The homeobox gene Six3 regulates forebrain development. Here we show that Six3 is also crucial for lens formation. Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was downregulated, and Sox2 was absent in the lens preplacodal ectoderm. Using ChIP, electrophoretic mobility shift assay, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain. Our results position Six3 at the top of the regulatory pathway leading to lens formation. We conclude that Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction.

Rationale: Endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) is an endoplasmic reticulum (ER) luminal glycoprotein that has a role in the formation of disulfide bonds of secreted proteins and membrane proteins. Emerging data identify ERO1L as a tumor promoter in a wide spectrum of human malignancies. However, its molecular basis of oncogenic activities remains largely unknown. Methods: Pan-cancer analysis was performed to determine the expression profile and prognostic value of ERO1L in human cancers. The mechanism by which ERO1L promotes tumor growth and glycolysis in pancreatic ductal adenocarcinoma (PDAC) was investigated by cell biological, molecular, and biochemical approaches. Results: ERO1L was highly expressed in PDAC and its precursor pancreatic intraepithelial neoplasia and acts as an independent prognostic factor for patient survival. Hypoxia and ER stress contributed to the overexpression pattern of ERO1L in PDAC. ERO1L knockdown or pharmacological inhibition with EN460 suppressed PDAC cell proliferation in vitro and slowed tumor growth in vivo. Ectopic expression of wild type ERO1L but not its inactive mutant form EROL-C394A promoted tumor growth. Bioinformatics analyses and functional analyses confirmed a regulatory role of ERO1L on the Warburg effect. Notably, inhibition of tumor glycolysis partially abrogated the growth-promoting activity of ERO1L. Mechanistically, ERO1L-mediated ROS generation was essential for its oncogenic activities. In clinical samples, ERO1L expression was correlated with the maximum standard uptake value (SUVmax) in PDAC patients who received 18F-FDG PET/CT imaging preoperatively. Analysis of TCGA cohort revealed a specific glycolysis gene expression signature that is highly correlated with unfolded protein response-related gene signature. Conclusion: Our findings uncover a key function for ERO1L in Warburg metabolism and indicate that targeting this pathway may offer alternative therapeutic strategies for PDAC.

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3'-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.

Filamin A, the first discovered non-muscle actin filament cross-linking protein, plays a crucial role in regulating cell migration that participates in diverse cellular and developmental processes. However, the regulatory mechanism of filamin A stability remains unclear. Here, we find that nuclear distribution gene C (NudC), a cochaperone of heat shock protein 90 (Hsp90), is required to stabilize filamin A in mammalian cells. Immunoprecipitation-mass spectrometry and western blotting analyses reveal that NudC interacts with filamin A. Overexpression of human NudC-L279P (an evolutionarily conserved mutation in NudC that impairs its chaperone activity) not only decreases the protein level of filamin A but also results in actin disorganization and the suppression of cell migration. Ectopic expression of filamin A is able to reverse these defects induced by the overexpression of NudC-L279P. Furthermore, Hsp90 forms a complex with filamin A. The inhibition of Hsp90 ATPase activity by either geldanamycin or radicicol decreases the protein stability of filamin A. In addition, ectopic expression of Hsp90 efficiently restores NudC-L279P overexpression-induced protein stability and functional defects of filamin A. Taken together, these data suggest NudC L279P mutation destabilizes filamin A by inhibiting the Hsp90 chaperoning pathway and suppresses cell migration.

The molecular defects which lead to multistep incidences of human T-cell leukemia have yet to be identified. The DNA-binding protein Helios (known as IKZF2), a member of the Ikaros family of Krüppel-like zinc-finger proteins, functions pivotally in T-cell differentiation and activation. In this study, we identify three novel short Helios splice variants which are T-cell leukemic specific, and demonstrate their dominant-negative function. We then test the cellular localization of distinct Helios isoforms, as well as their capability to form heterodimer with Ikaros, and the association with complexes comprising histone deacetylase (HDAC). In addition, the ectopic expression of T-cell leukemic Helios isoforms interferes with T-cell proliferation and apoptosis. The gene expression profiling and pathway analysis indicated the enrichment of signaling pathways essential for gene expression, translation, cell cycle checkpoint, and response to DNA damage stimulus. These data indicate the molecular function of Helios to be involved in the leukemogenesis and phenotype of T-cell leukemia, and also reveal Helios deregulation as a novel marker for T-cell leukemia.

