Potential role of nimbolide in chemoprevention and therapy of prostate cancer

OBJECTIVE Prostate cancer is one of the most commonly diagnosed cancers worldwide.Current therapies for metastatic prostate cancer are only marginally effective,and hence novel treatment modalities are urgently needed.Considerable evidence suggests that chronic inflammation plays a pivotal role in the development and progression of prostate cancer.Thus agents that can suppress these inflammatory mediators could form the basis of novel therapy for prostate cancer patients.In our study,we focused on analyzing the potential anticancer effects of nimbolide,a terpenoid lactone derived from Azadirachta indica(Neem tree)against prostate cancer.METHODS Molecular biology techniques such as western blot analysis,DNA binding,luciferase assays,and immunohistochemistry were used for both in vitro and in vivo experiments.RESULTS Data from the in vitrostudies indicated that nimbolide could inhibit cell proliferation,induce apoptosis and suppress cellular invasion and migration.Interestingly,nimbolide also abrogated the activation of pro-inflammatory STAT 3 transcription factor,and this effect was found to be mediated via an increased production of reactive oxygen species(ROS),whereas depletion of ROS attenuated pSTAT 3 inhibitory effects of the drug.The in vivo efficacy of nimbolide was also noted in transgenic adenocarcinoma of mouse prostate(TRAMP)model,in which this triterpenoid significantly suppressed the tumor progression and growth without exhibiting any substantial adverse effects.CONCLUSION Overall our findings indicate that nimbolide exhibits significant anticancer effects in prostate cancer,and these effects may be mediated at least in part through the modulation of STAT 3 signaling pathway.

RNA helicase DDX20 as a surrogate marker of statin activity in invasive breast cancer

OBJECTIVE To evaluate if RNA helicase DDX20,highly expressed in triple negative breast cancer(TNBC)cells,could serve as a surrogate marker for simvastatin treatment response.METHODS We first assessed correlation between 17 mevalonate pathway-related genes and expression of DDX20 in a cohort of 1325 breast cancer tumors.TNBC cells,MDA-MB-231,were then treated with simvastatin and mevalonate pathway intermediates to assess the alteration in DDX20 expression.In the mouse model,MDA-MB-231 cells were injected to tail veins of mice,groups of 8mice each were injected intraperioneally with vehicle or simvastatin 25mg·kg-1 3times a week for 6weeks.The number of metastatic colonies formed was quantified and immunohistochemical(IHC)staining of DDX20 was carried out in the lung tissues.RESULTS Among the 17 genes evaluated,positive correlation with DDX20 expression was observed in eight of them,with HMGCR having the highest correlation.Our in vitro experiments show exposure of breast cancer cells to simvastatin lead to a Rho-dependent decrease in gene expression of DDX20,leading to decreased tumor proliferation in a mevalonate pathway-dependent manner.Conversely,ectopic overexpression of DDX20 significantly abrogated the anti-metastatic activity of simvastatin.A similar observation is seen in the mouse model,where simvastatin-injected mice show significantly fewer visible lung metastases compared to placebo-fed mice.IHC staining on these lung tissues showed decreased DDX20 expression in simvastatin-injected group,corroborating our observations in vitro.CONCLUSION DDX20 is a potential surrogate marker for simvastatin treatment response in breast cancer and a long term implication of our findings is the possibility of an effective combinatorial therapeutic intervention using statins(to suppress DDX20 gene expression)and a suitable firstline agent″for the kill″of invasive breast cancer.

