CDKN2B Expression Research Articles

UC.183, UC.110, and UC.84 Ultra-Conserved RNAs Are Mutually Exclusive with miR-221 and Are Engaged in the Cell Cycle Circuitry in Breast Cancer Cell Lines

In the human genome, there are about 600 ultra-conserved regions (UCRs), long DNA sequences extremely conserved in vertebrates. We performed a large-scale study to quantify transcribed UCR (T-UCR) and miRNA levels in over 6000 cancer and normal tissue samples to find possible correlation between these kinds of regulatory molecules. Our analysis evidenced several non-coding RNAs showing negative co-regulation with miRNAs; among them, we focused on miR-221 to investigate any relationship with its pivotal role in the cell cycle. We have chosen breast cancer as model, using two cell lines with different phenotypes to carry out in vitro treatments with siRNAs against T-UCRs. Our results demonstrate that the expression of uc.183, uc.110, and uc.84 T-UCRs is mutually exclusive with miR-221 and is engaged in the regulation of CDKN1B expression. In addition, tests with a set of anticancer drugs, including BYL719, AZD5363, AZD8055, AZD7762, and XL765, revealed the modulation of specific T-UCRs without alteration of miR-221 levels.

RAAV-delivered PTEN therapeutics for prostate cancer

Effective treatments for prostate cancer (PCa) require further development, and previous studies have reported that PTEN and its downstream target CDKN1B are significantly downregulated in PCa cells compared with normal cells. Therefore, modulation of PTEN and CDKN1B expression might be a promising therapeutic approach for PCa treatment. Expression of PTEN and CDKN1B was verified in specimens from PCa patients and transgenic adenocarcinoma mouse prostate (TRAMP) mice. The effect of PTEN on PCa cell migration, apoptosis, and the cell cycle was analyzed in vitro using a wound-healing assay and flow cytometry. We assessed the ability of intraprostatic and intratumoral injections of recombinant adeno-associated virus (rAAV) 9 expressing Pten or Cdkn1b into TRAMP mice and a subcutaneous tumor xenograft mouse model, respectively, to inhibit PCa progression. PTEN and CDKN1B were significantly downregulated in human and mouse PCa samples, and CDKN1B expression correlated positively with PTEN expression. PTEN overexpression significantly inhibited cell migration and cell-cycle progression and promoted apoptosis in PCa cells by decreasing Ccnd1 expression and increasing that of Cdkn1b. Importantly, treatment with the rAAV9.Pten or rAAV9.Cdkn1b extended the lifespan of TRAMP mice and inhibited the growth rate of tumor xenografts by regulating downstream gene expression. Moreover, neoplasia in treated prostates was significantly diminished compared with that in control prostates, and apoptosis was markedly observed in xenografts treated with Pten or Cdkn1b. These data indicate that rAAV-based PTEN/CDKN1B delivery is promising for the development of novel therapeutics for PCa.

Abstract 13218: CITED4 is Required for Exercise-Induced Cardiac Growth and Cardiomyogenesis in Mice

Introduction: CITED4 (CBP/p300-interacting transactivators with E [glutamic acid]/D [aspartic acid]-rich-carboxylterminal domain 4) is a transcriptional regulator which limits pathologic remodeling after ischemic and pressure overload models of cardiac stress. It is also upregulated in the heart with exercise, an effective intervention which induces physiological hypertrophy and cardiomyogenesis in adult mice. We hypothesized that CITED4 is required for exercise-induced cardiac growth and cardiomyogenesis. Methods and Results: Eight- to ten-week-old mice with cardiomyocyte-specific CITED4 knockout (C4KO) and littermate controls underwent 8 weeks of voluntary wheel running in comparison to sedentary controls. 5-Ethynyl-2′-deoxyuridine (EdU) was injected subcutaneously every two days for the first two weeks. There were no significant differences in cardiac size and function at baseline between C4KO and control mice. In control mice, running increased heart weight to tibial length ratio (HW/TL, 6.26±0.25 vs 7.37±0.75, p=0.01, n=5-9 per group), heart weight to body weight ratio (HW/BW, 4.08±0.40 vs 4.93±0.48, p=0.01), and cardiomyocyte (CM) size without affecting fractional shortening (FS), consistent with physiologic remodeling. There was also a nonsignificant trend to increased EdU+ CMs in the control exercised mice (2.96±2.34% vs 5.23±6.28%, p=0.44), suggestive of previously documented exercise-induced cardiomyogenesis. In contrast, running did not change HW/TL and CM size in C4KO mice (HW/TL, 6.44±0.48 vs 6.66±0.29, p=0.22, n= 0-13 per group) but reduced %FS (58.78±3.27 vs 53.67±4.59, p=0.03) and increased chamber size (LVIDs, 1.32±0.13 vs 1.50±0.17, p=0.032), suggesting maladaptive cardiac remodeling with exercise, confirming prior findings observed in a more extreme swim-exercise model. Rather than the expected increase, hearts from exercised C4KO mice had fewer EdU+ CMs compared to sedentary C4KO mice (3.76±2.60% vs 2.11±0.76%, p=0.05) and reduced expression of cell cycle genes Cdkn1b, Cyclin B1, Cdkn2a, and Ki-67. Conclusions: CITED4 is required for exercise-induced cardiomyogenesis, CM hypertrophy, and adaptive remodeling. CITED4 deficiency leads to maladaptive cardiac remodeling with physiologic stress.

