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Миодистрофия Ландузи-Дежерина (МЛД) - распространённое наследственное заболевание, вызываемое эктопической экспрессией гена DUX4 в мышечных клетках. Метод подавления экспрессии генов при помощи siРНК зарекомендовал себя как эффективный и безопасный способ генной терапии без вмешательства непосредственно в геном. В данной работе мы продемонстрировали возможность трансфекции миобластов человека целевыми siРНК без ущерба жизнеспособности и дальнейшей дифференцировке этих клеток в миотубы. Также мы показали различия в профиле экспрессии генов-мишеней DUX4 между миобластами, полученными от здоровых людей и от пациентов с МЛД. В дальнейшем эти данные могут помочь при разработке эффективных и специфичных siРНК для терапии МЛД. Facioscapulohumeral dystrophy (FSHD) is a common hereditary disease caused by ectopic expression of the DUX4 gene in muscle cells. The method of suppressing gene expression using siRNA has been shown to be an effective and safe method of gene therapy without interfering directly with the genome. In this work, we demonstrated the possibility of transfecting human myoblasts with targeted siRNAs without compromising the viability and further differentiation of these cells into myotubes. We also showed differences in the expression profile of DUX4 target genes between healthy and patient-derived myoblasts. In the future, these data can be used to develop effective and specific siRNAs for the treatment of FSHD. more...

The ten-eleven translocation (Tet) family of dioxygenases convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Previous studies have shown that 5hmC-mediated epigenetic modifications play essential roles in diverse biological processes and diseases. Here, we show that Tet proteins and 5hmC display dynamic features during postnatal cardiac development and that Tet2 is the predominant dioxygenase present in heart. Tet2 knockout results in abnormal cardiac function, progressive cardiac hypertrophy and fibrosis. Mechanistically, Tet2 deficiency leads to reduced hydroxymethylation in the cardiac genome and alters the cardiac transcriptome. Mechanistically, Tet2 loss leads to a decrease of Hspa1b expression, a regulator of the extracellular signal-regulated protein kinase (Erk) signaling pathway, which leads to over-activation of Erk signaling. Acute Hspa1b knock down (KD) increased the phosphorylation of Erk and induced hypertrophy of cardiomyocytes, which could be blocked by Erk signaling inhibitor. Consistently, ectopic expression of Hspa1b was able to rescue the deficits of cardiomyocytes induced by Tet2 depletion. Taken together, our study’s results reveal the important roles of Tet2-mediated DNA hydroxymethylation in cardiac development and function. more...

Coronary artery disease (CAD) is one of the heavy health burdens worldwide. Aberrant proliferation of vascular smooth muscle cells (VSMCs) contributes to the occurrence and development of CAD. This study aimed at exploring differentially expressed microRNAs (miRNAs) and their regulatory mechanisms in the development of CAD.The miRNA expression profile of GSE28858 was obtained from the Gene Expression Omnibus database. Differentially expressed miRNAs (DEmiRNAs) between CAD and healthy control samples were analyzed using limma package in R. Target genes of DEmiRNAs were predicted, and a miRNA-target gene network was constructed. The relationship between miR-665 and transforming growth factor beta receptor 1 (TGFBR1) was selected for further analysis. The interaction between miR-665 and TGFBR1 was confirmed by dual luciferase reporter assay. Effects of miR-665 on cell viability and apoptosis of VSMCs were evaluated by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Besides, western blot assays for BCL2L11 and caspase 3 were also conducted.A total of 38 upregulated miRNAs and 28 downregulated miRNAs were identified. The expression level of miR-665 was significantly downregulated in patients with CAD. TGFBR1 was proved to be a target gene of miR-665. Besides, ectopic expression of miR-665 obviously inhibited VSMC growth and promoted VSMC apoptosis. TGFBR1 overexpression in VSMCs transfected with miR-665 mimic could restore the effect of miR-665 on the proliferation and apoptosis of VSMCs.MiR-665 might participate in the proliferation and apoptosis of VSMCs by targeting TGFBR1. more...

Background Ephrin-A2, a member of the Eph receptor subgroup, is used in diagnosing and determining the prognosis of prostate cancer. However, the role of ephrin-A2 in prostate cancer is still unclear. Methods We established stable clones overexpressing or silencing ephrin-A2 from prostate cancer cells. Then, CCK-8 was used in analyzing the proliferation ability of cells. CD31 staining was used in evaluating angiogenesis. Migration and invasion assay were conducted in vivo and in vitro. The expression of EMT-related markers was evaluated in prostate cancer cells through Western blotting. Results We revealed that the ectopic expression of ephrin-A2 in prostate cancer cells facilitated cell migration and invasion in vitro and promoted tumor metastasis and angiogenesis in vivo and that the silencing of ephrin-A2 completely reversed this effect. Although ephrin-A2 did not affect tumor cell proliferation in vitro, ephrin-A2 significantly promoted primary tumor growth in vivo. Furthermore, to determine the biological function of ephrin-A2, we assayed the expression of EMT-related markers in stable established cell lines. Results showed that the overexpression of ephrin-A2 in prostate cancer cells down-regulated the expression of epithelial markers (ZO-1, E-cadherin, and claudin-1) and up-regulated the expression of mesenchymal markers (N-cadherin, β-catenin, vimentin, Slug, and Snail), but the knocking out of ephrin-A2 opposed the effects on the expression of EMT markers. Conclusions These findings indicate that ephrin-A2 promotes prostate cancer metastasis by enhancing angiogenesis and promoting EMT and may be a potentially therapeutic target in metastatic prostate cancer. more...

miR156/157 plays multiple pivotal roles during plant growth and development. In this study, we identified 11 miR156- and 5 miR157-encoding loci from the genome of Petunia axillaris and Petunia inflata, designated as PaMIR0156/157s and PiMIR0156/157s, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis indicated that PhmiR156/157 was expressed predominantly in cotyledons, germinating seeds, flower buds, young fruits and seedlings. PhmiR156/157 levels declined in shoot apical buds and leaves of petunia before flowering as the plant ages; moreover, the temporal expression patterns of most miR156/157-targeted PhSPLs were complementary to that of PhmiR156/157. Ectopic expression of PhMIR0157a in Arabidopsis and petunia resulted in delayed flowering, dwarf plant stature, increased branches and reduced organ size. However, PhMIR0156f-overexpressing Arabidopsis and petunia plants showed only delayed flowering. In addition, downregulation of PhmiR156/157 level by overexpressing STTM156/157 led to taller plants with less branches, longer internodes and precocious flowering. qRT-PCR analysis indicated that PhmiR156/157 modulates these traits mainly by downregulating their PhSPL targets and subsequently decreasing the expression of flowering regulatory genes. Our results demonstrate that the PhmiR156/157-PhSPL module has conserved but also divergent functions in growth and development, which will help us decipher the genetic basis for the improvement of flower transition, plant architecture and organ development in petunia. more...

Calcium-dependent protein kinases (CDPKs) play vital roles in metabolic regulations and stimuli responses in plants. However, little is known about their function in grapevine. Here, we report that VpCDPK9 and VpCDPK13, two paralogous CDPKs from Vitis pseudoreticulata accession Baihe-35-1, appear to positively regulate powdery mildew resistance. The transcription of them in leaves of 'Baihe-35-1' were differentially induced upon powdery mildew infection. Overexpression of VpCDPK9-YFP or VpCDPK13-YFP in the V. vinifera susceptible cultivar Thompson Seedless resulted in enhanced resistance to powdery mildew (YFP, yellow fluorescent protein). This might be due to elevation of SA and ethylene production, and excess accumulation of H2 O2 and callose in penetrated epidermal cells and/or the mesophyll cells underneath. Ectopic expression of VpCDPK9-YFP in Arabidopsis resulted in varied degrees of reduced stature, pre-mature senescence and enhanced powdery mildew resistance. However, these phenotypes were abolished in VpCDPK9-YFP transgenic lines impaired in SA signaling (pad4sid2) or ethylene signaling (ein2). Moreover, both of VpCDPK9 and VpCDPK13 were found to interact with and potentially phosphorylate VpMAPK3, VpMAPK6, VpACS1 and VpACS2 in vivo (ACS, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase; MAPK, mitogen-activated protein kinase). These results suggest that VpCDPK9 and VpCDPK13 contribute to powdery mildew resistance via positively regulating SA and ethylene signaling in grapevine. more...

