Discovery of a Novel Bloom's Syndrome Protein (BLM) Inhibitor Suppressing Growth and Metastasis of Prostate Cancer.

Prostate cancer (PCa) is a common cancer and a major cause of cancer-related death worldwide in men, necessitating novel targets for cancer therapy. High expression of Bloom's syndrome protein (BLM) helicase is associated with the occurrence and development of PCa. Therefore, the identification and development of new BLM inhibitors may be a new direction for the treatment of PCa. Here, we identified a novel inhibitor by molecular docking and put it to systematic evaluation via various experiments, AO/854, which acted as a competitive inhibitor that blocked the BLM-DNA interaction. Cellular evaluation indicated that AO/854-suppressed tumor growth and metastasis in PC3 cells by enhancing DNA damage, phosphorylating Chk1/Chk2, and altering the p53 signaling pathway. Collectively, the study highlights the potential of BLM as a therapeutic target in PCa and reveals a distinct mechanism by which AO/854 competitively inhibits the function of BLM.

ML216-Induced BLM Helicase Inhibition Sensitizes PCa Cells to the DNA-Crosslinking Agent Cisplatin.

Using standard DNA-damaging medicines with DNA repair inhibitors is a promising anticancer tool to achieve better therapeutic responses and reduce therapy-related side effects. Cell viability assay, neutral comet assay, western blotting (WB), and cell cycle and apoptosis analysis were used to determine the synergistic effect and mechanism of ML216, a Bloom syndrome protein (BLM) helicase inhibitor, and cisplatin (CDDP), a DNA-crosslinking agent, in PCa cells. Based on the online database research, our findings revealed that BLM was substantially expressed in PCa, which is associated with a bad prognosis for PCa patients. The combination of ML216 and CDDP improved the antiproliferative properties of three PCa cell lines. As indicated by the increased production of γH2AX and caspase-3 cleavage, ML216 significantly reduced the DNA damage-induced high expression of BLM, making PC3 more susceptible to apoptosis and DNA damage caused by CDDP. Furthermore, the combination of ML216 and CDDP increased p-Chk1 and p-Chk2 expression. The DNA damage may have triggered the ATR-Chk1 and ATM-Chk2 pathways simultaneously. Our results demonstrated that ML216 and CDDP combination therapy exhibited synergistic effects, and combination chemotherapy could be a novel anticancer tactic.

In Situ Growth of MAPbBr3 Nanocrystals on Few-Layer MXene Nanosheets with Efficient Energy Transfer.

The performance of perovskite nanocrystals (NCs) in optoelectronics and photocatalysis is severely limited by the presence of large amounts of crystal boundaries in NCs film that greatly restricts energy transfer. Creating heterostructures based on perovskite NCs and 2D materials is a common approach to improve the energy transport at the perovskite/2D materials interface. Herein, methylamine lead bromide (MAPbBr3 , MA: CH3 NH3 + ) perovskite NCs are homogeneously deposited on highly conductive few-layer MXene (Ti3 C2 Tx ) nanosheets to form heterostructures through an in situ solution growth method. An optimal mixed solvent ratio is essential to realize the growth of perovskite NCs on Ti3 C2 Tx nanosheets. Time-resolved photoluminescence spectroscopy, transient absorption spectroscopy, and the photoresponse of electron- and hole-only photoelectric conversion devices reveal the interfacial energy transfer behavior within MAPbBr3 /Ti3 C2 Tx heterostructures. The present investigation may provide a useful guide toward use of halide perovskite/2D material heterostructures in applications such as photocatalysis as well as optoelectronics.

Nuclear localization of Newcastle disease virus matrix protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription.