Abstract P231: Aspirin Reverses Placental Gene Expression Changes Associated With Preeclampsia

Aspirin is the only medication recommended for preventing preeclampsia, a hypertensive disorder of pregnancy. However, the mechanism(s) by which aspirin prevents preeclampsia are unknown. To better understand the mechanism by which aspirin prevents preeclampsia, we compared expression of genes involved in signal transduction, cardiac remodeling, ion transport, stress and immune response, apoptosis, sarcomere structure, transcriptional regulation, and cell cycle regulation between placental tissues from 4 different cohorts: preeclamptic women, preeclamptic women who took aspirin during pregnancy, non-preeclamptic women, and non-preeclamptic women who took aspirin (N=3 per cohort). Tissues were obtained from the University of Iowa Maternal Fetal Tissue Bank (IRB# 200910784). When comparing whole thickness samples, 27 genes had a greater than 5-fold change in expression in the tissues of women with preeclampsia compared to those without. For example, CDKN1B whose protein inhibits the cell cycle, is increased in preeclampsia (-3.7ΔΔCt, 13.4-fold increase). However, in women who took aspirin, both those that developed preeclampsia and those who did not, CDKN1B expression was similar to women without preeclampsia who did not take aspirin indicating a decrease in gene expression in response to aspirin. However, aspirin also increases expression of several genes associated with preeclampsia. For example, we identified that RGS2 and PLAC1 in the decidual layer of the placenta were increased in response to aspirin. Decreases in expression of both genes is associated with preeclampsia. However, 19 genes whose expression is increased in preeclampsia compared to controls had no response to aspirin. Identifying specific genes and pathways that serve as targets of aspirin during pregnancy provides valuable insight into the mechanisms by which aspirin reduces the occurrence of preeclampsia and may help to identify who would benefit most from its use. Additionally, it may identify targets for the development of other preeclampsia preventative or therapeutic medications.

Targeting BRD9 by I-BRD9 efficiently inhibits growth of acute myeloid leukemia cells.

BackgroundAcute myeloid leukemia (AML) is characterized by genetic and epigenetic mutations that lead to a block in differentiation as well as unrestrained proliferation. Many epigenetic regulators have been shown to be important for AML initiation and development. Among these, bromodomain-containing protein 9 (BRD9), an epigenetic regulator, was recently identified as a critical factor required for AML development. Hence targeting BRD9 may provide a new therapeutic strategy. Thus, we investigated the role of BRD9 inhibitor I-BRD9 in AML cells and its potential mechanisms.MethodsCell Counting Kit-8 (CCK-8) assays were performed to explore the growth inhibitory effects of I-BRD9 on AML cells. Flow cytometry was used to examine the effects of I-BRD9 on apoptosis, Edu incorporation, and cell differentiation. Apoptotic pathway activation was confirmed by western blot. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was employed to analyze cell death and cell cycle-related gene expression.ResultsI-BRD9 significantly reduced AML cells growth. This is accompanied by decreased Edu incorporation and dramatic cell death. Mechanistically, cell death induced by I-BRD9 was largely blocked by the pan-caspase inhibitor Z-VAD-FMK and, to a lesser extent, by Ferrostatin-1.Furthermore, apoptotic markers including the cleavage of PARP, Capase9, and Capsese3, were induced by I-BRD9, which were rescued by pretreatment with Z-VAD-FMK. In addition, I-BRD9 treatment increased IRE3, CDKN1A, and CDKN2B expression in AML cells, possibly leading to the observed decrease in Edu incorporation. Together, these data strongly suggested that I-BRD9 induced growth inhibition in AML cells was dependent on apoptosis and cell cycle inhibition.ConclusionsOur data support the important role of BRD9 in AML cells; moreover, the BRD9 inhibitor I-BRD9 could be potentially useful in the treatment of AML .