Molecular mechanisms underlying intratumoral androgenesis and aberrant androgen receptor (AR) activation in prostate cancer remain poorly understood. Here we demonstrate that ectopic expression of the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger domain protein (SPOP) stabilizes 17βHSD4. SPOP bound a functional substrate-binding consensus (SBC) motif 315RATST319 in 17βHSD4 and promoted nondegradable K27- and K29-linked polyubiquitination of 17βHSD4. The effect of SPOP was antagonized by serum- and glucocorticoid kinase-3 (SGK3)-mediated phosphorylation of serine 318 (S318) in the SBC and S318 phosphorylation-dependent binding of SKP2 E3 ligase and subsequent K48-linked polyubiquitination and proteasomal degradation of 17βHSD4. Prostate cancer–associated SPOP mutations impaired the SPOP-17βHSD4 interaction, caused 17βHSD4 protein destruction in prostate cancer cells in culture and patient specimens, and increased testosterone production and prostate cancer cell growth in vitro and in mouse models. Thus, we have identified SPOP and SKP2 as two essential E3 ubiquitin ligases that exert opposite effects on 17βHSD4 protein degradation and intratumoral androgenesis in prostate cancer cells. We further demonstrate that SPOP mutations or SKP2 overexpression contribute to prostate cancer progression by decreasing 17βHSD4 expression and increasing intratumoral androgen synthesis. Significance: This study reveals a novel mechanism of aberrant AR activation in SPOP-mutated prostate cancer and uncovers putative biomarkers for effective treatment by AR-targeted therapies. more...

Cultivated grapevine (Vitis) is a highly valued horticultural crop, and cold stress affects its growth and productivity. Wild Amur grape (Vitis amurensis) PAT1 (Phytochrome A signal transduction 1, VaPAT1) is induced by low temperature, and ectopic expression of VaPAT1 enhances cold tolerance in Arabidopsis (Arabidopsis thaliana). However, little is known about the molecular mechanism of VaPAT1 during the cold stress response in grapevine. Here, we confirmed the overexpression of VaPAT1 in transformed grape calli enhanced cold tolerance. Yeast two-hybrid and bimolecular fluorescence complementation assays highlighted an interaction between VaPAT1 with INDETERMINATE-DOMAIN 3 (VaIDD3). A role of VaIDD3 in cold tolerance was also indicated. Transcriptome analysis revealed VaPAT1 and VaIDD3 overexpression and cold treatment coordinately modulate the expression of stress-related genes including lipoxygenase 3 (LOX3), a gene encoding a key jasmonate biosynthesis enzyme. Co-expression network analysis indicated LOX3 might be a downstream target of VaPAT1. Both electrophoretic mobility shift and dual luciferase reporter assays showed the VaPAT1-IDD3 complex binds to the IDD-box (AGACAAA) in the VaLOX3 promoter to activate its expression. Overexpression of both VaPAT1 and VaIDD3 increased the transcription of VaLOX3 and JA levels in transgenic grape calli. Conversely, VaPAT1-SRDX (dominant repression) and CRISPR/Cas9-mediated mutagenesis of PAT1-ED causing the loss of the C-terminus in grape calli dramatically prohibited the accumulation of VaLOX3 and JA levels during cold treatment. Together, these findings point to a pivotal role of VaPAT1 in the cold stress response in grape by regulating JA biosynthesis. more...

Sex determination is a crucially important developmental event that is pervasive throughout nature and enhances the adaptation of species. Among plants, cucumber (Cucumis sativus L.) can generate both unisexual and bisexual flowers, and the sex type is mainly controlled by several 1-aminocyclopropane-1-carboxylic acid synthases (CsACSs). However, the regulatory mechanism of these synthases remains elusive. Here, we used gene expression analysis, protein–DNA interaction assays, and transgenic plants to study the function of a gynoecium-specific gene, ETHYLENE RESPONSE FACTOR31 (CsERF31), in female flower differentiation. We found that in a predetermined female flower, ethylene signaling activates CsERF31 by CsEIN3, and then CsERF31 stimulates CsACS2, which triggers a positive feedback loop to ensure female rather than bisexual flower development. A similar interplay is functionally conserved in melon (Cucumis melo L.). Knockdown of CsERF31 by RNAi causes defective bisexual flowers to replace female flowers. Ectopic expression of CsERF31 suppresses stamen development and promotes pistil development in male flowers, demonstrating that CsERF31 functions as a sex switch. Taken together, our data confirm that CsERF31 represents the molecular link between female–male determination and female–bisexual determination, and provide mechanistic insight into how ethylene promotes female flowers, rather than bisexual flowers, in cucumber sex determination. more...

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development. more...

Previously, we elucidated the function of flotilin-2 (FLOT2) and branched-chain amino acid transaminase 1(BCAT1) in nasopharyngeal carcinoma (NPC). However, the relationship between FLOT2 and BCAT1 in promoting NPC progression remains unknown. Here, we observed that FLOT2 upregulated BCAT1 expression in NPC cells. Ectopic expression of BCAT1 significantly antagonized the inhibitory effects on NPC cell proliferation induced by FLOT2 depletion. Consequently, BCAT1 knockdown markedly inhibited the pro-proliferative effects of FLOT2 overexpression in NPC cells. FLOT2 expression was positively correlated with BCAT1 expression in NPC tissues and was inversely correlated with the prognosis of NPC patients. Mechanistically, FLOT2 maintains the expression level of c-Myc, a positive transcription factor of BCAT1, and subsequently promote BCAT1 transcription. FLOT2 inhibited miR-33b-5p in NPC cells and attenuated its inhibitory effects on c-Myc. Further, experimental validation of the function of the FLOT2/miR-33b-5p/c-Myc/BCAT1 axis in regulating NPC cell proliferation was performed. Our results revealed that FLOT2 promotes NPC cell proliferation by suppressing miR-33b-5p, to maintain proper levels of c-Myc, and upregulate BCAT1trancription. Therefore, the FLOT2/miR-33b-5p/c-Myc/BCAT1 axis is a potential therapeutic target for NPC. more...