In vitro and in vivo effect of artesunate against prostate cancer:Targetting STAT3 pathway to combat disease progression

OBJECTIVE In prostate cancer(PCa),signal transducer and activator of transcription factor3(STAT3)has been strongly associated with tumor progression,through numerous means.Hence this allows STAT3 to be an important target for therapeutic action.Artesunate(ART),a well know antimalarial agentis making its way as an anticancer drug.In the present study,we investigated whether ART can control aberrant STAT3 signaling,and thereby take a toll on PCa development.METHODS Various PCa cell lines(DU145,PC3,LNCaP)and in vivo xenograft mouse model are used.Cytotoxic effects of ART against various PCa cell lines were evaluated by MTT assay.Flow cytometry cell cycle analysis and DNA fragmentation assay was done to detect the apoptotic effect of ART.Expression of STAT3 and its regulated gene in the presence and absence of ART were measured by WB,IHC and RT PCR.STAT3 DNA binding activities was analyzed by ELISA.RESULTS ART was found to dephosphorylate STAT3 at Tyr 405,thereby reducing its nuclear translocation and DNA binding efficiency in DU145 PCa cells.We proclaim that ART can prevent the PCa development,as it can inhibit proliferation,bring about cell cycle arrest at G0/G1 phase,AnnexinⅤ positive staining,DNA fragmentation,caspase 3activation and PARP cleavage in PCa cell lines.Furthermore,inhibition of constitutive STAT3 expression was associated with the ability of ART to suppress its upstream kinases such as Janus kinase 1and 2(JAK1and JAK2).SHP-1,protein tyrosine phosphatases which are considered to be one of the major regulators of STAT3 phosphorylation was upregulated in the presence of ART.We observed reversal in ART mediated inhibition of STAT3 in the presence of pervanadate,tyrosine phosphates inhibitor and during SHP-1 knock down.ART was able to inactivate STAT3 in DU145 cells exposed to conditioned media(CM)rich in cytokines.In the presence of ART we observed the down regulation of various STAT3 regulated gene products which are involved in proliferation,survival,and angiogenesis.ART even blocked the motility and invasion of PCa cells.ART substantially decreased the tumor volume in xenograft mouse which is implanted with DU145 cells.Also ability of ART to control aberrant STAT3 signaling was in accordance with its invitrostudies.CONCLUSION Over all through our findings,we have disclosed for the first time that ART could possibly exerts it antitumor effect by interrupting deregulated expression of STAT3 in PCa,both in vitro and in vivo.

Impact of cytochrome P450 2D6 polymorphisms on decision-making and clinical outcomes in adjuvant hormonal therapy for breast cancer

BACKGROUND There are concerns that tamoxifen is less effective in Asian women because of the high prevalence of impaired function cytochrome P4502D6(CYP2D6)polymorphisms.AIM To evaluate how knowledge of CYP2D6 genotype impacted the choice of hormonal agent and how CYP2D6 genotype and agent were associated with clinical outcomes.METHODS Eighty-two women were recruited.Seventy-eight completed CYP2D6 genotyping and were categorized into poor,intermediate(IM)and extensive or ultra metabolizer phenotypes.Women with poor metabolizer and IM phenotypes were recommended aromatase inhibitors as the preferred agent.RESULTS More than 70%of the women had an IM phenotype,32%an extensive or ultra metabolizer phenotype,and 0%had a poor metabolizer phenotype.Regardless of genotype,more women opted for aromatase inhibitors.Overall,80%of women completed 5 years of hormonal therapy.Five women developed recurrence,3 contralateral breast cancer,5 died,and 1 was diagnosed with a second primary cancer.Five-year recurrence-free and overall survival were slightly better in women with the extensive or ultra metabolizer phenotype compared to those with the IM phenotype,though not statistically significant[P=0.743,hazard ratio(HR):1.441,95%confidence interval(CI):0.191 to 10.17 and P=0.798,HR:1.327,95%CI:0.172 to 9.915,respectively].Women receiving aromatase inhibitors also appeared to have a better,but also nonsignificant,5-year recurrence-free and overall survival(P=0.253,HR:0.368,95%CI:0.031 to 0.258 and P=0.292,HR:0.252,95%CI:0.005 to 4.951,respectively).CONCLUSION The IM phenotype was highly prevalent but was not associated with clinical outcome.

p53 mutations in colorectal cancer-molecular pathogenesis and pharmacological reactivation