BCL6 Enables RAS-Driven Pre-B Cell Transformation in Acute Lymphoblastic Leukemia

Background and Hypothesis: The transcriptional repressor and proto-oncogene BCL6 was previously identified to be a factor that bypasses oncogene-induced senescence (OIS) caused by activation of oncogenic RAS (Shvarts A. et al. 2002). Recently, we discovered a novel BCL6-dependent mechanism of drug resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL; Duy et al., 2011). Given that activating genetic lesions in the RAS pathway are found in ~50% of high-risk childhood pre-B ALL at diagnosis (Zhang et al., 2012), we tested the concept that BCL6 may facilitate RAS-mediated transformation of pre-B cells by suppressing the OIS-program.Results: To examine the consequences of acute activation of RAS-ERK signaling on Bcl6 expression, we engineered murine pre-B cells to carry a doxycycline (Dox)-inducible TetOn- NRASG12D vector system. Following induction with Dox, strong upregulation of Bcl6 expression was observed at both the mRNA and protein levels, which were abrogated upon treatment with a MEK inhibitor (PD325901). These findings suggest that increased Bcl6 levels were a consequence of ERK activation. Similarly, inducible expression of BRAF V600E mutant resulted in elevated levels of Bcl6 in parallel with increased phosphorylation of ERK1/2- T202/Y204. Furthermore, studying a diagnostic (KRAS wild-type) and a relapsed (KRASG12V) sample from one pre-B ALL patient revealed hyperactivation of ERK in association with increased BCL6 levels in KRASG12V relapsed ALL cells.Previously, we showed that BCL6 expression is repressed by STAT5 in pre-B ALL (Duy C. et al. 2011). Interestingly, while overexpression of oncogenic NRASG12D resulted in hyperactivation of ERK in pre-B cells, phosphorylation of STAT5-Y694 was drastically inhibited. Reverse Phase Protein Array (RPPA) measurements revealed that phosphorylation levels of MEK-S217/221 and ERK1/2-T202/Y204 were inversely correlated with phosphorylation of STAT5-Y694 in newly diagnosed cases of adult ALL (MDACC, 1983-2007). In addition, genetic lesions causing activation of the RAS-ERK and STAT5 pathways were found to be mutually exclusive in the majority of high-risk childhood pre-B ALL cases studied (COG P9906). Mechanistically, expression of oncogenic NRASG12D resulted in activation of PTPN6 (SHP1), an inhibitory protein tyrosine phosphatase. Loss of Ptpn6 function resulted in elevated levels of phospho-STAT5-Y694 and suppressed imatinib-mediated induction of Bcl6 expression in BCR-ABL1 -driven pre-B ALL. Taken together, our findings suggest that oncogenic RAS induces BCL6 expression through PTPN6-mediated dephosphorylation of STAT5.Our observations that RAS-ERK signaling induces BCL6 expression prompted us to examine the role of BCL6 in RAS-mediated malignant transformation of pre-B cells. Activation of oncogenic RAS increased expression of cell cycle checkpoint regulators CDKN1A (p21) and CDKN1B (p27). BCL6 has previously been shown to mediate transcriptional repression of CDKN1A and CDKN1B (Duy C. et al. 2011). Notably, ChIP-seq analysis of patient-derived KRASG12V pre-B ALL cells revealed binding of BCL6 across the loci of cell cycle checkpoint regulators CDKN1A, CDKN1B, CDKN2D and TP53BP1, as well as pro-apoptotic molecules BIM and BMF . Consistent with these findings, Cre-mediated deletion of Bcl6 in pre-B cells expressing NRASG12D led to depletion of cells in competitive-growth assays and impaired the ability to form colonies in semi-solid methylcellulose. Moreover, expression of NRASG12Din Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in transplant recipients.Conclusions: Our findings suggest that activation of oncogenic RAS enables the switch from STAT5-driven signaling to activation of BCL6, and that RAS-induced dephosphorylation of STAT5 is mediated by PTPN6. Furthermore, we demonstrated that BCL6 is required for RAS -mediated transformation of pre-B cells, suggesting that inhibition of BCL6 may provide a new therapeutic avenue. DisclosuresHurtz:Incyte: Research Funding.

Genetic and epigenetic bases of prostate tumor cell radioresistance.