The G‐protein‐coupled receptor 126 (GPR126) may play an important role in tumor development, although its role remains poorly understood. We found that GPR126 had higher expression in most colorectal cancer cell lines than in normal colon epithelial cell lines, and higher expression levels in colorectal cancer tissues than in normal adjacent colon tissues. GPR126 knockdown induced by shRNA inhibited cell viability and colony formation in HT‐29, HCT116, and LoVo cells, decreased BrdU incorporation into newly synthesized proliferating HT‐29 cells, led to an arrest of cell cycle progression at the G1 phase in HCT‐116 and HT‐29 cells, and suppressed tumorigenesis of HT‐29, HCT116, and LoVo cells in nude mouse xenograft models. GPR126 knockdown engendered decreased transcription and translation of histone deacetylase 2 (HDAC2), previously implicated in the activation of GLI1 and GLI2 in the Hedgehog signaling pathway. Ectopic expression of HDAC2 in GPR126‐silenced cells restored cell viability and proliferation, GLI2 luciferase reporter activity, partially recovered GLI2 expression, and reduced the cell cycle arrest. HDAC2 regulated GLI2 expression and, along with GLI2, it bound to the PTCH1 promoter, as evidenced by a chip assay with HT‐29 cells. Purmorphamine, a hedgehog agonist, largely restored the cell viability and expression of GLI2 proteins in GPR126‐silenced HT‐29 cells, whereas GANT61, a hedgehog inhibitor, further enhanced the GPR126 knockdown‐induced inhibitory effects. Our findings demonstrate that GPR126 regulates colorectal cancer cell proliferation by mediating the expression of HDAC2 and GLI2, therefore it may represent a suitable therapeutic target for colorectal cancer treatment., GLI2 binding with HDAC2 to the PTCH1 promoter is regulated by HDAC2 and its expression recovery restores cell viability in GPR126‐silenced cells. A, Western blotting of HDAC2 and GLI2 protein levels in NC‐ and HDAC2‐shRNA‐infected HT‐29 cells. β‐actin was used as a loading control. B, Chromatin from HT‐29 cells was immunoprecipitated with anti‐HDAC2 antibodies and then eluted and immunoprecipitated again with normal mouse IgG(IgG) or anti‐Gli2(GLI2 Ab). Eluted DNA was PCR‐amplified using PTCH1 and GAPDH primers (B1). Quantitative PCR(qPCR) was used to examine the abundance of eluted DNA from B1, with the DNAs from input control as the internal control (B2). C, Ectopic expression of HDAC2 in GPR126‐silenced cells restores transcription activity of GLI2 promoter (Gli2‐Luc). NC or Sh1, cell infected by control virus or GPR126 shRNA virus; pGL3, empty luciferase vector; Gli2‐Luc, luciferase vector with Gli2 promoter; zs‐HDAC2, HDAC2 ectopic expression construct; zs, empty vector for cloning HDAC2 expression vector; ‘‐’, not used in co‐transfection; ‘+’, used in co‐transfection. D‐E, the effect of Purmorphamine (hedgehog agonist) and GANT61 (GLI2 inhibitor), at indicated concentration, on cell viability and GLI2 protein expression in indicate colorectal cancer cells. DMSO, the solvent of Purmorphamine and GANT61. β‐actin was used as a loading control. F, Diagram illustrating the putative mechanisms of GPR126 function in colorectal cancer cells. GPR126 regulates SMO, GLI1, and HDAC2 expression (GSE106696). GLI1 and GLI2, as the downstream components of GPR126 signaling, regulate the expression of hedgehog (HH) target genes, including PTCH1, regulating tumor growth. HDAC2 is regulated by GPR126 regulated HDAC2, mediating GLI2 expression, and binds with GLI2 to the PTCH1 promoter or other promoters of HH target genes. The dotted line indicating ‘Not reported in this article’; solid line indicating ‘Has support in this article’; ‘?’ meaning ‘through unknown mechanism’. more...

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 1 (NLRP1) is an inflammasome sensor that mediates the activation of caspase-1 to induce cytokine maturation and pyroptosis1–4. Gain-of-function mutations of NLRP1 cause severe inflammatory diseases of the skin4–6. NLRP1 contains a function-to-find domain that auto-proteolyses into noncovalently associated subdomains7–9, and proteasomal degradation of the repressive N-terminal fragment of NLRP1 releases its inflammatory C-terminal fragment (NLRP1 CT)10,11. Cytosolic dipeptidyl peptidases 8 and 9 (hereafter, DPP8/DPP9) both interact with NLRP1, and small-molecule inhibitors of DPP8/DPP9 activate NLRP1 by mechanisms that are currently unclear10,12–14. Here we report cryo-electron microscopy structures of the human NLRP1–DPP9 complex alone and with Val-boroPro (VbP), an inhibitor of DPP8/DPP9. The structures reveal a ternary complex that comprises DPP9, full-length NLRP1 and the NLRPT CT. The binding of the NLRP1 CT to DPP9 requires full-length NLRP1, which suggests that NLRP1 activation is regulated by the ratio of NLRP1 CT to full-length NLRP1. Activation of the inflammasome by ectopic expression of the NLRP1 CT is consistently rescued by co-expression of autoproteolysis-deficient full-length NLRP1. The N terminus of the NLRP1 CT inserts into the DPP9 active site, and VbP disrupts this interaction. Thus, VbP weakens the NLRP1–DPP9 interaction and accelerates degradation of the N-terminal fragment10 to induce inflammasome activation. Overall, these data demonstrate that DPP9 quenches low levels of NLRP1 CT and thus serves as a checkpoint for activation of the NLRP1 inflammasome. Structures of NLRP1–DPP9 alone and with a small-molecule inhibitor of DPP9 reveal the mechanisms through which NLRP1 is regulated, providing insights into the role of this complex in inflammasome regulation. more...

Although skeletal muscle cells and adipocytes are derived from the same mesoderm, they do not transdifferentiate in vivo and are strictly distinct at the level of gene expression. To elucidate some of the regulatory mechanisms underlying this strict distinction, Pax7, a myogenic factor, was ectopically expressed in 3T3-L1 adipose progenitor cells to perturb their adipocyte differentiation potential. Transcriptome analysis showed that ectopic expression of Pax7 repressed the expression of some adipocyte genes and induced expression of some skeletal muscle cell genes. We next profiled the epigenomic state altered by Pax7 expression using H3K27ac, an activating histone mark, and H3K27me3, a repressive histone mark, as indicators. Our results show that ectopic expression of Pax7 did not result in the formation of H3K27ac at loci of skeletal muscle-related genes, but instead resulted in the formation of H3K27me3 at adipocyte-related gene loci. These findings suggest that the primary function of ectopic Pax7 expression is the formation of H3K27me3, and muscle gene expression results from secondary regulation. more...

Ectopic expression of Glycine max two-component system member GmHP08 in Arabidopsis enhanced drought tolerance of transgenic plants, possibly via ABA-dependent pathways. Phosphorelay by two-component system (TCS) is a signal transduction mechanism which has been evolutionarily conserved in both prokaryotic and eukaryotic organisms. Previous studies have provided lines of evidence on the involvement of TCS genes in plant perception and responses to environmental stimuli. In this research, drought-associated functions of GmHP08, a TCS member from soybean (Glycine max L.), were investigated via its ectopic expression in Arabidopsis system. Results from the drought survival assay showed that GmHP08-transgenic plants exhibited higher survival rates compared with their wild-type (WT) counterparts, indicating better drought resistance of the former group. Analyses revealed that the transgenic plants outperformed the WT in various regards, i.e. capability of water retention, prevention of hydrogen peroxide accumulation and enhancement of antioxidant enzymatic activities under water-deficit conditions. Additionally, the expression of stress-marker genes, especially antioxidant enzyme-encoding genes, in the transgenic plants were found greater than that of the WT plants. In contrary, the expression of SAG13 gene, one of the senescence-associated genes, and of several abscisic acid (ABA)-related genes was repressed. Data from this study also revealed that the ectopic expression lines at germination and early seedling development stages were hypersensitive to exogenous ABA treatment. Taken together, our results demonstrated that GmHP08 could play an important role in mediating plant response to drought, possibly via an ABA-dependent manner. more...