Nuclear localization of paramyxovirus proteins is crucial for virus life cycle, including the regulation of viral replication and the evasion of host immunity. We previously showed that a recombinant Newcastle disease virus (NDV) with nuclear localization signal mutation in the matrix (M) protein results in a pathotype change and attenuates viral pathogenicity in chickens. However, little is known about the nuclear localization functions of NDV M protein. In this study, the potential functions of the M protein in the nucleus were investigated. We first demonstrate that nuclear localization of the M protein could not only promote the cytopathogenicity of NDV but also increase viral RNA synthesis and transcription efficiency in DF-1 cells. Using microarray analysis, we found that nuclear localization of the M protein might inhibit host cell transcription, represented by numerous up-regulating genes associated with transcriptional repressor activity and down-regulating genes associated with transcriptional activator activity. The role of representative up-regulated gene prospero homeobox 1 (PROX1) and down-regulated gene aryl hydrocarbon receptor (AHR) in the replication of NDV was then evaluated. The results show that siRNA-mediated knockdown of PROX1 or AHR significantly reduced or increased the viral RNA synthesis and viral replication, respectively, demonstrating the important roles of the expression changes of these genes in NDV replication. Together, our findings demonstrate for the first time that nuclear localization of NDV M protein promotes virus replication by affecting viral RNA synthesis and transcription and inhibiting host cell transcription, improving our understanding of the molecular mechanism of NDV replication and pathogenesis.

Characterization of the nuclear import pathway for BLM protein.

Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin ß1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin ß1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin ß1 through its NLS domain with the 14-16 HEAT repeats of importin ß1. Furthermore, importin ß1, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin ß1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase.

[The nuclear import of Newcastle disease virus matrix protein depends on KPNB1 and Ran protein].

Objective: The aim of this study was to identify the transport proteins that mediates the nuclear import of Newcastle disease virus (NDV) matrix (M) protein. Methods: Chicken KPNA1 to KPNA6 gene and KPNB1 gene were cloned from DF-1 cells and then inserted into eukaryotic expression vectors. The constructed recombinant plasmids with a combination of grouping were transfected into HEK-293T cells to identify the transport proteins interacting with NDV M protein by co-immunoprecipitation (Co-IP) assay. Moreover, fluorescent co-localization assay was used to verify the transport proteins by co-expressing M and Ran protein mutant or M and its interactive protein deletant. Results: The recombinant proteins could normally express in plasmid-transfected HEK-293T cells. Indirect immunofluorescence detection showed that the recombinant proteins except for Myc-KPNA2 displayed the same nuclear localization as NDV M protein. The results of Co-IP revealed that M protein could interact with KPNA1 and KPNB1. Further fluorescent co-localization indicated that co-expression of M and DN-KPNA1 did not change the nuclear localization of M, whereas co-expression of M and DN-KPNB1 or M and Ran-Q69L disrupted the nuclear localization of M, demonstrating that the nuclear import of M protein was dependent on KPNB1 and Ran protein. Conclusion: KPNB1 and Ran protein jointly mediated the nuclear import of NDV M protein, showing that KPNB1 protein interacted with NDV M protein to form binary complex and then entered into the nucleus with the assistance of Ran protein.

miR-29b-3p inhibits 22Rv1 prostate cancer cell proliferation through the YWHAE/BCL-2 regulatory axis.

Prostate cancer (PCa) is one of the most common malignant tumours in the world and seriously affects health of men. Studies have shown that microRNA (miR)-29b-3p and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) play important roles in influencing the proliferation and apoptosis of PCa cells. However, the molecular mechanism of miR-29b-3p and YWHAE in the proliferation and apoptosis of PCa cells remains unclear. In the present study, bioinformatics as well as in vivo and in vitro experiments were used to predict and verify the targeting relationship between YWHAE and mir-29B-3p and investigate the potential roles of YWHAE and mir-29b-3p in the proliferation and apoptosis of 22RV1 cells. Using bioinformatics and a double luciferase system assay, it was confirmed that miR-29b-3p can target YWHAE 3'untranslated region and affect the expression of YWHAE, suggesting that miR-29b-3p may be a potential miRNA of YWHAE. Reverse transcription-quantitative PCR, Cell Counting Kit-8, Transwell and cell scratch assays showed that miR-29b-3p significantly inhibited the proliferation, invasion and migration of 22Rv1 cells (P<0.01). Rescue experiments demonstrated that YWHAE gene introduction reversed the inhibitory effect of miR-29b-3p on 22Rv1 cells. Western blotting revealed that the upregulation of miR-29b-3p inhibited YWHAE expression, resulting in a very significant decrease in the ratio of p-BAD/BAD and full-length caspase 3/cleaved caspase 3 (P<0.01) and an extremely significant increase in the ratio of BAX/BCL-2 (P<0.01). A tumourigenesis test in nude mice in vivo confirmed that the upregulation of miR-29b-3p inhibited tumour growth by targeting YWHAE. The present experiments confirmed that miR-29b-3p plays a tumour suppressor role in 22Rv1 PCa cells, and the YWHAE/BCL-2 regulatory axis plays a vital role in miR-29b-3p regulating the proliferation and apoptosis of 22Rv1 cells. These results may provide a theoretical basis for the diagnosis and targeted treatment of PCa.