Radiation therapy plays aleading role in the treatment of prostate cancer, but the emergence of radioresistant forms of this disease dictates the need for apersonalized ap-proach based on the data from genetic and epigenetic markers. Such markers include the copy number variation as well as gene and microRNA expression. The aim of the study was to validate the list of potential predictors of radioresistance of prostate tumor cells in amodel experiment based on the determination of gene copy number variation, gene transcriptional activity and microRNA expression. The study used aPC-3prostate cancer cell culture. The determination of the relative copy number variation and expression of 32genes (BRCA1, BRCA2, PTEN, CASP3, CASP8, BAX, BCL2, CASP9, P53, MDM2, AKT1, ATM, BRIP1, CDK1, CDKN1B, CCND1, CCND3, FGFR2, KU70, RAD50, RAP80, Rif1, RNF168, TopBP1, HIST, H2AX, EXO1, XRCC4, RBBP8, EP300, LIG4, C-FLIP), as well as 15microRNAs (let-7, miR15a/ 16, miR-17, miR-18a, miR-21, miR-24, miR-26b, miR-99a, miR-100, miR-101, miR-106a, miR-663a, miR-143, miR-145) was performed using the real-time quantitative polymerase chain reaction method. It was found that daily irradiation of PC-3cells on aNovalis TX linear accelerator at doses of 6and 7Gy for 5days leads to asignificant decrease in the total number of cells and the number of viable cells. Nevertheless, after 5days of irradiation, about 15% of the initial number of prostate tumor cells retained their viability, which is due to their special genetic and epigenetic characteristics: increased copy number and expression of genes BRCA2, CDK1, CDKN1B, H2AX, RAD50, XRCC4, RBBP8and EP300and reduced copy number and expression of CCND3, TP53, and BCL2genes, as well as differential expression of sixmicroRNAs (hsa-miR-18a-5p, hsa-miR-24-5p, hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-145-5p, hsa-let-7a-3p). This study enabled to identify genetic and epigenetic markers of prostate tumor cells resistance to radiation therapy.

MiR-29b restrains cholangiocarcinoma progression by relieving DNMT3B-mediated repression of CDKN2B expression.

Numerous studies have reported the important role of microRNAs (miRNAs) in human cancers. Although abnormal miR-29b expression has been linked to tumorigenesis in several cancers, its role in cholangiocarcinoma remains largely unknown. We found that miR-29b expression is frequently downregulated in human cholangiocarcinoma QBC939 cells and in clinical tumor samples. In cholangiocarcinoma patients, low miR-29b expression predicts poor overall survival. Overexpression of miR-29b in QBC939 cells inhibited proliferation, induced G1 phase cycle arrest, and promoted apoptosis. Methylation-specific PCR (MSP) analysis revealed a decreased methylation imprint at the promoter of the cell cycle inhibitor gene CDKN2B in cells overexpressing miR-29b. After identifying the DNA methyltransferase DNMT3B as a putative miR-29b target, luciferase reporter assays confirmed a suppressive effect of miR-29b on DNMT3B expression. Accordingly, we detected an inverse correlation between miR-29b and DNMT3B expression in clinical cholangiocarcinoma specimens. In QBC939 cells, DNMT3B overexpression promoted proliferation and inhibited apoptosis. DNMT3B silencing, in turn, led to increased CDKN2B expression. We also observed significant growth arrest in subcutaneous tumors formed in nude mice by QBC939 cells overexpressing miR-29b. These findings suggest miR-29b functions as a tumor suppressor in cholangiocarcinoma by relieving DNMT3B-mediated repression of CDKN2B expression.

PATZ1 (MAZR) Co-occupies Genomic Sites With p53 and Inhibits Liver Cancer Cell Proliferation via Regulating p27.

Liver cancer is the third most common cause of cancer death in the world. POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1/MAZR) is a transcription factor associated with various cancers. However, the role of PATZ1 in cancer progression remains controversial largely due to lack of genome-wide studies. Here we report that PATZ1 regulates cell proliferation by directly regulating CDKN1B (p27) in hepatocellular carcinoma cells. Our PATZ1 ChIP-seq and gene expression microarray analyses revealed that PATZ1 is strongly related to cancer signatures and cellular proliferation. We further discovered that PATZ1 depletion led to an increased rate of colony formation, elevated Ki-67 expression and greater S phase entry. Importantly, the increased cancer cell proliferation was accompanied with suppressed expression of the cyclin-dependent kinase inhibitor CDKN1B. Consistently, we found that PATZ1 binds to the genomic loci flanking the transcriptional start site of CDKN1B and positively regulates its transcription. Notably, we demonstrated that PATZ1 is a p53 partner and p53 is essential for CDKN1B regulation. In conclusion, our study provides novel mechanistic insights into the inhibitory role of PATZ1 in liver cancer progression, thereby yielding a promising therapeutic intervention to alleviate tumor burden.