Glioblastoma (GBM) is the most malignant form of glioma. Glioma stem cells (GSCs) contribute to the initiation, progression, and recurrence of GBM as a result of their self-renewal potential and tumorigenicity. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in gliomagenesis remain largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in GSCs. The genetic inhibition of CDK8 by shRNA or CRISPR interference resulted in an abrogation of the self-renewal potential and tumorigenicity of patient-derived GSCs, which could be significantly rescued by the ectopic expression of c-MYC, a stem cell transcription factor. Moreover, we demonstrated that the pharmacological inhibition of CDK8 significantly attenuated the self-renewal potential and tumorigenicity of GSCs. CDK8 expression was significantly higher in human GBM tissues than in normal brain tissues, and its expression was positively correlated with stem cell markers including c-MYC and SOX2 in human GBM specimens. Additionally, CDK8 expression is associated with poor survival in GBM patients. Collectively, these findings highlight the importance of the CDK8-c-MYC axis in maintaining stemness and tumorigenicity in GSCs; these findings also identify the CDK8-c-MYC axis as a potential target for GSC-directed therapy. more...

Bardet-Biedl syndrome (BBS) is a rare ciliopathy for which there are no current effective treatments. BBS is a genetically heterogeneous disease, though the M390R mutation in BBS1 is involved in ~25% of all genetic diagnoses of BBS. The principle features of BBS include retinal degeneration, obesity, male infertility, polydactyly, intellectual disability, and renal abnormalities. Patients with mutations in BBS genes often present with night blindness within the first decade of life, which progresses to complete blindness. This is due to progressive loss of photoreceptor cells. Male infertility is caused by a lack of spermatozoa flagella, rendering them immobile. In this study, we have crossed the wild-type human BBS1 gene, driven by the CAG promoter, onto the Bbs1M390R/M390R mouse model to determine if ectopic expression of BBS1 rescues male infertility and retinal degeneration. qRT-PCR indicates that the BBS1 transgene is expressed in multiple tissues throughout the mouse, with the highest expression seen in the testes, and much lower expression in the eye and hypothalamus. Immunohistochemistry of the transgene in the eye showed little if any expression in the photoreceptor outer nuclear layer. When male Bbs1M30R/M390R;BBS1TG+ mice are housed with WT females, they are able to sire offspring, indicating that the male infertility phenotype of BBS is rescued by the transgene. Using electroretinography (ERGs) to measure retinal function and optical coherence tomography to measure retinal thickness, we show that the transgene does not confer protection against retinal degeneration in Bbs1M300R/M390R;BBS1TG+ mice. The results of this study indicate that the male infertility aspect of BBS is an attractive target for gene therapy. more...

Superoxide dismutase (SOD) is a crucial reactive oxygen species (ROS) scavenger, which converts superoxide radical to H2O2, so it is thought to enhance abiotic stress tolerance by reducing ROS and thus avoiding oxidative damage. In this study, we isolated a salt- and oxidative stress-responsive copper-zinc (Cu/Zn) SOD encoding gene TaSOD5 from wheat. The ectopic overexpression of TaSOD5 in Arabidopsis thaliana increased total SOD and Cu/Zn SOD activities and enhanced tolerance to salt stress. Arabidopsis ectopically expressing TaSOD5 possessed a superior resistance to oxidative stress stimulated by exogenous H2O2. Ectopic overexpression of TaSOD5 elevated the activities of both ROS scavengers and an O2.- producer - NADPH oxidase. These findings show that Cu/Zn SOD enhanced salt tolerance via regulating the machinery of redox homeostasis rather than improving SOD activity alone. more...

Summary In grape, MYBA1 and MYBA2 at the colour locus are the major genetic determinants of grape skin colour, and the mutation of two functional genes (VvMYBA1 and VvMYBA2) from these loci leads to white skin colour. This study aimed to elucidate the regulation of grape berry coloration by isolating and characterizing VvMYBA2w and VvMYBA2r alleles. The overexpression of VvMYBA2r up‐regulated the expression of anthocyanin biosynthetic genes and resulted in higher anthocyanin accumulation in transgenic tobacco than wild‐type (WT) plants, especially in flowers. However, the ectopic expression of VvMYBA2w inactivated the expression of anthocyanin biosynthetic genes and could not cause obvious phenotypic modulation in transgenic tobacco. Unlike in VvMYBA2r, CA dinucleotide deletion shortened the C‐terminal transactivation region and disrupted the transcriptional activation activity of VvMYBA2w. The results indicated that VvMYBA2r positively regulated anthocyanin biosynthesis by forming the VvMYBA2r‐VvMYCA1‐VvWDR1 complex, and VvWDR1 enhanced anthocyanin accumulation by interacting with the VvMYBA2r‐VvMYCA1 complex; however, R44L substitution abolished the interaction of VvMYBA2w with VvMYCA1. Meanwhile, both R44L substitution and CA dinucleotide deletion seriously affected the efficacy of VvMYBA2w to regulate anthocyanin biosynthesis, and the two non‐synonymous mutations were additive in their effects. Investigation of the colour density and MYB haplotypes of 213 grape germplasms revealed that dark‐skinned varieties tended to contain HapC‐N and HapE2, whereas red‐skinned varieties contained high frequencies of HapB and HapC‐Rs. Regarding ploidy, the higher the number of functional alleles present in a variety, the darker was the skin colour. In summary, this study provides insight into the roles of VvMYBA2r and VvMYBA2w alleles and lays the foundation for the molecular breeding of grape varieties with different skin colour. more...

Growing lncRNAs have been noted to involve in the initiation and development of several tumours including tongue squamous cell carcinomas (TSCCs). However, the biological role and mechanism of lncRNA RPSAP52 were not well‐explained. We indicated that RPSAP52 was higher in TSCC samples compared with that in control samples. The higher expression of RPSAP52 was positively correlated with higher T stage and TNM stage. Ectopic expression of RPSAP52 induced TSCC cell growth and cycle and induced cytokine secretion including IFN‐γ, IL‐1β and IL‐6, IL‐8, IL‐10 and TGF‐β. We found that the overexpression of RPSAP52 suppressed miR‐423‐5p expression in SCC‐4 cell. miR‐423‐5p was lower in TSCC samples compared with that in control samples, and miR‐423‐5p level was negatively correlated with higher T stage and TNM stage. Pearson's correlation indicated that miR‐423‐5p was negatively associated with that of RPSAP52 in TSCC tissues. Furthermore, MYBL2 was one direct gene of miR‐423‐5p and elevated expression of miR‐423‐5p suppressed MYBL2 expression and ectopic expression of RPSAP52 increased MYBL2 expression in SCC‐4 cell. Finally, we illustrated that RPSAP52 overexpression promoted TSCC cell growth and cycle and induced cytokine secretion including IFN‐γ, IL‐1β and IL‐6, IL‐8, IL‐10 and TGF‐β via modulating MYBL2. These data provided new insight into RPSAP52, which may be one potential treatment target for TSCC. more...

Primary open angle glaucoma (POAG) is the leading cause of irreversible blindness. Dysfunction of the trabecular meshwork (TM), resulting in decreased outflow of aqueous humor and increased intraocular pressure (IOP), plays an important role in the pathogenesis of POAG. However, the underlying mechanisms still remain unclear. In this study, we demonstrated that the eIF2-α/ATF4/CHOP branch of unfolded protein response (UPR) was activated in human trabecular meshwork cells (HTMCs) upon tert-butyl hydroperoxide (TBHP) exposure. Inhibition of ATF4 ameliorated TBHP-induced apoptosis and inflammatory cytokine production, while ectopic expression of ATF4 increased the expression of endothelial leukocyte adhesion molecule (ELAM)-1 and IL-8 in HTMCs. Furthermore, we found that ATF4 inhibition reduced tunicamycin-induced caspase-3 activation, ROS production, ELAM-1 expression, and HTMCs phagocytosis impairment. By an in vivo study in mice, we showed that overexpression of ATF4 in the TM induced C/EBP homologous protein (CHOP) expression and TM cells apoptosis, contributing to inflammatory cytokine production, and probably IOP elevation. More importantly, upregulation of ATF4 and CHOP, and colocalization of ATF4 with ELAM-1 were found in the TM of POAG patients. These results suggest that ATF4 is a critical mediator of oxidative stress and ER stress-induced TM cell dysfunction and apoptosis in POAG. more...