Effects of N-Acetylcysteine on the Proliferation, Hormone Secretion Level, and Gene Expression Profiles of Goat Ovarian Granulosa Cells.

The purpose of this paper was to investigate the effects of N-acetylcysteine (NAC) on the proliferation, hormone secretion, and mRNA expression profiles of ovarian granulosa cells (GCs) in vitro. A total of 12 ovaries from 6 follicular-stage goats were collected for granulosa cell extraction. The optimum concentration of NAC addition was determined to be 200 µM via the Cell Counting Kit 8 (CCK-8) method. Next, GCs were cultured in a medium supplemented with 200 µM NAC (200 µM NAC group) and 0 µ M NAC (control group) for 48 h. The effects of 200 µM NAC on the proliferation of granulosa cells and hormones were studied by 5-ethynyl-2'-deoxyuridine (EdU) assay and enzyme-linked immunosorbent assay (ELISA). mRNA expression was analyzed by transcriptome sequencing. The results indicate that 200 µM NAC significantly increased cell viability and the proportion of cells in the S phase but promoted hormone secretion to a lesser degree. Overall, 122 differentially expressed genes (DEGs) were identified. A total of 51 upregulated and 71 downregulated genes were included. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the most DEGs were enriched in terms of cell growth regulation, cell growth, neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, the cAMP-signaling pathway, and the Wnt-signaling pathway. Seven genes related to granulosa cell proliferation were screened, IGFBP4, HTRA4, SST, SSTR1, WISP1, DAAM2, and RSPO2. The above results provide molecular theoretical support for NAC as a feed additive to improve follicle development and improve reproductive performance in ewes.

Nano-Sized Calcium Copper Titanate for the Fabrication of High Dielectric Constant Functional Ceramic-Polymer Composites.

A novel calcium copper titanate (CaCu3Ti4O12)-polyvinylidene fluoride composite (CCTO@PVDF) with Cu-deficiency was successfully prepared through the molten salt-assisted method. The morphology and structure of polymer composites uniformly incorporated with CCTO nanocrystals were characterized. At the same volume fraction, the CCTOs with Cu-deficiency displayed higher dielectric constants than those without post-treatment. A relatively high dielectric constant of 939 was obtained at 64% vol% CCTO@PVDF content, 78 times that of pure PVDF. The high dielectric constants of these composites were attributed to the homogeneous dispersion and interfacial polarization of the CCTO into the PVDF matrix. These composites also have prospective applications in high-frequency regions (106 Hz). The enhancement of the dielectric constant was predicted in several theoretical models, among which the EMT and Yamada models agreed well with the experimental results, indicating the excellent distribution in the polymer matrix.

ZIF-67 Derivative Decorated MXene for a Highly Integrated Flexible Self-Powered Photodetector.

The rapid development of portable and wearable electronics has promoted the integration of multifunction techniques. Although flexible energy storage systems have been successfully investigated, the compact configuration with photodetector and energy storage components has received less attention. As a new member of the 2D material class, MXene exhibits remarkable electronic and optical properties. Here, through the intentional introduction of ZIF-67 derivatives deposited on the Mo2CTx nanosheets, the synthesized Co-CoOx/NC/Mo2CTx heterostructure not only provided a straightforward pathway for photogenerated electrons to transport but also enhanced the structural stability of Mo2CTx, leading to a high responsivity and short rise/decay time under the illumination of simulated light in the photoelectrochemical (PEC) configuration. The integrated flexible device based on a zinc ion battery and Co-CoOx/NC/Mo2CTx heterostructure shows outstanding photodetection function and retains the intrinsic charge/discharge behaviors, which could monitor 1 day sunlight changes in real time. The paradigm presented here paves the way for realizing the development of miniaturization and multifunction toward next-generation portable and wearable technologies.

A Proteomic Study of the Effect of N-acetylcysteine on the Regulation of Early Pregnancy in Goats.