Smad4 deficiency substitutes Cdkn2b but not Cdkn2a downregulation in pancreatic cancer following induction of genetic events in adult mice

BackgroundMinor progress in pancreatic cancer treatment and prognosis implies that more reliable animal models are urgently needed to decipher its molecular mechanisms and preclinical research. We recently reported a genetically engineered adult mouse model where Cdkn2b downregulation was required together with Cdkn2a downregulation to inactivate the Rb pathway. Besides, the role of Smad4, which is mutated more frequently than Cdkn2b in human pancreatic cancer, was determined critical on the development of the pancreas tumor by some reports. However, the impact of Smad4 deficiency in combination with PDAC-relevant mutations, such as Cdkn2a when induced in adult pancreas has not been completely elucidated in mice. MethodsLentiviral delivered oncogene/tumor suppressors in adult pancreas. The development of pancreatic cancer was monitored. Hematoxylin and eosin staining and immunofluorescence were performed for pathological identification of the pancreatic cancer. Real-time polymerase chain reaction, immunofluorescence and western blot were used to test gene expression. ResultsLoss of Smad4 could cooperate with alterations of KRAS, Trp53, and Cdkn2a to induce pancreatic cancer in adult mice. The role of Smad4 was mainly in downregulating the expression of Cdkn2b and further inducing phosphorylation of the Rb1 protein. ConclusionsThese findings show an essential role of Smad4 deficiency in pancreatic ductal adenocarcinoma (PDAC) formation. This model better recapitulates the adult onset, clonal origin, and genetic alterations in human PDAC and can be simply generated on a large-scale.

PRMT5 functionally associates with EZH2 to promote colorectal cancer progression through epigenetically repressing CDKN2B expression.

Background: Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that symmetrically di-methylates arginine residues on both histone and non-histone protein substrates. Accumulating evidence suggests that PRMT5 exerts its oncogenic properties in a wide spectrum of human malignancies. However, the underlying mechanisms by which PRMT5 contributes to the progression of colorectal cancer (CRC) remain to be defined.Methods: Western blot and real-time PCR were used to analyze the expression of CDKN2B. Co-immunoprecipitation (Co-IP), immunofluorescence and GST pulldown assays were employed to investigate the interaction between PRMT5 and EZH2. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were performed to validate CDKN2B as a direct target of PRMT5/EZH2. DNA methylation status at the CpG islands of promoter region of CDKN2B gene was analyzed by bisulfite sequencing. The effect of PRMT5/EZH2 on malignant phenotypes was examined through in vitro and in vivo assays. PRMT5 and EZH2 protein expression levels in CRC tissues were analyzed by immunohistochemistry (IHC) staining.Results: We observed that PRMT5-deficient CRC cells exhibit proliferation defects in vitro. PRMT5 was identified as a major transcriptional repressor of CDKN2B (p15INK4b) for determining CRC progression. Mechanistically, PRMT5-mediated histone marks H4R3me2s and H3R8me2s were predominantly deposited at the promoter region of CDKN2B gene in CRC cells. Knockdown of PRMT5 in CRC cells decreased the accumulation of H4R3me2s and H3R8me2s marks and reduced the CpG methylation level of CDKN2B promoter, then re-activated CDKN2B expression. Strikingly, silencing of CDKN2B partially abrogated the proliferation defects caused by PRMT5 depletion in vitro and in vivo. Furthermore, we proved that PRMT5 interacted with Enhancer of zeste homolog 2 (EZH2), leading to enhanced EZH2 binding and H3K27me3 deposition together with decreased transcriptional output of CDKN2B gene. Importantly, we found that the combined interventions exerted a synergistic inhibitory effect of combined treatment with PRMT5i (GSK591) and EZH2i (GSK126) on the growth of CRC cells/xenografts in vitro and in vivo. Moreover, PRMT5 and EZH2 were found to be significantly elevated and associated with poor prognosis in CRC patients.Conclusion: PRMT5 functionally associates with EZH2 to promote CRC progression through epigenetically repressing CDKN2B expression. Thus, our findings raise the possibility that combinational intervention of PRMT5 and EZH2 may be a promising strategy for CRC therapy.