Conversion of astrocytes into neurons in vivo offers an alternative therapeutic approach for neuronal loss after injury or disease. However, not only the efficiency of the conversion of astrocytes into functional neurons by single Neurog2, but also the conundrum that whether Neurog2-induced neuronal cells (Neurog2-iNs) are further functionally integrated into existing matured neural circuits remains unknown. Here, we adopted the AAV(2/8) delivery system to overexpress single factor Neurog2 into astrocytes and found that the majority of astrocytes were successfully converted into neuronal cells in multiple brain regions, including the midbrain and spinal cord. In the midbrain, Neurog2-induced neuronal cells (Neurog2-iNs) exhibit neuronal morphology, mature electrophysiological properties, glutamatergic identity (about 60%), and synapse-like configuration local circuits. In the spinal cord, astrocytes from both the intact and lesioned sources could be converted into functional neurons with ectopic expression of Neurog2 alone. Notably, further evidence from our study also proves that Neurog2-iNs in the intact spinal cord are capable of responding to diverse afferent inputs from dorsal root ganglion (DRG). Together, this study does not merely demonstrate the feasibility of Neurog2 for efficient in vivo reprogramming, it gives an indication for the Neurog2-iNs as a functional and potential factor in cell-replacement therapy. more...

As an effective anticancer drug, the clinical limitation of doxorubicin (Dox) is the time- and dose-dependent cardiotoxicity. Yes-associated protein 1 (YAP1) interacts with transcription factor TEA domain 1 (TEAD1) and plays an important role in cell proliferation and survival. However, the role of YAP1 in Dox-induced cardiomyopathy has not been reported. In this study, the expression of YAP1 was reduced in clinical human failing hearts with dilated cardiomyopathy and Dox-induced in vivo and in vitro cardiotoxic model. Ectopic expression of Yap1 significantly blocked Dox-induced cardiomyocytes apoptosis in TEAD1 dependent manner. Isorhapontigenin (Isor) is a new derivative of stilbene and responsible for a wide range of biological processes. Here, we found that Isor effectively relieved Dox-induced cardiomyocytes apoptosis in a dose-dependent manner in vitro. Administration with Isor (30 mg/kg/day, intraperitoneally, 3 weeks) significantly protected against Dox-induced cardiotoxicity in mice. Interestingly, Isor increased Dox-caused repression in YAP1 and the expression of its target genes in vivo and in vitro. Knockout or inhibition of Yap1 blocked the protective effects of Isor on Dox-induced cardiotoxicity. In conclusion, YAP1 may be a novel target for Dox-induced cardiotoxicity and Isor might be a new compound to fight against Dox-induced cardiotoxicity by increasing YAP1 expression. more...

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens. more...

Accumulating evidence shows that circRNAs play critical roles in the development of human tumors. We observed that circ_0000527 was overexpressed in osteosarcoma cells (SAOS-2, HOS, MG-63 and U2OS) compared in hFOB1.19 cells. We demonstrated that the circ_0000527 level was higher in osteosarcoma specimens than in non-tumor specimens. The ectopic expression of circ_0000527 was shown to induce cell growth, cell cycle progression and the secretion of inflammatory mediators, including IL-1β, IL-6, IL-8 and TNF-α. We demonstrated that circ_0000527 sponges miR-646 in osteosarcoma cells and that ARL2 is a target gene of miR-646. MiR-646 expression was decreased and ARL2 was overexpressed in osteosarcoma cells (SAOS-2, HOS, MG-63 and U2OS) compared to hFOB1.19 cells. Overexpression of circ_0000527 was demonstrated to induce ARL2 expression in MG-63 cells. We showed that miR-646 was downregulated in osteosarcoma specimens compared to that of non-tumor specimens and that the level of circ_0000527 was negatively correlated with miR-646 expression in osteosarcoma specimens. The elevated expression of circ_0000527 was shown to promote cell growth and cell cycle progression by modulating miR-646 expression. The ectopic expression of circ_0000527 was shown to promote cell growth, cell cycle progression and the secretion of inflammatory mediators by modulating ARL2. The present study suggested that the circ_0000527/miR-646/ARL2 axis may be a potential treatment target for osteosarcoma. more...

Adoptive transfer of antitumor cytotoxic T cells is an emerging form of cancer immunotherapy. A key challenge to expanding the utility of adoptive cell therapies is how to enhance the survival and function of the transferred T cells. Immune-cell survival requires adaptation to different microenvironments and particularly to the hypoxic milieu of solid tumors. The hypoxia-inducible factor (HIF) transcription factors are an essential aspect of this adaptation. In this study, we undertook experiments to define structural determinants of HIF that potentiate antitumor efficacy in cytotoxic T cells. We first created retroviral vectors to deliver ectopic expression of HIF1α and HIF2α in mouse CD8+ T cells, together or individually and with or without sensitivity to the oxygen-dependent HIFα inhibitors Von Hippel–Lindau and factor-inhibiting HIF (FIH). HIF2α, but not HIF1α, drove broad transcriptional changes in CD8+ T cells, resulting in increased cytotoxic differentiation and cytolytic function against tumor targets. A specific mutation replacing the hydroxyl group–acceptor site for FIH in HIF2α gave rise to the most effective antitumor T cells after adoptive transfer in vivo. In addition, codelivering an FIH-insensitive form of HIF2α with an anti-CD19 chimeric antigen receptor greatly enhanced cytolytic function of human CD8+ T cells against lymphoma cells both in vitro and in a xenograft adoptive transfer model. These experiments point to a means to increase the antitumor efficacy of therapeutic CD8+ T cells via ectopic expression of the HIF transcription factor. See related Spotlight on p. 364 more...

Polycomb repressive complex 1 (PRC1) and PRC2 are critical epigenetic developmental regulators. PRC1 and PRC2 largely overlap in their genomic binding and cooperate to establish repressive chromatin domains demarcated by H2AK119ub and H3K27me3. However, the functional contribution of each complex to gene repression has been a subject of debate, and understanding of its physiological significance requires further studies. Here, using the developing murine epidermis as a paradigm, we uncovered a previously unappreciated functional redundancy between Polycomb complexes. Coablation of PRC1 and PRC2 in embryonic epidermal progenitors resulted in severe defects in epidermal stratification, a phenotype not observed in the single PRC1-null or PRC2-null epidermis. Molecular dissection indicated a loss of epidermal identity that was coupled to a strong derepression of nonlineage transcription factors, otherwise repressed by either PRC1 or PRC2 in the absence of its counterpart. Ectopic expression of subsets of PRC1/2-repressed nonepidermal transcription factors in wild-type epidermal stem cells was sufficient to suppress epidermal identity genes, highlighting the importance of functional redundancy between PRC1 and PRC2. Altogether, our studies show how PRC1 and PRC2 function as two independent counterparts, thereby providing a repressive safety net that protects and preserves lineage identity. more...