Dietary supplementation with N-acetyl-L-cysteine (NAC) may support early pregnancy regulation and fertility in female animals. The purpose of this study was to investigate the effect of supplementation with 0.07% NAC on the expression of the uterine keratin gene and protein in Qianbei-pockmarked goats during early pregnancy using tandem mass spectrometry (TMT) relative quantitative proteomics. The results showed that there were significant differences in uterine keratin expression between the experimental group (NAC group) and the control group on day 35 of gestation. A total of 6271 proteins were identified, 6258 of which were quantified by mass spectrometry. There were 125 differentially expressed proteins (DEPs), including 47 upregulated and 78 downregulated proteins, in the NAC group. Bioinformatic analysis showed that these DEPs were mainly involved in the transport and biosynthesis of organic matter and were related to the binding of transition metal ions, DNA and proteins and the catalytic activity of enzymes. They were enriched in the Jak-STAT signalling pathway, RNA monitoring pathway, amino acid biosynthesis, steroid biosynthesis and other pathways that may affect the early pregnancy status of does through different pathways and thus influence early embryonic development. Immunohistochemistry, real-time quantitative PCR and Western blotting were used to verify the expression and localization of glial fibrillary acidic protein (GFAP) and pelota mRNA surveillance and ribosomal rescue factor (PELO) in uterine horn tissue. The results showed that both PELO and GFAP were localized to endometrial and stromal cells, consistent with the mass spectrometry data at the transcriptional and translational levels. Moreover, NAC supplementation increased the levels of the reproductive hormones follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestradiol (E2), progesterone (P4), superoxide dismutase (SOD), glutamate peroxidase (GSH-Px) and nitric oxide (NO) in the serum of does. These findings provide new insight into the mechanism by which NAC regulates early pregnancy and embryonic development in goats.

BLM interaction with EZH2 regulates MDM2 expression and is a poor prognostic biomarker for prostate cancer.

Prostate cancer (PCa) is one of the major causes of cancer death among males worldwide. Our previous studies indicated that the proliferation of prostate cancer cells was reduced after BLM knockdown, however, the mechanism is still not clear. In this study, we identified a direct interaction between BLM and EZH2, which had extremely significantly positive correlations (P<0.001). In vitro, our research revealed that tumor growth was inhibited after EZH2 knockdown and that inhibition could be reversed by BLM overexpression; conversely, tumor growth was promoted after EZH2 overexpression, and promotion could be reversed by BLM knockdown. This suggests that BLM and EZH2 play important roles in the progression of prostate cancer cells. In vivo, the impact of BLM and EZH2 was investigated in mouse xenograft models, and the results showed that EZH2 could be regulated by BLM, which was consistent with our in vitro observations. Our results demonstrated that the expression of P53 is affected by the binding of BLM and EZH2 to the MDM2 promoter region. This finding indicated that EZH2 regulates the expression of MDM2 at the transcriptional level by interacting with BLM.

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats.

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (BMPR-IB), follicle-stimulating hormone (FSHR), and inhibin α (INHA), were determined in granulosa cells. Results showed that CTSD was expressed in corpus luteum, follicle, and granulosa cells. Notably, CTSD expression in the monotocous group was significantly higher than that in the polytocous group. In addition, CTSD knockdown could improve granulosa cell proliferation, inhibit cell apoptosis, and significantly elevate the expression of proliferating cell nuclear antigen (PCNA) and B cell lymphoma 2 (Bcl-2), but it lowered the expression of Bcl-2-associated X (Bax) and caspase-3. Furthermore, CTSD knockdown significantly reduced the ratios of cells in the G0/G1 and G2/M phases but substantially increased the ratio of cells in the S phase. The expression levels of cyclin D2 and cyclin E were elevated followed by the obvious decline of cyclin A1 expression. However, the expression levels of BMPR-IB, FSHR, and INHA clearly increased as a result of CTSD knockdown. Hence, our findings demonstrate that CTSD is an important factor affecting the litter size trait in goats by regulating the granulosa cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific trait.

miR-31-5p Regulates 14-3-3 ɛ to Inhibit Prostate Cancer 22RV1 Cell Survival and Proliferation via PI3K/AKT/Bcl-2 Signaling Pathway.