Colorectal cancer(CRC) is one of the most common malignancies with high prevalence and low 5-year survival.CRC is a heterogeneous disease with a complex,genetic and biochemical background.It is now generally accepted that a few important intracellular signaling pathways,including Wnt/β-catenin signaling,Ras signaling,and p53 signaling are frequently dysregulated in CRC.Patients with mutant p53 gene are often resistant to current therapies,conferring poor prognosis.Tumor suppressor p53 protein is a transcription factor inducing cell cycle arrest,senescence,and apoptosis under cellular stress.Emerging evidence from laboratories and clinical trials shows that some small molecule inhibitors exert anti-cancer effect via reactivation and restoration of p53 function.In this review,we summarize the p53 function and characterize its mutations in CRC.The involvement of p53 mutations in pathogenesis of CRC and their clinical impacts will be highlighted.Moreover,we also describe the current achievements of using p53 modulators to reactivate this pathway in CRC,which may have great potential as novel anti-cancer therapy.

Dickkopfs and Wnt/β-catenin signalling in liver cancer

Liver cancer is the fifth and seventh most common cause of cancer in men and women,respectively.Wnt/β-catenin signalling has emerged as a critical player in both the development of normal liver as well as an oncogenic driver in hepatocellular carcinoma(HCC).Based on the current understanding,this article summarizes the possible mechanisms for the aberrant activation of this pathway with specific focus on HCC.Furthermore,we will discuss the role of dickkopfs(DKKs)in regulating Wnt/β-catenin signalling,which is poorly understood and understudied.DKKs are a family of secreted proteins that comprise at least four members,namely DKK1-DKK4,which act as inhibitors of Wnt/β-catenin signalling.Nevertheless,not all members antagonize Wnt/β-catenin signalling.Their functional significance in hepatocarcinogenesis remains to be further characterized for which these studies should provide new insights into the regulatory role of DKKs in Wnt/β-catenin signalling in hepatic carcinogenesis.Because of the important oncogenic roles,there are an increasing number of therapeutic molecules targetingβ-catenin and the Wnt/β-catenin pathway for potential therapy of HCC.

Novel mechanism of drug resistance to proteasome inhibitors in multiple myeloma

Multiple myeloma(MM) is a cancer caused by uncontrolled proliferation of antibody-secreting plasma cells in bone marrow, which represents the second most common hematological malignancy. MM is a highly heterogeneous disease and can be classified into a spectrum of subgroups based on their molecular and cytogenetic abnormalities. In the past decade, novel therapies, especially, the first-in-class proteasome inhibitor bortezomib, have been revolutionary for the treatment of MM patients. Despite these remarkable achievements, myeloma remains incurable with a high frequency of patients suffering from a relapse, due to drug resistance. Mutation in the proteasome β5-subunit(PSMB5) was found in a bortezomib-resistant cell line generated via long-term coculture with increasing concentrations of bortezomib in 2008, but their actual implication in drug resistance in the clinic has not been reported until recently. A recent study discovered four resistance-inducing PSMB5 mutations from a relapsed MM patient receiving prolonged bortezomib treatment. Analysis of the dynamic clonal evolution revealed that two subclones existed at the onset of disease, while the other two subclones were induced. Protein structural modeling and functional assays demonstrated that all four mutations impaired the binding of bortezomib to the 20 S proteasome, conferring different degrees of resistance. The authors further demonstrated two potential approaches to overcome drug resistance by using combination therapy for targeting proteolysis machinery independent of the 20 S proteasome.

Garcinol sensitizes human head and neck carcinoma to cisplatin in a xenograft mouse model despite downregulation of proliferative biomarkers

OBJECTIVE Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma(HNSCC).In the present study,we investigated whether garcinol,apolyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin.METHODS The effect of garcinol and cisplatin on HNSCC was assessed by MTT,Western blotting,real time PCR,FACS,immunohistochemistry,DNA binding assay and xenograft mouse model.RESULTS We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines,enhanced the apoptotic effect of cisplatin,suppressed constitutive as well as cisplatin-induced NF-κB activation,and downregulated the expression of various oncogenic gene products(cyclin D1,Bcl-2,survivin and VEGF).In vivo study showed that administration of garcinol alone(0.5 mg·kg-1,ip five times/week)significantly suppressed the growth of the tumor,and this effect was further increased by cisplatin.Both the markers of proliferation index(Ki-67)and microvessel density(CD31)were downregulated in tumor tissues by the combination of cisplatin and garcinol.The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after ip administration of garcinol at 0.5and 2mg·kg-1 with mean peak concentration(cmax)of 1825.4 and 6635.7nmol·L-1 in the mouse serum,respectively.CONCLUSION Overall,our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.