Laryngeal squamous cell cancer (LSCC) is a common carcinoma with high morbidity and mortality. Metastasis constitutes the major cause of death and poor prognosis among patients with LSCC. Recent evidence confirms critical function of Wnt1-inducible signaling protein 1 (WISP1) in several cancers. However, its contribution in LSCC metastasis remains unclear. Specimens of tumor tissues and adjacent normal mucosa were collected from patients with LSCC. The mRNA and protein levels were determined using quantitative real-time PCR and Western blot, respectively. RNA interference was applied to silence the expression of WISP1 and TGF-β, and recombinant adenovirus was used to overexpress WISP1 in human LSCC cell line TU212 cells. Cell invasion and migration were determined by transwell assay. High expression of WISP1 was observed in LSCC tissues, especially in those from metastatic groups. Ectopic expression of WISP1 enhanced invasion and migration of TU212 cells. On the contrary, WISP1 knockdown reduced numbers of invasive and migrated cells. Additionally, elevation of WISP1 depressed the expression of epithelial marker E-cadherin and increased levels of mesenchymal marker vimentin in TU212 cells, whereas WISP suppression yielded the opposite effects. Further analysis corroborated that WISP1 overexpression enhanced activation of TGF-β-Smad signaling by increasing expression of TGF-β1, p-Smad2, and p-Smad3, which was abrogated following WISP1 down-regulation. Moreover, TGF-β1 exposure facilitated LSCC cell invasion and migration. Notably, blockage of the TGF-β-Smad pathway by si-TGF-β overturned WISP-1-evoked epithelial-to-mesenchymal transition (EMT), and subsequent cell invasion and migration. These findings highlight the pro-metastatic function of WISP1 in LSCC by regulating cell invasion and migration via TGF-β-Smad-mediated EMT, supporting a promising invention target for LSCC therapy. more...

Introduction Protein kinase C theta (PKCθ) is expressed in ER-negative breast cancer and promotes cancer stem cells (CSCs) phenotype. PKCθ gene (PRKCQ) is predicted to be a target for tumor suppressor miR-203. Herein, we aim to validate this prediction and evaluate the ability of miR-203 to inhibit migration of breast cancer cell line enriched with CSCs, MDA-MB-231, via PRKCQ targeting. Methods Cells were transfected with miR-203 mimic, PRKCQ siRNA and negative control; then real-time PCR, migration assay, western blotting, reporter assay, and chromatin accessibility assay were performed. Results Our findings displayed significant decrease in PRKCQ mRNA level and luciferase signals in cells with restored miR-203 expression, therefore, validated PRKCQ as a direct target of miR-203. Additionally, inhibiting PRKCQ by siRNA led to significant inhibition of miR-203 expression and significant decrease of chromatin accessibility at miR-203 promoter region 466-291 upstream TSS. Both of miR-203 re-expression and PRKCQ suppression resulted in altering migration ability of MDA-MB-231 through regulating AKT pathway and genes involved in breast cancer stem cells, CD44 and ALDH1A3. Expression of CDK5, GIV, and NANOG was significantly downregulated in miR-203 mimic-transfected cells, while PRKCQ siRNA-transfected cells displayed downregulation of OCT3/4, SOX2, and NANOG. Furthermore, we found that miR-224 expression was enhanced while miR-150 was downregulated after ectopic expression of miR-203. Conclusion The study highlighted the negative feedback loop between miR-203 and its target PRKCQ and the interplay between them in regulating genes involved in BCSCs. The study also concluded "microRNA-mediated microRNA regulation" as an event in breast cancer cells. more...

Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4–p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins. more...

Legumes control their nodule numbers through the autoregulation of nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development and also acts to shape root system architecture. more...

The expression level of CEP55, a centrosome and midbody-associated protein is pivotal for cell cytokinesis and is significantly correlated with tumor stage. Our previous study demonstrated that ectopic expression of TSG101 can decrease androgen receptor expression level through the lysosome degradation pathway. Here, we further extended the investigation of TSG101 in modulating protein levels through lysosomes, and identified ubiquitously expressed transcript (UXT) to be a novel TSG101 interaction partner associated with TSG101-containing cytoplasmic vesicles. We also demonstrated that CEP55 can be recruited to TSG101 cytoplasmic vesicles resulting in downregulation of CEP55 through lysosome degradation. Moreover, UXT depletion promoted TSG101 vesicle-lysosome association and elevated autophagic carrier flux to enhance CEP55 degradation upon TSG101 overexpression. In summary, we identified a novel CEP55 regulation pathway mediated by TSG101 overexpression via lysosome degradation and revealed that UXT plays a role in the late endosome/autophagosome-lysosome fusion event, engaging in TSG101-mediated lysosome degradation. more...

Epithelial junctions ensure cell–cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli–BMP–miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3′UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. Here, we show that ectopic expression of Gli interferes with Tkv–Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv–pMad signaling activity, and this effect is continuous through larval and pupal wing formation. more...

Protocadherin 10 (PCDH10) is identified as a tumor suppressor in multiple cancers. The molecular mechanisms that mediate the functions of PCDH10 have yet to be fully elucidated. Here, we demonstrated that ectopic expression of PCDH10 in colorectal cancer (CRC) cells induced cell cycle retardation and increased apoptosis through regulation of the p53/p21/Rb axis and Bcl-2 expression. Overexpression of PCDH10 reversed the epithelial-mesenchymal transition (EMT) process with morphological changes and EMT marker alterations. Mechanistic study revealed that PCDH10 inhibited AKT/GSK3β signaling pathway which in turn reduced β-catenin activity and thus attenuated Snail and Twist1 expression. Furthermore, PCDH10 inhibited the stemness of CRC cells, including spheroid formation and stem cell markers. A proteomics approach revealed that PCDH10 could interact with EGFR, which was further verified by co-immunoprecipitation. Moreover, restoration of PCDH10 expression reduced EGFR phosphorylation. Accordingly, our work proposes a novel pathway by which PCDH10 directly engages in the negative regulation of EGFR/AKT/β-catenin signaling pathway, resulting in tumor suppression. more...

Graphical abstract, JUP, a homologue of β-catenin, is a cell-cell junction protein involved in adhesion junction and desmosome composition. JUP may have a controversial role in different malignancies dependence of its competence with or collaboration with β-catenin as a transcription factor. In this study, we reveal that the function of JUP is related to its cellular location in GC development process from epithelium-like, low malignant GC to advanced EMT-phenotypic GC. Gradual loss of membrane and/or cytoplasm JUP is closely correlated with GC malignancy and poor prognostics. Knockdown of JUP in epithelium-like GC cells causes EMT and promotes GC cell migration and invasion. Ectopic expression of wild JUP in malignant GC cells leads to an attenuated malignant phenotype such as reduced cell invasive potential. In mechanism, loss of membrane and/or cytoplasm JUP abolishes the restrain of JUP to EGFR at cell membrane and results in increased p-AKT levels and AKT/GSK3β/β-catenin signaling activity. In addition, nuclear JUP interacts with nuclear β-catenin and TCF4 and plays a synergistic role with β-catenin in promoting TCF4 transcription and its downstream target MMP7 expression to fuel GC cell invasion. more...

Purpose: Luminal B breast tumors are more aggressive estrogen receptor–positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies. Experimental Design: We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms. Results: Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition. Conclusions: This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion. more...

The synaptonemal complex (SC) is a proteinaceous structure that is transiently formed during meiosis to promote homologous recombination between maternal and paternal chromosomes. As this structure is required only for meiotic recombination, the proteins constituting the complex are almost undetectable in normal somatic cells, but they can be expressed under the conditions in which the transcriptional machinery is deregulated. Accumulating evidence indicates that they are epigenetically expressed in cancers of various origin. Not surprisingly, in contrast to their meiotic roles, the somatic roles of the SC proteins remain to be investigated. However, it has recently been reported that SYCP3 and SYCE2 control DNA double‐strand break repair negatively and positively, respectively, suggesting that the ectopic expression of the SC proteins in somatic cells could be associated with the maintenance of genomic instability. Thus, it is highly likely that the investigation of the somatic roles of the SC proteins would improve our understanding of the mechanisms underlying tumor development., The synaptonemal complex (SC) is a proteinaceous structure that is transiently formed during meiosis to promote homologous recombination. Although the proteins constituting the complex are highly expressed in meiotic cells and almost undetectable in normal somatic cells, accumulating evidence indicates that they are epigenetically expressed in cancers of various origin. Recent studies report that the SC proteins regulate the endogenous cellular machinery that maintains genome integrity in somatic cells. In this review, we focus on the clinical significance of the SC proteins and the possible mechanism underlying their ectopic expression in human cancers. more...