Ubiquitination is a post-translational regulatory mechanism that controls a variety of biological processes in plants. The E3 ligases confer specificity by recognizing target proteins for ubiquitination. Here, we identified SEVEN IN ABSENTIA (SINA) ubiquitin ligases, which belong to the RING-type E3 ligase family, in upland cotton (Gossypium hirsutum). Twenty-four GhSINAs were characterized, and the expression levels of GhSINA7, GhSINA8, and GhSINA9 were upregulated at 24 h after inoculation with Verticillium dahliae. In vitro ubiquitination assays indicated that the three GhSINAs possessed E3 ubiquitin ligase activities. Transient expression in Nicotiana benthamiana leaves showed that they localized to the nucleus. And yeast two-hybrid (Y2H) screening revealed that they could interact with each other. The ectopic overexpression of GhSINA7, GhSINA8, and GhSINA9 independently in Arabidopsis thaliana resulted in increased tolerance to V. dahliae, while individual knockdowns of GhSINA7, GhSINA8, and GhSINA9 compromised cotton resistance to the pathogen. Thus, GhSINA7, GhSINA8, and GhSINA9 act as positive regulators of defense responses against V. dahliae in cotton plants.

Mature microRNA (miRNA) 34a-5p, which is a well-known tumor suppressor in hepatitis virus-associated hepatocellular carcinoma (HCC), plays an important role in cell processes, such as cell proliferation and apoptosis, and is therefore an optimal biomarker for future clinical use. However, the role of miRNA-34a-5p in chemoresistance has yet to be identified. In the present study, the expression of miRNA-34a-5p was assessed by an in situ hybridization assay in HCC tissues and was found to be significantly decreased compared with the pericarcinomatous areas of the tissue specimens, which consisted of samples obtained from 114 patients with HCC. High expression of miRNA-34a-5p was found to be associated with a favorable overall survival time in HCC patients. Functional tests performed by transfecting miRNA-34a-5p mimics or inhibitors into MHCC-97L cells illustrated that miRNA-34a-5p inhibited proliferation, elevated apoptosis and decreased chemoresistance to cisplatin in HCC cells. AXL is the direct target of miRNA-34a-5p, as confirmed by sequence analysis and luciferase assay. Transfection of the cells with small interfering RNA for AXL (siAXL) increased the apoptosis ratio of the MHCC-97L cell line. Transfection with siAXL led to similar biological behaviors in the MHCC-97L cells to those induced by ectopic expression of miRNA-34a-5p. Thus, it was concluded that miRNA-34a-5p enhanced the sensitivity of the cells to chemotherapy by targeting AXL in hepatocellular carcinoma. In addition, low expression of miRNA-34a-5p in HCC tissues yielded an unfavorable prognosis for patients with HCC that received radical surgery, due to the promotion of proliferation and an increase in chemoresistance in HCC cells.

A major challenge for cancer chemotherapy is the development of safe and clinically effective chemotherapeutic agents. With its low toxicity profile, sophocarpine (SC), a naturally occurring tetracyclic quinolizidine alkaloid derived from Sophora alopecuroides L, has shown promising therapeutic properties, including anti-inflammatory, anti-nociceptive, and antivirus activities. However, the antitumor efficacy of SC and its underlying mechanisms have not been completely delineated. In the present study, the inhibitory effect of SC on head and neck squamous cell carcinoma (HNSCC) progression and possible mechanisms for this effect involving microRNA-21 (miR-21) regulation were investigated. By cell viability, Transwell, and wound healing assays, we show that SC effectively inhibited proliferation, invasion, and migration of HNSCC cells. Moreover, SC exerted its growth-inhibitory effect via the downregulation of miR-21 expression by blocking Dicer-mediated miR-21 maturation. Furthermore, SC treatment led to the increased expression of PTEN and p38MAPK phosphorylation as well as the reversal of epithelial-mesenchymal transition (EMT), which was rescued by ectopic expression of miR-21 in cells. Notably, SC dramatically repressed tumor growth without observable tissue cytotoxicity in a mouse xenograft model of HNSCC. Our findings offer a preclinical proof of concept for SC as a leading natural agent for HNSCC cancer therapy.