NF-κB promotes the stem-like properties of leukemia cells by activation of LIN28B

AIM To examine whether nuclear factor kappa B(NF-κB) activity regulates LIN28 B expression and their roles in leukemia stem cell(LSC)-like properties. METHODS We used pharmacological inhibitor and cell viability assays to examine the relation between NF-κB and LIN28 B. Western blot and q RT-PCR was employed to determine their protein and m RNA levels. Luciferase reporter was constructed and applied to explore the transcriptional regulation of LIN28 B. We manipulated LIN28 B level in acute myeloid leukemia(AML) cells and investigated LSC-like properties with colony forming and serial replating assays. RESULTS This study revealed the relationship between NF-κB and LIN28 B in AML cells through drug inhibition and overexpression experiments. Notably,inhibition of NF-κB by pharmacological inhibitors reduced LIN28 B expression and decreased cell proliferation. We demonstrated that NF-κB binds to the-819 to-811 region of LIN28 B promoter,and transcriptionally regulates LIN28 B expression. LIN28 B protein was significantly elevated in NFκB1 transfected cells compared to vector control. Importantly,ectopic expression of LIN28 B partially rescued the self-renewal capacity impaired by pharmacological inhibition of NF-κB activity. CONCLUSION These results uncover a regulatory signaling,NF-κB/LIN28 B,which plays a pivotal role in leukemia stem cell-like properties and it could serve as a promising intervening target for effective treatment of AML disease.

Garcinol suppresses the growth of human hepatocellular carcinoma by inducing abrogation of STAT3 phosphorylation,acetylation and dimerization

OBJECTIVE Hepatocellular carcinoma(HCC)is the fifth most common malignancy worldwide and the third cause of global cancer mortality.Activation of signal transducer and activator of transcription 3(STAT3)is commonly observed in tumor cells and is a critical mediator of on cogenic signaling in HCC and controls the expression of several genes involved in proliferation,survival,metastasis and angiogenesis.Current drug-targeted therapies,besides being expensive,are associated with serious side effects and morbidity.Thus,novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC.In the present report,we investigated whether the potent HAT/KAT inhibitor,garcinol,(apolyisoprenylatedbenzophenone),could suppress STAT3 activation in HCC cells and in nude mice model.METHODS The effect of garcinol on HCC cell lines wasdetermined by MTT assay,immunoblotting,DNA binding assays,immuno-fluorescenceand immune-histochemical analysis.The effect of garcinolon the inhibition of tumor growth in vivo was also investigated using HCCxenograft tumor modelin athymic nu/nu mice.RESULTS We found that garcinol could inhibit constitutive STAT3 activation in a dose-and time-dependent manner both by inhibiting STAT3 phosphorylation and acetylation in HCC cells.When investigated for molecular mechanism(s),we found that garcinol interferes with the dimer formation of STAT3 thereby inhibits its nuclear localization.Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppresses its dimerization in vitro.To understand the cellular mechanism(s)of inhibition of STAT3 function by garcinol,we observed that upon inhibition of STAT3 dimerization bygarcinol,STAT3 DNA binding ability gets repressed.The inhibition of STAT3 activation by garcinol led to the suppression of various gene products involved in proliferation,survival,and angiogenesis.Finally,when administered i.p.,garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice.CONCLUSION Results frominvitroand in vivo studies suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling cascade in HCC by inhibiting its phosphorylation,acetylation and ultimately dimerization.