There is a continued need for investigating the roles of microRNAs (miRNAs) and their targets on the progression of gastric cancer (GC), especially metastasis. Here, we performed an integrated study to identify dysregulated miRNAs critical for GC development and progression. miR-135b was determined as a promising biomarker for GC. The expression level of miR-135b was increased among GC cell lines, patient tumor tissues, serum samples, and correlation with aggravation of the GC patients. The in vitro functional assays demonstrated overexpression of miR-135b promoted cell proliferation, migration and invasion in GC, while miR-135b inhibition led to the opposite results. CAMK2D was found to be the direct target of miR-135b, serving as a tumor suppressor in GC cells. Based on our and public datasets, we confirmed the attenuation of CAMK2D expression in GC tissues. And, the expression levels of miR-135b and CAMK2D were closely associated with prognosis of GC patients. Ectopic expression of miR-135b resulted in the down-regulation of CAMK2D. Additionally, CAMK2D was a prerequisite for miR-135b to promote GC cells proliferation and migration by regulating the EMT process, which was confirmed by the in vivo experiments. Importantly, in vivo injection of miR-135b antagomir significantly repressed the tumor growth and metastasis of xenograft models, which suggested that the miR-135b antagomir were promising for clinical applications. Taken together, these results indicate that miR-135b/CAMK2D axis drives GC progression by EMT process remodeling, suggesting that miR-135b may be utilized as a new therapeutic target and prognostic marker for GC patients. more...

Melatonin is involved in plant responses to various environmental stresses. Although many studies have demonstrated that the tolerance of plants to stress is improved by exogenous melatonin, the role of endogenous melatonin metabolism in the response of apples to UV-B stress remains unclear. Here, the human melatonin biosynthesis-related enzyme genes AANAT or HIOMT were transformed into 'GL-3' apple, and the transgenic lines were treated with UV-B stress. The ectopic expression of AANAT or HIOMT significantly increased the melatonin content in apples. After UV-B stress, the tolerance of apple lines with ectopic expression of AANAT or HIOMT was markedly improved. The decrease in chlorophyll fluorescence, the generation of reactive oxygen species and the shrinkage of stomata caused by UV-B stress were alleviated by AANAT or HIOMT ectopic expression. In addition, the total phenolic content was markedly increased in the transgenic lines compared with the WT (wild type). The increase in phenolic compounds was related to the increase in benzoic acid, hydroxycinnamic acid, dihydrochalcones and flavanols, among which increases in chlorogenic acid, phloridzin and procyanidin B1 content were most prominent. Furthermore, the transgenic lines did not only promote the expression of genes related to phenolic synthesis under UV-B stress, but they also increased the accumulation of phenolic compounds by inhibiting the expression of MdPPO and MdPOD related to phenolic degradation. In summary, our results demonstrate that AANAT- or HIOMT-mediated melatonin synthesis improved the tolerance of apples to UV-B stress, mainly by scavenging reactive oxygen species, increasing photosynthetic capacity and increasing total phenolic content. more...

Fibrotic lesions accompany several pathological conditions, including tumors. We show that expression of a dominant-active form of the Ras oncogene in Drosophila salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components, including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apicobasal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide drosomycin (Drs), which also restores the secretory activity of the SGs. In addition to extracellular matrix components, both Drs and F-actin localize to fibrotic lesions. more...

Background/aim Activation-induced cytidine deaminase (AID) is a DNA modifying enzyme which has an essential function in promoting antibody diversification. Its overexpression is strongly associated with B-cell derived malignancies including Burkitt lymphoma, where AID is required for the characteristic c-MYC/IGH translocation. This study aimed at defining AID's oncopathogenic role which is still poorly understood. Materials and methods We created over-expressing and knock-down cell culture models of AID, and used cellular assays to provide insight into its contribution to lymphomagenesis. Results We showed that AID expression is highly specific to, and abundantly expressed in B-cell-derived cancers and that ectopic overexpression of AID leads to rapid cell death. Using a knock-down model, we revealed that AID expression significantly impacts genomic stability, proliferation, migration and drug resistance. Conclusion AID is an important driver of lymphoma, impacting multiple cellular events, and is potentially a strong candidate for targeted therapy in lymphoma. more...

Background Ectopic expression of transcription elongation factor A (SII)-like 7 (TCEAL7) has been observed in several kinds of cancers, but its role in melanoma is still unclear. This study was carried out to investigate TCEAL7 role in melanoma progression, and uncover the underlying mechanisms. Methods TCEAL7 expression levels in melanoma tissues and cells were determined by using real-time quantitative PCR (RT-PCR) and western blotting. CCK-8, transwell chambers, flow cytometry, starch assay and tumorigenesis assay were applied to detect cell growth, invasion, apoptosis, migration and tumorigenesis, respectively. Results A low expression level of TCEAL7 was observed in melanoma tissues and cells, which was associated with malignant clinical process and poor prognosis. TCEAL7 negatively modulated AKT1, AKT2, c-Myc, N-cadherin and PCNA expression and inhibited cancer progression via decreasing AKT1 and c-Myc levels. In addition, TCEAL7 was negatively modulated by miR-758-3p which promoted melanoma progression. Moreover, overexpression of TCEAL7 abolished miR-758-3p role in promoting melanoma progression. Conclusion This study demonstrated that TCEAL7, regulated by miR-758-3p inhibited melanoma progression through decreasing the expression levels of c-Myc and AKT1. more...

Hepatocellular carcinoma (HCC) is one of the common malignancy and lacks effective therapeutic targets. Here, we demonstrated that ectopic expression of trophinin-associated protein (TROAP) dramatically drove HCC cell growth assessed by foci formation in monolayer culture, colony formation in soft agar and orthotopic liver transplantation in nude mice. Inversely, silencing TROAP expression with short-hairpin RNA attenuated the malignant proliferation of HCC cells in vitro and in vivo. Next, mechanistic investigation revealed that TROAP directly bound to dual specificity tyrosine phosphorylation regulated kinase 1A/B (DYRK1A/B), resulting in the cytoplasmic retention of proteins DYRK1A/B and promoting cell cycle process via activation of Akt/GSK-3β signaling. Combination of cisplatin with an inhibitor of DYRK1 AZ191 effectively inhibited tumor growth in mouse model for HCC cells with high level of TROAP. Clinically, TROAP was significantly upregulated by miR-142-5p in HCC tissues, which predicted the poor survival of patients with HCC. Therefore, TROAP/DYRK1/Akt axis may be a promising therapeutic target and prognostic indicator for patients with HCC. more...

SOX9 (SRY-related HMG box gene 9) is an essential regulator of male sex determination and testis differentiation in many vertebrate species. However, the functional role of Sox9 in testis differentiation has not yet been identified in any reptilian species. Herein, Sox9 knockdown in the red-eared slider turtle (Trachemys scripta) embryos at a male-producing temperature led to complete male-to-female sex reversal, characterized by the formation of an ovary-like structure, disappearance of male marker AMH, and ectopic expression of ovarian regulator FOXL2, as well as a female distribution pattern of germ cells. Conversely, in-ovo overexpression of Sox9 at a female-producing temperature resulted in partial masculinization of putative female embryos, with the co-existence of AMH and FOXL2. Our study provides the first direct evidence that Sox9 is indispensable for testicular differentiation in a reptilian species, further confirming the conserved role of Sox9 in vertebrate sexual development. more...