Epithelial ovarian cancer (EOC) still remains the most lethal gynaecological malignancy in women, despite the recent progress in the management, including surgery and chemotherapy. According to the microarray data of the GSE18520 and GSE54388 datasets, LINC01215 was identified as an upregulated long noncoding RNA (lncRNA) in EOC. Therefore, this study aimed to figure out the involvement of LINC01215 in the progression of EOC. RT-qPCR was conducted to select the EOC cell line with the highest expression of LINC01215. Methylation of RUNX3 was then examined in EOC cells by MS-PCR. Furthermore, the interaction between LINC01215 and methylation-related proteins was revealed according to the results of RIP and RNA pull down assays. Subsequently, the involvement of LINC01215 and RUNX3 in regulating biological behaviors of EOC cells was investigated. Finally, the effects of the ectopic expression of LINC01215 and RUNX3 on the tumor formation and lymph node metastasis (LNM) of EOC cells were assessed in the xenograft tumors of nude mice. Overexpressing LINC01215 contributed to downregulated levels of RUNX3, as demonstrated by the recruitment of methylation-related proteins. Silencing of LINC01215 elevated the expression of RUNX3, thus suppressing cell proliferation, migration, invasion and EMT and decreasing the expressions of MMP-2, MMP-9 and Vimentin, but increased the expression of E-cadherin. The tumor growth and LNM were suppressed by downregulated levels of LINC01215 through inducing the expression of RUNX3. Collectively, the down-regulating LINC01215 could upregulate the expression of RUNX3 by promoting its methylation, thus suppressing EOC cell proliferation, migration and invasion, EMT, tumor growth and LNM.

Although sunitinib contributes to prolonging the progression-free survival of metastatic renal cell carcinoma significantly, the universal presence of resistance limits the initial response rate and restricts durable responses. The mechanisms involved in sunitinib resistance vary and need further investigation. We found long non-coding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) overexpressed in sunitinib-resistant cells while declined in the parental cells. Moreover, lncRNA CCAT1 increased significantly in samples with resistance to sunitinib compared with those with responses to sunitinib. The reduction of CCAT1 suppressed cell growth and colony formation while triggering apoptosis. Inversely, the ectopic expression of c-Myc reversed the inhibition of cell growth and enhancement of apoptosis by the knockdown of CCAT1. We also verified that anti-apoptosis protein B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia 1 (Mcl-1) decreased along with the deregulation of CCAT1, whereas the expression of Bcl-2 and Mcl-1 restored in cells that were transfected sh-CCAT1 and c-Myc simultaneously. Apart from the in vitro experiments, we demonstrated that knockdown of CCAT1 boosted response to sunitinib by performing sunitinib-resistant ACHN mouse models. Briefly, lncRNA CCAT1 conferred renal cell carcinoma resistance to sunitinib in a c-Myc-dependent manner, providing a novel target for improvement of sunitinib therapy.

Primary cilia are antenna-like subcellular structures to act as signaling platforms to regulate many cellular processes and embryonic development. m1A RNA modification plays key roles in RNA metabolism and gene expression; however, the physiological function of m1A modification remains largely unknown. Here we find that the m1A demethylase ALKBH3 significantly inhibits ciliogenesis in mammalian cells by its demethylation activity. Mechanistically, ALKBH3 removes m1A sites on mRNA of Aurora A, a master suppressor of ciliogenesis. Depletion of ALKBH3 enhances Aurora A mRNA decay and inhibits its translation. Moreover, alkbh3 morphants exhibit ciliary defects, including curved body, pericardial edema, abnormal otoliths, and dilation in pronephric ducts in zebrafish embryos, which are significantly rescued by wild-type alkbh3, but not by its catalytically inactive mutant. The ciliary defects caused by ALKBH3 depletion in both vertebrate cells and embryos are also significantly reversed by ectopic expression of Aurora A mRNA. Together, our data indicate that ALKBH3-dependent m1A demethylation has a crucial role in the regulation of Aurora A mRNA, which is essential for ciliogenesis and cilia-associated developmental events in vertebrates.

