small interfering RNA. BCAR4 encoded a functional protein and its overexpression resulted in the enhanced migration than controls cells. This result proposed a similar role of BCAR4 in lung cancer considering its previously reported role in breast cancer metastasis. We also detected a fusion gene of BCAR4 in a lung cancer tissue by analyzing RNA-sequencing data. BCAR4 protein as well as its fusion form showed the improved tumorigenic feature in the mouse xenograft model with lung cancer cells. We compared the gene expression pattern in BCAR4 overexpressed cells to identify the possible mechanism of the oncogenic effect of BCAR4. Cyclin D1, Cyclin E, and MMP1 mRNA and protein levels were significantly increased after BCAR4 overexpression. However, downstream genes of GLI2 signaling were not altered by protein expression of BCAR4 arguing an alternative role for lncRNA BCAR4. Our results suggest a novel oncogenic role of BCAR4 protein in lung cancer. Citation Format: Kieun Bae, Minkyong Lee, Daseul Yoon, Yun-Hee Kim, Kyong-Ah Yoon. Breast cancer anti-estrogen resistance 4 (BCAR4) is a novel oncogene in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2017-3137

Suppression of the Wnt signaling pathway may contribute to the inhibition of proliferation of human hepatocellular carcinoma SMMC-7721 cells by esculetin.

The aims of the present study were to investigate the effects of esculetin on the proliferation of human hepatocellular carcinoma SMMC-7721 cells and to determine the underlying mechanism behind this activity. An MTT assay was used to assess cell proliferation, reverse transcription-quantitative polymerase chain reaction was used to determine the relative mRNA expression levels of β-catenin, c-Myc and cyclin D1, and western blot analysis was utilized to determine the levels of the associated proteins. Compared with the dimethyl sulfoxide control, esculetin reduced the cell viability of SMMC-7721 and HL-7702 cells in a dose- and time-dependent manner. Treatment of SMMC-7721 cells with esculetin resulted in downregulation of the mRNA and protein levels of β-catenin, c-Myc and cyclin D1. Esculetin increased the phosphorylation of β-catenin at Ser33/Ser37/Thr41 and inhibited the proliferation of human hepatoma SMMC-7721 cells by suppressing the Wnt signaling pathway. The results of the present study suggest that esculetin inhibited the Wnt/β-catenin signaling pathway in SMMC-7721 cells and may have potential as an effective anti-cancer drug, acting to inhibit the Wnt/β-catenin signaling pathway.

Anti-cancer activity of Bacillus amyloliquefaciens AK-0 through cyclin D1 proteasomal degradation via GSK3β-dependent phosphorylation of threonine-286.

Microorganisms have been regarded as important sources of novel bioactive natural products. In this study, we evaluated the anti-cancer activity and the potential mechanism of Bacillus amyloliquefaciens AK-0 newly isolated from the rhizosphere soil of Korean ginseng. The ethyl acetate fraction from the culture medium of B. amyloliquefaciens AK-0 (EA-AK0) inhibited markedly the proliferation of human colorectal cancer cells such as HCT116, SW480, LoVo and HT-29. EA-AK0 effectively decreased cyclin D1 protein level in human colorectal cancer cells, while cyclin D1 mRNA level was not changed by EA-AK0 treatment. Inhibition of proteasomal degradation by MG132 blocked EA-AK0-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, EA-AK0 increased threonine-286 (T286) phosphorylation of cyclin D1, and a point mutation of T286 to alanine attenuated cyclin D1 degradation by EA-AK0. Inhibition of GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by EA-AKO. From these results, EA-AK0 may suppress the proliferation of human colorectal cancer cells by inducing cyclin D1 proteasomal degradation through GSK3β-dependent T286 phosphorylation. These results indicate that EA-AK0 could be used for treating colorectal cancer and serve as a potential candidate for anticancer drug development. In addition, these findings will be helpful for expanding the knowledge on the molecular anti-cancer mechanisms of EA-AK0.

Tyr23 phosphorylation of Anxa2 enhances STAT3 activation and promotes proliferation and invasion of breast cancer cells.

Overexpression of Annexin A2 (Anxa2) is positively correlated with breast cancer progression, drug resistance, and poor prognosis of patients with breast cancer. Tyr23 Phosphorylation by Src-family tyrosine kinase is an important post-translational modification of Anxa2. This modification regulates the subcellular localization and functions of Anxa2 and has significant effects on cell proliferation, migration, and invasion. This study aims at revealing the association of Anxa2-Tyr23 phosphorylation in Anxa2-mediated acceleration of breast cancer progression and their elaborate molecular mechanisms. Cell biological function experiments were performed to determine the effects of Anxa2-Tyr23 Phosphorylation on breast cancer cell proliferation and invasion in vitro and metastasis in vivo. The interaction of Tyr23 phosphorylated Anxa2 and STAT3 was verified by co-immunoprecipitation assay. Related mRNA and protein expression levels of cyclin D1 and MMP2/9 and phosphorylation level of STAT3 were detected. Anxa2-Tyr23 phosphorylation is necessary for proliferation, invasion, and metastasis of breast cancer cells in vitro and in vivo. Tyr23 phosphorylated Anxa2 binds and enhances the sensitivity of STAT3 activation in response to IL-6, thereby increasing the protein and mRNA expression levels of cyclin D1 and MMP2/9 which are STAT3 key target genes and serve pivotal regulatory functions in cell proliferation and invasion, respectively. Our findings further confirmed the regulatory role of Anxa2 and revealed the direct relationship between Anxa2-Tyr23 phosphorylation and activation of STAT3. Moreover, this study provides novel insights into the function of Anxa2-Tyr23 phosphorylation in signal transduction for further understanding of the mechanism through which Anxa2 promotes the progression of breast cancer.