Ascochlorin overcomes chemoresistance and regulates the plasticity of doxorubicin induced EMT via modulation of the NF-кB pathway in hepatocellular carcinoma

OBJECTIVE Doxorubicin-based therapy has been found to be not significantly effective for the treatment of advanced stage hepatocellular carcinomas(HCCs),which often undergo epithelial-mesenchymal transition(EMT)during tumor progression.Increasing evidence suggest(s)that epithelial cell transformation to mesenchymal state canenhance the ability to self-renew and confer greater resistance to the conventional chemotherapeutic drugs.The aim of this study was to examine the potential efficacyof ascochlorin,an isoprenoid antibiotic to overcome drug resistance induced by doxorubicin in HCC cell lines and to elucidate its underlying mechanism(s)of action.METHODS The effect of doxorubicin and ascochlorin on HCC cell lines was determined by MTT,Western blotting,immunofluorescence and NF-кB DNA binding assays.RESULTS Our results indicate that HCC cells that show a mesenchymal-like phenotype,are resistance to the doxorubicin therapy which directly correlated with an increased slug expression.We also observed that activation of NF-кB pathway plays an essential role in doxorubicin induced-chemoresistance and pharmacological inhibition of this pathway with ascochlorin can significantly reverse drug-induced invasion/migration and resistance in HCC cells.CONCLUSION Our results indicate that combination treatment of doxorubicin with ascochlorin has the potential to inhibit HCC growth and metastasis.

Roles, Functions, and Mechanisms of Long Non-coding RNAs in Cancer

Long non-coding RNAs （ineRNAs） play important roles in cancer. They are involved in chromatin remodeling, as well as transcriptional and post-transcriptional regulation, through a vari- ety of chromatin-based mechanisms and via cross-talk with other RNA species, lncRNAs can func- tion as decoys, scaffolds, and enhancer RNAs. This review summarizes the characteristics of lncRNAs, including their roles, functions, and working mechanisms, describes methods for identi- fying and annotating lncRNAs, and discusses future opportunities for lncRNA-based therapies using antisense oligonucleotides.

Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine?

Frequent activation of phosphatidylinositol-3 kinases(PI3K)/Akt/m TOR signaling pathway in gastric cancer(GC) is gaining immense popularity with identification of mutations and/or amplifications of PIK3 CA gene or loss of function of PTEN,a tumor suppressor protein,to name a few; both playing a crucial role in regulating this pathway. These aberrations result in dysregulation of this pathway eventually leading to gastric oncogenesis,hence,there is a need for targeted therapy for more effective anticancer treatment. Several inhibitors are currently in either preclinical or clinical stages for treatment of solid tumors like GC. With so many inhibitors under development,further studies on predictive biomarkers are needed to measure the specificity of any therapeutic intervention. Herein,we review the common dysregulation of PI3K/Akt/m TOR pathway in GC and the various types of single or dual pathway inhibitors under development that might have a superior role in GC treatment. We also summarize the recent developments in identification of predictive biomarkers and propose use of predictive biomarkers to facilitate more personalized cancer therapy with effective PI3K/Akt/m TOR pathway inhibition.

Identification and targeting leukemia stem cells: The path to the cure for acute myeloid leukemia

Accumulating evidence support the notion that acute myeloid leukemia(AML) is organized in a hierarchical system, originating from a special proportion of leukemia stem cells(LSC). Similar to their normal counterpart, hematopoietic stem cells(HSC), LSC possess selfrenewal capacity and are responsible for the continued growth and proliferation of the bulk of leukemia cells in the blood and bone marrow. It is believed that LSC are also the root cause for the treatment failure and relapse of AML because LSC are often resistant to chemotherapy. In the past decade, we have made significant advancement in identification and understanding the molecular biology of LSC, but it remains a daunting task to specifically targeting LSC, while sparing normalHSC. In this review, we will first provide a historical overview of the discovery of LSC, followed by a summary of identification and separation of LSC by either cell surface markers or functional assays. Next, the review will focus on the current, various strategies for eradicating LSC. Finally, we will highlight future directions and challenges ahead of our ultimate goal for the cure of AML by targeting LSC.