Background/Aims: LncRNAs are a new modulator in the development of intervertebral disc degeneration. However, the functional role and mechanism of HOXC13-AS in intervertebral disc degeneration remain unclear. Methods: qRT-PCR analysis was performed to measure the relative expression levels of HOXC13-AS and miR-497-5p, and the levels of IL-1β, IL-6, and TNF-α in the medium supernatant were analyzed by ELISA. The related mechanism between HOXC13-AS and miR-497-5p was detected by luciferase assays. Results: The results revealed that TNF-α and IL-1β induced HOXC13-AS expression in NP cells. HOXC13-AS was overexpressed in IDD specimens compared to control specimens, and higher expression of HOXC13-AS was correlated with high Pfirrmann scores. Ectopic expression of HOXC13-AS promoted MMP-3 and ADAMTS4 and inhibited aggrecan and collagen II expression in NP cells. Furthermore, overexpression of HOXC13-AS increased the expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Our results demonstrated that TNF-α and IL-1β induced ADAMTS5 expression and suppressed miR-497-5p expression. miR-497-5p was downregulated in IDD specimens compared to control specimens, and the lower expression of miR-497-5p was correlated with high Pfirrmann scores. The miR-497-5p level was negatively proportional to HOXC13-AS expression in IDD specimens. Luciferase analysis data indicated that ADAMTS5 was a direct target gene of miR-497-5p. HOXC13-AS induced inflammatory cytokine expression and ECM degradation by modulating miR-497-5p/ADAMTS5. Conclusion: HOXC13-AS may be a treatment target for IDD.

Vascular endothelial growth factor receptor-1/fms-related tyrosine kinase-1 (VEGFR-1/Flt-1) is a tyrosine kinase receptor that binds placental growth factor (PlGF). Flt-1 is also highly expressed in breast-cancer tissues and breast-cancer cell lines. However, the molecular mechanism by which Flt-1 promotes breast-cancer invasion and metastasis by binding to PlGF-1 is unclear. In this study, we discovered that PlGF-1 and Flt-1 played a key role in the migration and invasion of breast cancer. Flt-1 promoted the migration and chemotaxis of breast-cancer cells by binding to PlGF-1. In addition, Flt-1 was confirmed to be a direct target gene of miR-507. miR-507 up-regulation inhibited the invasion and metastasis of breast-cancer cells in vitro and in vivo. Flt-1 overexpression rescued the invasion partially caused by the ectopic expression of miR-507. miR-507 expression in breast-cancer tissues and cell lines was lower than that in adjacent non-neoplastic tissues and normal cells. Clinical analysis indicated that miR-507 was negatively correlated with tumor differentiation, lymphatic metastasis, and the expression of Flt-1 in breast cancer. Furthermore, we showed that miR-507 down-regulation was due to the hypermethylation of its promotor region. Our results indicated that miR-507 represented potential therapeutic targets in breast cancer by modulating Flt-1.