MicroRNA‑146a promotes the proliferation of rat vascular smooth muscle cells by downregulating p53 signaling.

The present study aimed to detect and verify gene expression profile differences for microRNA (miR)‑146a and its role in the proliferation of vascular smooth muscle cells (VSMCs). Artificially synthesized miR‑146a mimics, miR‑146 inhibitor, scramble‑miRNA or PBS was transfected into cultured primary rat VSMCs invitro. Reverse transcription‑quantitative polymerase chain reaction confirmed that the miR‑146a expression level was significantly decreased in VSMCs treated with miR‑146a inhibitor (P<0.01). Cell Counting Kit‑8 was used to determine the proliferation ability, which demonstrated that proliferation was significantly decreased in VSMCs treated with miR‑146a inhibitor (P<0.01). Microarray expression profiling analysis revealed that the p53 signal pathway was upregulated in VSMCs treated with the miR‑146a inhibitor. Compared with untransfected VSMCs, the mRNA and protein expression levels of caspase‑3 and phosphatase and tensin homolog (PTEN) in p53 signal transduction pathway did not exhibit a significant difference (P>0.05); however, the mRNA and protein expression levels of p53 were significantly decreased in cells transfected with miR‑146a mimics and increased in miR‑146a inhibitor transfected cells (both P<0.01). The mRNA and protein expression levels of cyclin D1 significantly increased in miR‑146a mimics transfected cells and decreased in cells transfected with the miR‑146a inhibitor (both P<0.05). The present data indicated that miR‑146a may promote the proliferation of rat VSMCs by downregulating p53 and upregulating cyclin D1 expression.

Cibotium barometz polysaccharides stimulate chondrocyte proliferation invitro by promoting G1/S cell cycle transition.

Cibotium barometz polysaccharides (CBPS) are one of the most important bioactive components extracted from the Cibotium barometz plant, which belongs to the Dicksoniaceae family. It has been widely used for the treatment of orthopedic diseases in traditional Chinese medicine. However, the molecular mechanisms behind the therapeutic effects of CBPS remain to be clarified. In the present study, the concentration of CBPS was detected by phenol-vitriol colorimetry. Furthermore, the effects stimulated by CBPS on the viability and G1/S cell cycle transition in primary chondrocytes from Sprague-Dawley rats were investigated. A cell viability assay demonstrated that chondrocyte proliferation may be enhanced by CBPS in a dose- and time-dependent manner. The mechanism underlying the promotion of chondrocyte cell cycle was suggested to involve the stimulation of G1 to S phase transition. To further confirm the results, reverse transcription-quantitative polymerase chain reaction and western blot analyses were used to detect the expression of mRNA and protein levels of cyclin D1, cyclin-dependent kinase 4 and retinoblastoma protein. The results suggested that CBPS may stimulate chondrocyte proliferation via promoting G1/S cell cycle transition. Since osteoarthritis is characterized by deficient proliferation in chondrocytes, the present study indicates that CBPS may potentially serve as a novel method for the treatment of osteoarthritis.

Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases

ABSTRACTFlaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21WAF1/CIP1, a negative regulator of the G1 phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G1-phase cyclins and CDKs, and upregulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

Identification and analysis of ZFPM2 as a target gene of miR-17-92 cluster in chicken.