Resistance to FLT3 inhibitors in acute myeloid leukemia: Molecular mechanisms and resensitizing strategies

FMS-like tyrosine kinase 3(FLT3) is classified as a type Ⅲ receptor tyrosine kinase, which exerts a key role in regulation of normal hematopoiesis. FLT3 mutation is the most common genetic mutation in acute myeloid leukemia(AML) and represents an attractive therapeutic target. Targeted therapy with FLT3 inhibitors in AML shows modest promising results in current ongoing clinical trials suggesting the complexity of FLT3 targeting in therapeutics. Importantly, resistance to FLT3 inhibitors may explain the lack of overwhelming response and could obstruct the successful treatment for AML. Here, we summarize the molecular mechanisms of primary resistance and acquired resistance to FLT3 inhibitors and discuss the strategies to circumvent the emergency of drug resistance and to develop novel treatment intervention.

γ-生育三烯酚可能用做新型胃癌化疗增敏剂(英文)

Gamma-tocotrienol,a member of vitamin E superfamily has attracted great attention of late for its anti-proliferative and anti-carcinogenic potential against different cancers.For example,our group had previously reported that anti-proliferative and chemosensitizing effects ofγ-tocotrienol are associated with its ability to suppress activation of signal transducers and activator of transcription 3(STAT3),apro-inflammatory transcription factor that plays a pivotal role in the survival,proliferation,angiogenesis and chemoresistance of hepatocellular carcinoma.However,the potential of gamma-tocotrienol to overcome chemoresistance in gastric cancer,which is one of the deadliest cancers in Asia-pacific region,has never been explored before.Hence,we analyzed the efficacy of gamma-tocotrienol in combination with capecitabine to modulate tumor growth and survival in gastric cancer cell lines and xenograft mouse model.Cell proliferation and apoptosis assays indicated that gamma-tocotrienol potentiated capecitabine induced programmed cell death in various gastric cancer cell lines.Gamma-tocotrienol also inhibited expression of Bcl-2,Bcl-xL,cyclin-D1,COX-2,ICAM-1,VEGF,CXCR4,MMP-9 proteins,induced PARP cleavage and inhibited constitutive and capecitabine-induced NF-κB activation in gastric cancer cells.In vivo studies using xenograft model of human gastric cancer demonstrated that gamma-tocotrienol alone suppressed tumor growth and this effect was further potentiated in conjunction with capecitabine.Also the markers of proliferation index Ki-67 and the micro vessel density CD31 were significantly downregulated in tumor tissues by the combination of capecitabine and gamma-tocotrienol.As compared to the vehicle control,gamma-tocotrienol further suppressed the NF-κB activation and expression of cyclin D1,COX-2,ICAM-1,MMP-9 and survivin in tumor tissues obtained from treatment groups.Overall our results suggest for the first time that gamma-tocotrienol can potentiate the effects of capecitabine through modulation of multiple markers of proliferation,invasion,angiogenesis and metastasis in gastric cancer.

Safety evaluation of nanodiamond-doxorubicin complexes in a Naïve Beagle canine model using hematologic,histological,and urine analysis

While doxorubicin(DOX)is one of the most common chemotherapeutic drugs for treating cancer,use of DOX must be managed carefully due to dose-related toxicity.Nanodiamond(ND)drug delivery system conjugated with DOX(NDX)has been reported to enhance treatment efficacy and attenuate toxicity in murine cancer models.In addition,extensive biocompatibility studies indicate that NDs seem to be well tolerated in non-human primates.Before the clinical translation of NDX,it is necessary to verify the safety of ND in large mammals.Studies of nanomedicine drug safety for large animal are not commonly reported,and this work represents a key milestone in bridging earlier advances towards clinical assessment.Herein,NDs’safety as a drug-delivery platform was evaluated in Naïve Beagle dogs.The study is performed with DOX,ND,and NDX in a dual-gender animal model using intravenous(IV)injection and hepatic portal vein(HPV)injection methods.The dogs are monitored for their health phenotype changes in continuous 5 days.Blood and urine obtained are for clinical pathology research.The results indicate that ND drug delivery platform significantly relieves DOX toxicity for Naïve Beagle dog model.This study provides guidance for the pre-clinical safety assessment of NDX therapy at large animal level.