Rationale: Breast cancer (BrCa) is the most common cancer worldwide, and the 5-year relative survival rate has declined in patients diagnosed at stage IV. Advanced BrCa is considered as incurable, which still lack effective treatment strategies. Identifying and characterizing new tumor suppression genes is important to establish effective prognostic biomarkers or therapeutic targets for late-stage BrCa. Methods: RNA-seq was applied in BrCa tissues and normal breast tissues. Through analyzing differentially expressed genes, DRD2 was selected for further analysis. And expression and promoter methylation status of DRD2 were also determined. DRD2 functions were analyzed by various cell biology assays in vitro. Subcutaneous tumor model was used to explore DRD2 effects in vivo. A co-cultivated system was constructed to investigate interactions of DRD2 and macrophages in vitro. WB, IHC, IF, TUNEL, qRT-PCR, Co-IP, Antibody Array, and Mass Spectrum analysis were further applied to determine the detailed mechanism. Results: In BrCa, DRD2 was found to be downregulated due to promoter methylation. Higher expression of DRD2 positively correlated with longer survival times especially in HER2-positive patients. DRD2 also promoted BrCa cells sensitivity to Paclitaxel. Ectopic expression of DRD2 significantly inhibited BrCa tumorigenesis. DRD2 also induced apoptosis as well as necroptosis in vitro and in vivo. DRD2 restricted NF-κB signaling pathway activation through interacting with β-arrestin2, DDX5 and eEF1A2. Interestingly, DRD2 also regulated microenvironment as it facilitated M1 polarization of macrophages, and triggered GSDME-executed pyroptosis. Conclusion: Collectively, this study novelly manifests the role of DRD2 in suppressing BrCa tumorigenesis, predicting prognosis and treatment response. And this study further reveals the critical role of DRD2 in educating M1 macrophages, restricting NF-κB signaling pathway and triggering different processes of programmed cell death in BrCa. Taking together, those findings represent a predictive and therapeutic target for BrCa. more...

WRKY proteins participate in various plant physiological processes, especially in response to stress. However, limited information is available for WRKY proteins in tea plants. Camellia sinensis WRKY26 (CsWRKY26), a group I WRKY member, was characterized in this study. Tissue specificity of CsWRKY26 expression revealed that its transcripts are abundant in leaves and roots. Moreover, CsWRKY26 transcripts were markedly induced by treatments with sodium chloride (NaCl), polyethylene glycol (PEG), drought and different hormones. Ectopic expression of CsWRKY26 conferred ABA insensitivity and tolerance to transgenic Arabidopsis plants subjected to PEG treatments. When exposed to 15% PEG, the expression levels of some stress-tolerant genes, such as responsive to dehydration 22 (RD22), 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), and Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1), were up-regulated in transgenic plants compared to wild-type (WT) plants. Under natural drought stress, the growth performance of transgenic lines was better than those of WT plants, consistent with their significantly lower relative electrolyte leakage (REL) and higher proline content. These findings indicate that CsWRKY26 could enhance drought tolerance in Arabidopsis. This study provides a potential strategy for future crop improvement research. more...

Human leukocyte antigen G (HLA-G) is a non-classical MHC class I molecule that regulates many immune functions. The physiologic HLA-G expression is restricted to foetal tissues such as: amniotic cells, erythroid precursors, and cytotrophoblasts, and, in adults, to immune-privileged organs. The ectopic expression in tumours could point out to a strategy used by malignant cells to escape the immune surveillance. There are two forms of HLA-G, membrane-bound and soluble. The structure of the soluble and membrane bound isoforms differs at the C-terminus. The extracellular domain and the intracytoplasmic tail are replaced in the secreted isoforms by a short hydrophilic tail. These differences could serve as a marker to distinguish shed or proteolytically cleaved HLA-G isoforms from secreted HLA-G isoforms. HLA-G induces tolerance by inhibiting different cells and this function is mediated by binding of both soluble and membrane-bound HLA-G to the inhibitory receptors. There exists a consistent evidence in literature that HLA-G represents an important factor in determining prognosis in various types of cancer. In this review, we will focus on soluble form of HLA-G (sHLA-G) in cancers and its association with the prognosis of cancer patients, because this immune check-point molecule appears as a promising relevant target for cancer immunotherapy (Fig. 2, Ref. 115). Keywords: cancer, diagnosis, HLA-G, soluble HLA-G, tumour. more...

Rhizoctonia solani is a necrotrophic fungal pathogen that causes disease in diverse plant species. In recent years, the genomic and transcriptomic studies have identified several candidate pathogenicity determinants of R. solani; however, most of them remain to be validated. In this study, we report a viral vector-based host-induced gene silencing (HIGS) as well as a dsRNA (double-stranded RNA)-based approach to effectively downregulate genes of R. solani AG1-IA (BRS1 strain) during pathogenesis in tomato. We tested a few of the in-planta upregulated R. solani genes and observed that silencing of one of them, i.e., RS_CRZ1 (a C2H2 type zinc finger transcription factor) significantly compromises the pathogenesis of R. solani in tomato. The RS_CRZ1-silenced plants not only exhibited significant reduction in disease symptoms, but the depth of pathogen colonization was also compromised. Furthermore, we identified the R. solani genes that were coregulated with RS_CRZ1 during the pathogenicity process. The HIGS-mediated silencing of a few of them [CL1756Contig1; subtilisin-like protease and CL1817Contig2; 2OG-Fe(II) oxygenase] compromised the pathogenesis of R. solani in tomato. The ectopic expression of RS_CRZ1 complemented the crz1 mutant of yeast and restored tolerance against various metal ion stress. Overall, our study reveals the importance of RS_CRZ1 in managing the hostile environment encountered during host colonization. Also, it emphasizes the relevance of the HIGS and dsRNA-based gene silencing approach toward functional characterization of pathogenicity determinants of R. solani. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license . more...

WRINKLED1 belongs to AP2/EREB family of transcription factors whose role has been well established in seed oil biosynthesis. The objective of the study was to trace the role of fiber related Gbwri1 in seed development and fatty acid biosynthesis. In this study, we isolated a transcript from elite fiber producing cotton (Gossypium barbadense), which is over-expressed in G. barbadense fibers as compared to G. hirsutum and G. arboreum. The putative protein encoded by this transcript exhibited homology in specific domains and protein structure with WRINKLED1 of Arabidopsis thaliana and was thus designated as Gbwri1. In this study, we investigated the functional homology of fiber elongation related Gbwri1 with fatty acid biosynthesis regulator Atwri1. Ectopic expression of Gbwri1 in wri1-3 mutant of A. thaliana was analyzed. In the transgenic lines of A. thaliana, Gbwri1 resumed the seed weight, seed area, and surface morphology to the wild type. Gbwri1 transformation rescued the wrinkled phenotype of wri1-3 mutants by resuming the expression of fatty acid biosynthesis genes biotin carboxyl carrier protein isoform 2 (bccp2) and keto-ACP synthase 1 (kas1). Moreover, the seedling development of transgenic lines on non-sucrose medium demonstrated that the Gbwri1 was able to regulate the supply of sucrose for normal seedling establishment. Our results showed that the transformation of Gbwri1 in A. thaliana wri1-3 mutant was able to complement wri1-3 impaired phenotype. Thus, Gbwri1 is involved in cotton fiber development and fatty acid biosynthesis in seeds. © 2021 Friends Science Publishers more...