The miR-17-92 cluster plays important roles in a variety of physiological and pathological processes in mammals. Previously, we showed that miR-17-92 cluster promotes chicken preadipocyte proliferation; however, the mechanism for its action is unknown. In order to explore the mechanism by which miR-17-92 cluster promotes chicken preadipocyte proliferation, CCK8 proliferation assay was performed to determine the effect of ZFPM2 knockdown on chicken preadipocyte proliferation. The results showed that ZFPM2 knockdown significantly promoted chicken preadipocyte proliferation (P<0.01). Consistent with the CCK8 results, the mRNA levels of cell proliferation marker genes, i.e., Cyclin D1, PCNA and Ki67, were markedly increased in the si-ZFPM2-transfected preadipocytes (P<0.01 or P<0.05). Bioinformatics analysis showed that there were two potential miRNA binding sites for the four individual members of miR-17-92 cluster in the ZFPM2 3'UTR, one for miR-17-5p and miR-20a and the other for miR-19a and miR-19b. To test whether ZFPM2 is a target for the miR-17-92 cluster, the ZFPM2 3'UTR reporter (psi-CHECK2-ZFPM2-3'UTR-WT) and its mutant reporter (psi-CHECK2-ZFPM2-3'UTR-MUT) were constructed. Reporter assays showed that overexpression of miR-17-92 cluster significantly inhibited the luciferase reporter activity of psi-CHECK2-ZFPM2-3'UTR-WT (P<0.01), as compared with control vector (empty pcDNA3.1). Transfection of miR-17-5p, miR-19a and miR-20a inhibitors increased the reporter activities of psi-CHECK2-ZFPM2-3'UTR-WT (P<0.01 or P<0.05). In contrast, transfection of miR-17-5p, miR-19a, and miR-20a inhibitors had no obvious effect on reporter activity of psi-CHECK2-ZFPM2-3'UTR-MUT. Further qRT-PCR analysis showed that miR-17-5p, miR-20a and miR-19a inhibitors significantly elevated the endogenous ZFPM2 mRNA expression (P<0.01 or P<0.05). Cotransfection of either miR-17-5p or miR-19a inhibitor and siZFPM2 showed that both inhibitors tended to reduce only slightly the promoting effect of siZFPM2 on chicken preadipocyte proliferation. Taken together, these data demonstrated that ZFPM2 is a target of miR-17-5p, miR-20a, miR-19a, and miR-19b, and that miR-17-92 cluster promotes chicken preadipocyte proliferation at least in part by targeting ZFPM2 and inhibiting its expression.

Effects of SOST Gene Silencing on Proliferation, Apoptosis, Invasion, and Migration of Human Osteosarcoma Cells Through the Wnt/β-Catenin Signaling Pathway.

Our study explored the effects of SOST gene silencing on the proliferation, apoptosis, invasion, and migration of human osteosarcoma cells through Wnt/β-catenin signaling pathway. Fresh tissues were obtained from 108 patients with osteosarcoma and 46 patients with osteochondroma. Human osteosarcoma cells (MG-63, U2-OS, HOS, and Saos-2) and normal osteoblast (hFoB1.19) were selected and cultured. Osteosarcoma cells were grouped randomly into the blank group, the scrambled control group, and the SOST-siRNA group. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis were tested by flow cytometry. Transwell and scratch test were performed to determine cell invasion and migration. The qRT-PCR and Western blotting were used to detect mRNA and protein expression level of sclerostin, Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7. The activity of caspase-3 was assessed by immunocytochemistry. Alkaline phosphatase (ALP) activity was measured using P-nitrophenylphosphate as a substrate. Low SOST mRNA and sclerostin protein expression levels were observed in osteosarcoma tissues and cells. Compared with the blank and scrambled control groups, sclerostin expression, apoptotic cells, ALP activity, and caspase-3 activity were down-regulated, while the proliferation, invasion, and migration abilities of osteosarcoma cells were evidently enhanced in the SOST-siRNA group. After SOST gene silencing, the mRNA and protein expression levels of Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7 in osteosarcoma cells and β-catenin protein expression levels in the nucleus and cytoplasm were significantly elevated. SOST gene silencing promotes the proliferation, invasion, and migration, and inhibits apoptosis of osteosarcoma cells by activating Wnt/β-catenin signaling pathway.

LncRNA, TUG1 regulates the oral squamous cell carcinoma progression possibly via interacting with Wnt/β-catenin signaling

Oral squamous cell carcinoma (OSCC) is one the most common cancer affecting the head and neck region, and the molecular mechanisms underlying OSCC development is largely unknown. Long non-coding RNAs (lncRNAs) are emerging as key regulators in tumor development. The present study aimed to investigate the role of lncRNA, taurine upregulated gene 1 (TUG1) in OSCC development. The mRNA and protein expression levels were determined by qRT-PCR and western blotting; flow cytometry and ELISA experiments were employed to examine the cell apoptosis; CCK-8 assay, MTT assay, colony formation assay, and cell invasion assay was used to determine cell growth, cell proliferation and cell invasion, respectively. qRT-PCR results showed that TUG1 was up-regulated in both OSCC tissues and cell lines. The high expression level of TUG1 was significantly correlated with TNM stage, lymph node metastasis and tumor grade in OSCC patients. CCK-8 assay, MTT assay, colony formation assay, and cell invasion assay results showed that knock-down of TUG1 by siRNA transfection suppressed cell growth, cell proliferation, and cell invasion in OSCC cell lines (Tca8113 and TSCCA). The cell apoptosis was induced in Tca8113 and TSCCA cells transfected with TUG1 siRNA. In addition, knock-down of TUG1 in Tca8113 and TSCCA cells significantly suppressed the mRNA and protein expression levels of β-catenin, cyclin D1, and c-myc. Wnt/β-catenin pathway activator (LiCl) reversed the TUG1 knock-down effect on cell proliferation, cell invasion and cell apoptosis in Tca8113 and TSCCA cells. In summary, knock-down of TUG1 suppressed cell growth, proliferation and invasion, and also induced apoptosis of OSCC possibly via targeting Wnt/β-catenin signaling. Our data suggest that knock-down of TUG1 may represent a novel therapeutic target for the management of OSCC.