Accurate Identification of DNA Replication Origin by Fusing Epigenomics and Chromatin Interaction Information

DNA replication initiation is a complex process involving various genetic and epigenomic signatures.The correct identification of replication origins(ORIs)could provide important clues for the study of a variety of diseases caused by replication.Here,we design a computational approach named iORI-Epi to recognize ORis by incorporating epigenome-based features,sequencebased features,and 3D genome-based features.The iORI-Epi displays excellent robustness and generalization ability on both training datasets and independent datasets of K562 cell line.Further experiments confrm that iORI-Epi is highly scalable in other cell lines(MCF7 and HCT116).We also analyze and clarify the regulatory role of epigenomic marks,DNA motifs,and chromatin interaction in DNA replication initiation of eukaryotic genomes.Finally,we discuss gene enrichment pathways from the perspective of ORIs in different replication timing states and heuristically dissect the effect of promoters on replication initiation.Our computational methodology is worth extending to ORI identification in other eukaryotic species.

DeeReCT-APA:Prediction of Alternative Polyadenylation Site Usage Through Deep Learning

Alternative polyadenylation(APA)is a crucial step in post-transcriptional regulation.Previous bioinformatic studies have mainly focused on the recognition of polyadenylation sites(PASs)in a given genomic sequence,which is a binary classification problem.Recently,computational methods for predicting the usage level of alternative PASs in the same gene have been proposed.However,all of them cast the problem as a non-quantitative pairwise comparison task and do not take the competition among multiple PASs into account.To address this,here we propose a deep learning architecture,Deep Regulatory Code and Tools for Alternative Polyadenylation(DeeReCT-APA),to quantitatively predict the usage of all alternative PASs of a given gene.To accommodate different genes with potentially different numbers of PASs,DeeReCT-APA treats the problem as a regression task with a variable-length target.Based on a convolutional neural network-long short-term memory(CNN-LSTM)architecture,DeeReCT-APA extracts sequence features with CNN layers,uses bidirectional LSTM to explicitly model the interactions among competing PASs,and outputs percentage scores representing the usage levels of all PASs of a gene.In addition to the fact that only our method can quantitatively predict the usage of all the PASs within a gene,we show that our method consistently outperforms other existing methods on three different tasks for which they are trained:pairwise comparison task,highest usage prediction task,and ranking task.Finally,we demonstrate that our method can be used to predict the effect of genetic variations on APA patterns and sheds light on future mechanistic understanding in APA regulation.Our code and data are available at https://github.com/lzx325/DeeReCT-APA-repo.

Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells

Chromatin modification contributes to pluripotency maintenance in embryonic stem cells(ESCs).However,the related mechanisms remain obscure.Here,we show that Npac,a"reader"of histone H3 lysine 36 trimethylation(H3K36me3),is required to maintain mouse ESC(mESC)pluripotency since knockdown of Npac causes mESC differentiation.Depletion of Npac in mouse embryonic fibroblasts(MEFs)inhibits reprogramming efficiency.Furthermore,our chromatin immunoprecipitation followed by sequencing(ChIP-seq)results of Npac reveal that Npac co-localizes with histone H3K36me3 in gene bodies of actively transcribed genes in mESCs.Interestingly,we find that Npac interacts with positive transcription elongation factor b(p-TEFb),Ser2-phosphorylated RNA PolⅡ(RNA PolⅡSer2P),and Ser5-phosphorylated RNA PolⅡ(RNA PolⅡSer5 P).Furthermore,depletion of Npac disrupts transcriptional elongation of the pluripotency genes Nanog and Rif1.Taken together,we propose that Npac is essential for the transcriptional elongation of pluripotency genes by recruiting p-TEFb and interacting with RNA PolⅡSer2P and Ser5P.