The role of estrogen receptors in rat Sertoli cells at different stages of development

The aim of the study was to investigate the effects of estrogen receptors (ESR1 and ESR2) on the expression of the proteins involved with proliferation (CCND1) and differentiation (CDKN1B and CTNNB) of Sertoli cells from rat in different stages of development. ESR1-selective agonist PPT, but not ESR2-selective agonist DPN, increased CCND1 expression in Sertoli cells from 5- and 15-day old rats. PPT did not have any effect on CCND1 expression in Sertoli cells from 20- and 30-day-old rats. DPN, but not PPT, increased CDKN1B expression in Sertoli cells from 15-, 20-, 30-day-old rats. DPN did not have any effect on Sertoli cells from 5-day-old rats. 17β-estradiol (E2) and PPT enhanced the [Methyl-3H] thymidine incorporation in Sertoli cells from 15-day-old rats, whereas the treatment did not have any effect in 20-day-old rats. E2 and DPN, but not PPT, increased non-phosphorylated CTNNB expression in Sertoli cells from 20-day-old rats. This upregulation was blocked by ESR2-selective antagonist PHTPP. The activation of ESR1 and ESR2, respectively, plays a role in the proliferation and differentiation of Sertoli cells in a critical period of testicular development. Furthermore, in Sertoli cells from 20-day-old rats, upregulation of non-phosphorylated CTNNB by E2/ESR2, via c-SRC/ERK1/2 and PI3K/AKT, may play a role in the interaction between Sertoli cells and/or in cell-germ cell adhesion and/or in the stabilization and accumulation of CTNNB in the cytosol. CTNNB could be translocated to the nucleus and modulate the transcriptional activity of specific target genes. The present study reinforces the important role of estrogen in normal testis development.

Inhibition of MUC1 exerts cell-cycle arrest and telomerase suppression in glioblastoma cells

Mucin 1 (MUC1) is a transmembrane glycoprotein involved in tumorigenesis of diverse cancers. However, the role of MUC1 in glioblastoma (GBM) has not yet been fully explored. In this study, the anticancer mechanism of MUC1 suppression in GBM was investigated. The expression level of MUC1 was analyzed in human glioma and paired normal brain tissues. MUC1 was overexpressed in GBM and was negatively associated with overall survival. Moreover, we silenced MUC1 to investigate its effect in GBM cell lines and found that knockdown of MUC1 inhibited cell proliferation and resulted in cell cycle arrest at G1 phase. MUC1 silencing decreased the phosphorylation of RB1 and increased the expression of CDKN1B. Gene set enrichment analysis showed that a series of genes related to cell cycle, telomere maintenance and transforming growth factor Beta (TGF-β) signaling in epithelial mesenchymal transition (EMT) were influenced by MUC1 knockdown. Notably, the reduced TERT expression levels combined with impaired telomerase activity and the switching of telomere maintenance mechanism to alternative lengthening of telomeres (ALT) were observed after MUC1 knockdown. Our results support the role of MUC1 in oncological process in GBM which can be developed as a therapeutic target for cell cycle control and telomere maintenance mechanism.

Effects of rapamycin and AZD3463 combination on apoptosis, autophagy, and cell cycle for resistance control in breast cancer

Breast cancer is the most common cancer in women and the leading cause of cancer mortality in women over 40 it's the year. The existence of the PI3K/AKT/mTOR pathway aberrations in more than 70% of breast cancer has caused to become a therapeutic target. AZD3463 is an anti-cancer agent used as a potential inhibitor of ALK/IGF1R. It also induces apoptosis and autophagy of the PI3K/AKT/mTOR pathway in cancer cells. Although the mTOR signaling might be inhibited by rapamycin treatment, signals transmitted from the upstream pathway supports cell survival and proliferation.The WST-1 assay test was performed to evaluate the anti-proliferative effects of rapamycin and AZD3463. Besides, the effects of them on apoptosis, autophagy, cytostatic, and metabolism in MCF7 breast cancer cells were investigated. Also, changes in the expression of apoptotic regulatory genes, cell cycle, and metabolism in the PI3K/AKT/mTOR Pathway were determined by Quantitative RT-PCR.The results showed that rapamycin and AZD3463 treatments significantly reduced survival in MCF7 cells. Also, apoptosis, autophagy, and cell population in the G0/G1 stage in the MCF7 cell category in the treatment group showed an increase compared to the control group. The combination of rapamycin and AZD3463 (AZD-RAPA) was determined as an additive according to isobologram analysis. In the combination of rapamycin with AZD3463, the expression of CDKN1B, PTEN, FOXO3, and APC genes increases, and the expression of PRKCB and PIK3CG genes decreases. Our results showed that the use of AZD-RAPA reduced the resistance of cancer cells to treatment and it leads cancer cells to apoptosis.