INTRODUCTION: Prostate cancer (PCa) is one of the most common malignancies, and almost all patients with advanced PCa will develop castration-resistant prostate cancer (CRPC) after receiving endocrine therapy. Effective treatment for patients with CRPC has not been established. Novel approaches are needed to identify therapeutic targets for CRPC. PURPOSE: Recent research studies have found that members of the 14-3-3 family play an important role in the development and progression of PCa. Previous results have shown that 14-3-3 ɛ is significantly upregulated in several cancers. This study aimed to identify novel miRNAs that regulate 14-3-3 ɛ expression and therapeutic targets for CRPC. METHODS: In this study, we used computation and experimental approaches for the prediction and verification of the miRNAs targeting 14-3-3 ɛ, and investigated the potential roles of 14-3-3 ɛ in the survival and proliferation of 22RV1 cells. RESULTS: We confirm that mir-31-5p is downregulated in 22RV1 cells and acts as a tumor suppressor by regulating 14-3-3 ɛ. Ectopic expression of miR-31-5p or 14-3-3 ɛ interference significantly inhibits cell proliferation, invasion, and migration in 22RV1 cells, as well as promotes cell apoptosis via the PI3K/AKT/Bcl-2 signaling pathway. Moreover, 14-3-3 ɛ is required for the miR-31-5p-mediated upregulation of the PI3K/AKT/Bcl-2 signaling pathway. CONCLUSION: Our findings provide information on the underlying mechanisms of miR-31-5p/14-3-3 ɛ in 22RV1 cell proliferation and apoptosis through the PI3K/AKT/Bcl-2 signaling pathway. These results suggest that miR-31-5p and 14-3-3 ɛ may potentially be utilized as novel prognostic markers and therapeutic targets for PCa treatment.

TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein.

Nuclear localization of cytoplasmic RNA virus proteins mediated by intrinsic nuclear localization signal (NLS) plays essential roles in successful virus replication. We previously reported that NLS mutation in the matrix (M) protein obviously attenuates the replication and pathogenicity of Newcastle disease virus (NDV), but the attenuated replication mechanism remains unclear. In this study, we showed that M/NLS mutation not only disrupted M's nucleocytoplasmic trafficking characteristic but also impaired viral RNA synthesis and transcription. Using TMT-based quantitative proteomics analysis of BSR-T7/5 cells infected with the parental NDV rSS1GFP and the mutant NDV rSS1GFP-M/NLSm harboring M/NLS mutation, we found that rSS1GFP infection stimulated much greater quantities and more expression changes of differentially expressed proteins involved in host cell transcription, ribosomal structure, posttranslational modification, and intracellular trafficking than rSS1GFP-M/NLSm infection. Further in-depth analysis revealed that the dominant nuclear accumulation of M protein inhibited host cell transcription, RNA processing and modification, protein synthesis, posttranscriptional modification and transport; and this kind of inhibition could be weakened when most of M protein was confined outside the nucleus. More importantly, we found that the function of M protein in the cytoplasm effected the inhibition of TIFA expression in a dose-dependent manner, and promoted NDV replication by down-regulating TIFA/TRAF6/NF-κB-mediated production of cytokines. It was the first report about the involvement of M protein in NDV immune evasion. Taken together, our findings demonstrate that NDV replication is closely related to the nucleocytoplasmic trafficking of M protein, which accelerates our understanding of the molecular functions of NDV M protein.

Bloom Syndrome Protein Activates AKT and PRAS40 in Prostate Cancer Cells.

PURPOSE: Prostate cancer (PC) is a common malignant tumor and a leading cause of cancer-related death in men worldwide. In order to design new therapeutic interventions for PC, an understanding of the molecular events underlying PC tumorigenesis is required. Bloom syndrome protein (BLM) is a RecQ-like helicase, which helps maintain genetic stability. BLM dysfunction has been implicated in tumor development, most recently during PC tumorigenesis. However, the molecular basis for BLM-induced PC progression remains poorly characterized. In this study, we investigated whether BLM modulates the phosphorylation of an array of prooncogenic signaling pathways to promote PC progression. METHODS: We analyzed differentially expressed proteins (DEPs) using iTRAQ technology. Site-directed knockout of BLM in PC-3 prostate cancer cells was performed using CRISPR/Cas9-mediated homologous recombination gene editing to confirm the effects of BLM on DEPs. PathScan® Antibody Array Kits were used to analyze the phosphorylation of nodal proteins in PC tissue. Immunohistochemistry and automated western blot (WES) analyses were used to validate these findings. RESULTS: We found that silencing BLM in PC-3 cells significantly reduced their proliferative capacity. In addition, BLM downregulation significantly reduced levels of phosphorylated protein kinase B (AKT (Ser473)) and proline-rich AKT substrate of 40 kDa (PRAS40 (Thr246)), and this was accompanied by enhanced ROS (reactive oxygen species) levels. In addition, we found that AKT and PRAS40 inhibition reduced BLM, increased ROS levels, and induced PC cell apoptosis. CONCLUSIONS: We demonstrated that BLM activates AKT and PRAS40 to promote PC cell proliferation and survival. We further propose that ROS act in concert with BLM to facilitate PC oncogenesis, potentially via further enhancing AKT signaling and downregulating PTEN expression. Importantly, inhibiting the BLM-AKT-PRAS40 axis induced PC cell apoptosis. Thus, we highlight new avenues for novel anti-PC treatments.