High Expression of miR-196b Predicts Poor Prognosis in Patients with Ovarian Cancer.

Background/AimsTo analyze the clinical significance of miR-196b expression in ovarian cancer and predict the function and possible mechanism of miR-196b.MethodsBoth Kaplan–Meier (K-M) and Cox proportional hazards regression model were used to analyze the prognostic factors of patients with ovarian cancer. MiR196-b was modulated in ovarian cancer cells, and the cell viability, cell cycle, and cell cycle-related gene expression were analyzed. The target genes of miR-196b were then predicted and checked the relationship between the target genes.ResultsMiR-196b was an independent risk factor, while high expression of miR-196b was associated with poor prognosis of ovarian cancer. MiR-196b overexpression increased cancer cell proliferation. Cdkn1b, as one of the targets of miR-196b, was related to cell viability and mitosis.ConclusionHigh expression of miR-196b was significantly associated with poor prognosis of the patients with ovarian cancer. MiR-196b could increase the cell proliferation of ovarian cancer by modulating Cdkn1b expression.

Study on the ameliorating effect of miR-221-3p on the nerve cells injury induced by sevoflurane

Purpose Sevoflurane is a widely used anesthetics, however, it has been reported that sevoflurane has neurotoxic effects. Studies have shown that miR-221-3p can ameliorate neuron damage. This study was to investigate whether miR-221-3p could reduce the neurotoxic effect of sevoflurane on nerve cells. Materials and methods The rat hippocampal neuron cells were treated with sevoflurane or cultured normally. And we constructed neuron cells that overexpressed or low expression of miR-221-3p in the presence or absence of sevoflurane. The cells were transfected with CDKN1B or siCDKN1B, and co-transfected with miR-221-3p mimic and CDKN1B or miR-221-3p inhibitor and siCDKN1B. Cell viability and apoptosis were detected by CCK-8 and flow cytometer. Target gene of miR-221-3p were predicted by TargetScan and luciferase reporter assay. The expressions of related genes were detected by western blotting and quantitative real-time polymerase chain reaction. Results Sevoflurane decreased miR-221-3p level and increased CDKN1B level, inhibited cell viability and promoted apoptosis. Overexpress of miR-221-3p decreased CDKN1B level, up-regulated cell viability and inhibited apoptosis, and reversed the effects of sevoflurane on cell viability and apoptosis, while the effects low expression of miR-221-3p was contrary. CDKN1B was the target gene of miR-221-3p, which inhibited cell viability and promoted apoptosis, and reversed the effects of miR-221-3p mimic, whereas siCDKN1B did the opposite effects. Conclusions Sevoflurane can cause nerve cell injury, and miR-221-3p may promote cell activity and inhibit apoptosis by inhibiting CDKN1B expression, thereby ameliorating cell injury induced by sevoflurane.

Molecular Genetics and Functional Analysis Implicate CDKN2BAS1-CDKN2B Involvement in POAG Pathogenesis.

The genes in the 9p21 locus (CDKN2B-AS1 & CDKN2B) are widely associated with Primary open-angle glaucoma (POAG). However, the functional importance of this locus in POAG pathogenesis is still unexplored. This study investigated the role of CDKN2BAS1-CDKN2B axis in POAG. We observed significant association of CDKN2B-AS1 SNP rs4977756 with POAG and its endophenotypic traits (vertical cup-disc ratio (p = 0.033) and central corneal thickness (p = 0.008)) by screening African American POAG cases (n = 1567) and controls (n = 1600). A luciferase reporter assay in Human embryonic kidney 293T (HEK293T) cells revealed that the region surrounding rs4977756 likely serves as a transcriptional repressor. siRNA-mediated knockdown of CDKN2B-AS1 in HEK293T cells and trabecular meshwork (TM) cells resulted in significantly increased expression of CDKN2B, which was also observed in human POAG ocular tissues. Pathway focused qRT-PCR gene expression analysis showed increased cellular senescence, TGFβ signaling and ECM deposition in TM cells after CDKN2B-AS1 suppression. In conclusion, we report that CDKN2B-AS1 may act as a regulator, and it could function by modulating the expression of CDKN2B. In addition, increase in CDKN2B levels due to CDKN2B-AS1 suppression may result in the senescence of trabecular meshwork cells leading to POAG pathogenesis.

MIAT inhibits proliferation of cervical cancer cells through regulating miR-150-5p

BackgroundTo characterize the MIAT expression in cervical cancer and elucidate its mechanistic involvement in the tumor biology of this disease.MethodsThe relative expression of MIAT and miR-150 was determined by real-time PCR. Cell proliferation was measured by the CCK-8 and clonogenic assay. The anchorage-independent growth was evaluated by soft agar assay. The in vivo tumor progression was assayed with xenograft mice model. The regulatory effect of miR-150 on MIAT was interrogated by luciferase reporter assay. The endogenous CNKD1B protein was detected by western blotting.ResultsThe low expression of MIAT was characterized in cervical cancer, which associated with relatively poor prognosis. Ectopic expression of MIAT inhibited malignant growth of cervical cancer both in vitro and in vivo. Mechanistically, MIAT regulated CDKN1B expression via competition with miR-150, and miR-150-inhibition directly suppressed cervical cancer cell growth.ConclusionsOur study characterized the anti-tumor property of MIAT in cervical cancer and elucidated its competitively regulation of CDKN1B with miR-150. Our data highlighted the critical role of MIAT-miR-150-CDKN1B signaling axis in cervical cancer.

Pisosterol Induces G2/M Cell Cycle Arrest and Apoptosis via the ATM/ATR Signaling Pathway in Human Glioma Cells.

Pisosterol, a triterpene derived from Pisolithus tinctorius, exhibits potential antitumor activity in various malignancies. However, the molecular mechanisms that mediate the pisosterol-specific effects on glioma cells remain unknown. This study aimed to evaluate the antitumoral effects of pisosterol on glioma cell lines. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue exclusion assays were used to evaluate the effect of pisosterol on cell proliferation and viability in glioma cells. The effect of pisosterol on the distribution of the cells in the cell cycle was performed by flow cytometry. The expression and methylation pattern of the promoter region of MYC, ATM, BCL2, BMI1, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, MDM2, p14ARF and TP53 was analyzed by RT-qPCR, western blotting and bisulfite sequencing PCR (BSP-PCR). Here, it has been reported that pisosterol markedly induced G2/M arrest and apoptosis and decreased the cell viability and proliferation potential of glioma cells in a dose-dependent manner by increasing the expression of ATM, CASP3, CDK1, CDKN1A, CDKN2A, CDKN2B, CHEK1, p14ARF and TP53 and decreasing the expression of MYC, BCL2, BMI1 and MDM2. Pisosterol also triggered both caspase-independent and caspase-dependent apoptotic pathways by regulating the expression of Bcl-2 and activating caspase-3 and p53. It has been, for the first time, confirmed that the ATM/ATR signaling pathway is a critical mechanism for G2/M arrest in pisosterol-induced glioma cell cycle arrest and suggests that this compound might be a promising anticancer candidate for further investigation.

The long noncoding RNA Arrl1 inhibits neurite outgrowth by functioning as a competing endogenous RNA during neuronal regeneration in rats

The intrinsic regeneration ability of neurons is a pivotal factor in the repair of peripheral nerve injury. Therefore, identifying the key modulators of nerve regeneration may help improve axon regeneration and functional recovery after injury. Unlike for classical transcription factors and regeneration-associated genes, the function of long noncoding RNAs (lncRNAs) in the regulation of neuronal regeneration remains mostly unknown. In this study, we used RNA-Seq-based transcriptome profiling to analyze the expression patterns of lncRNAs and mRNAs in rat dorsal root ganglion (DRG) following sciatic nerve injury. Analyses using the lncRNA-mRNA co-expression network, gene ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway databases indicated that the lncRNA Arrl1 decreases neurite outgrowth after neuronal injury. shRNA-mediated Arrl1 silencing increased axon regeneration both in vitro and in vivo and improved functional recovery of the sciatic nerve. Moreover, inhibiting an identified target gene of Arrl1, cyclin-dependent kinase inhibitor 2B (Cdkn2b), markedly promoted neurite outgrowth of DRG neurons. We also found that Arrl1 acts as a competing endogenous RNA that sponges a Cdkn2b repressor, microRNA-761 (miR-761), and thereby up-regulates Cdkn2b expression during neuron regeneration. We conclude that the lncRNA Arrl1 affects the intrinsic regeneration of DRG neurons by derepressing Cdkn2b expression. Our findings indicate a role for an lncRNA-microRNA-kinase pathway in the regulation of axon regeneration and functional recovery following peripheral nerve injury in rats.