Efficiently Predicting Hot Spots in PPIs by Combining Random Forest and Synthetic Minority Over-Sampling Technique.

Hot spot residues bring into play the vital function in bioinformatics to find new medications such as drug design. However, current datasets are predominately composed of non-hot spots with merely a tiny percentage of hot spots. Conventional hot spots prediction methods may face great challenges towards the problem of imbalance training samples. This paper presents a classification method combining with random forest classification and oversampling strategy to improve the training performance. A strategy with an oversampling ability is used to generate hot spots data to balance the given training set. Random forest classification is then invoked to generate a set of forest trees for this oversampled training set. The final prediction performance can be computed recursively after the oversampling and training process. This proposed method is capable of randomly selecting features and constructing a robust random forest to avoid overfitting the training set. Experimental results from three data sets indicate that the performance of hot spots prediction has been significantly improved compared with existing classification methods.

miR­27b­3p and miR­607 cooperatively regulate BLM gene expression by directly targeting the 3'­UTR in PC3 cells.

BLM RecQ like helicase (BLM) has a pivotal role in genetic recombination, transcription, DNA replication and DNA repair, which presents the possibility of using BLM as an anti­cancer target for treatment. However, the post­transcriptional control regulation of BLM gene expression is not fully understood and limits the application of drugs targeting BLM for carcinoma therapy in the future. MicroRNAs (miRNAs) inhibit gene expression through interaction with the 3' untranslated region (3'­UTR) of mRNA at the post­transcriptional stage. Therefore, the current study screened for miRNAs that regulate BLM gene expression, with software predicting that miRNA (miR)­27b­3p, miR­607, miR­361­3p, miR­628­5p and miR­338­3p. BLM gene expression levels in the PC3 prostate cancer cell line and RWPE­2 normal prostate epithelium cell line were detected by reverse transcription­quantitative PCR. Additionally, BLM mRNA levels were following miRNA overexpression for 24 and 48 h. For further miRNA filtration and validation, a dual­luciferase reporter system and western blot analysis were performed, which demonstrated that miR­27b­3p and miR­607 reduce BLM gene expression by directly targeting the BLM mRNA 3'­UTR. A Box­Behnken design experiment suggested that miR­27b­3p and miR­607 have synergetic mutual effects on BLM gene expression. Finally, the suppressive effect of miR­27b­3p and miR­607 on PC3 cell proliferation, colony formation, migration and invasion indicated the benefit of studying BLM as a drug target in cancer. In conclusion, the findings of the current provide evidence that miR­27b­3p and miR­607 have an oncosuppressive function in PC3 cells and cooperatively downregulate BLM expression at the post­transcriptional level.

Importin α5 negatively regulates importin ß1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin ß1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin ß1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin ß1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin ß1. However, importin ß1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin ß1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

Characterization and Sequencing of an H6N6 Avian Influenza Virus Isolated from Sansui Sheldrake Ducks in Guizhou, Southwestern China.

Here, we report the complete genome sequence of an H6N6 avian influenza virus (AIV) isolated from Sansui Sheldrake ducks in Guizhou Province, China, in 2014. Phylogenetic analysis showed that the H6N6 virus was a reassortant virus derived from three different H6 subtype lineages. The finding of this study will help us understand the epidemiology and the evolutionary characteristics of H6 subtypes of AIV in ducks in southwestern China.