Hot spot gene mutations in splicing factor 3b subunit 1 (SF3B1) are observed in many types of cancer and create abundant aberrant mRNA splicing, which is profoundly implicated in tumorigenesis. Here, we identified that the SF3B1 K700E (SF3B1K700E ) mutation is strongly associated with tumor growth in pancreatic ductal adenocarcinoma (PDAC). Knockdown of SF3B1 significantly retarded cell proliferation and tumor growth in a cell line (Panc05.04) with the SF3B1K700E mutation. However, SF3B1 knockdown had no notable effect on cell proliferation in two cell lines (BxPC3 and AsPC1) carrying wild-type SF3B1. Ectopic expression of SF3B1K700E but not SF3B1WT in SF3B1-knockout Panc05.04 cells largely restored the inhibitory role induced by SF3B1 knockdown. Introduction of the SF3B1K700E mutation in BxPC3 and AsPC1 cells also boosted cell proliferation. Gene set enrichment analysis demonstrated a close correlation between SF3B1 mutation and aerobic glycolysis. Functional analyses showed that the SF3B1K700E mutation promoted tumor glycolysis, as evidenced by glucose consumption, lactate release, and extracellular acidification rate. Mechanistically, the SF3B1 mutation promoted the aberrant splicing of PPP2R5A and led to the activation of the glycolytic regulator c-Myc via post-translational regulation. Pharmacological activation of PP2A with FTY-720 markedly compromised the growth advantage induced by the SF3B1K700E mutation in vitro and in vivo. Taken together, our data suggest a novel function for SF3B1 mutation in the Warburg effect, and this finding may offer a potential therapeutic strategy against PDAC with the SF3B1K700E mutation.

Unexpectedly, we found that c-Myc-expressing porcine embryonic fibroblasts (PEFs) subcutaneously implanted into nude mice formed cartilage-like tissues in vivo, while previous studies revealed the direct conversion of mouse and human somatic cells into chondrocytes by the combined use of several defined factors, including c-Myc, which prompted us to explore whether PEFs can be reprogrammed to become pig induced chondrocyte-like cells (piCLCs) via ectopic expression of c-Myc alone. In this study, c-Myc-expressing PEFs, designated piCLCs, which exhibited a significantly enhanced proliferation ability in vitro, displayed a chondrogenic phenotypes in vitro, as shown by the cell morphology, toluidine blue staining, alcian blue staining and chondrocyte marker gene expression. Additionally, piCLCs with a polygonal chondrocyte-like morphology were readily and efficiently converted from PEFs by enforced c-Myc expression within 10 days, while piCLCs maintained the chondrocytic phenotype and normal karyotype during long-term subculture. piCLC-derived single clones with a chondrogenic phenotype in vitro exhibited homogeneity in cell morphology and staining intensity compared with mixed piCLCs. Although the mixtures of cartilaginous tissues and tumorous tissues accounted for ~12% (6/51) of all xenografts (51), piCLCs generated stable, homogenous, hyaline cartilage-like tissues without tumour formation at 45 out of the 51 injected sites when subcutaneously injected into nude mice. The hyaline cartilage-like tissues remained for at least 16 weeks. Taken together, these findings demonstrate for the first time the direct induction of chondrocyte-like cells from PEFs with only c-Myc.

Centriole duplication is tightly controlled to occur once per cell cycle, and disruption of this synchrony causes centriole amplification, which is frequently observed in many cancers. Our previous work showed that nuclear distribution gene C (NudC)-like protein 2 (NudCL2) localizes to centrosomes; however, little is known about the role of NudCL2 in the regulation of centrosome function. Here, we find that NudCL2 is required for accurate centriole duplication by stabilizing the E3 ligase HECT domain and RCC1-like domain-containing protein 2 (HERC2). Knockout (KO) of NudCL2 using CRISPR/Cas9-based genome editing or depletion of NudCL2 using small interfering RNA causes significant centriole amplification. Overexpression of NudCL2 significantly suppresses hydroxyurea-induced centriole overduplication. Quantitative proteomic analysis reveals that HERC2 is downregulated in NudCL2 KO cells. NudCL2 is shown to interact with and stabilize HERC2. Depletion of HERC2 leads to the similar defects to that in NudCL2-downregulated cells, and ectopic expression of HERC2 effectively rescues the centriole amplification caused by the loss of NudCL2, whereas the defects induced by HERC2 depletion cannot be reversed by exogenous expression of NudCL2. Either loss of NudCL2 or depletion of HERC2 leads to the accumulation of ubiquitin-specific peptidase 33 (USP33), a centrosomal protein that positively regulates centriole duplication. Moreover, knockdown of USP33 reverses centriole amplification in both NudCL2 KO and HERC2-depleted cells. Taken together, our data suggest that NudCL2 plays an important role in maintaining the fidelity of centriole duplication by stabilizing HERC2 to control USP33 protein levels, providing a previously undescribed mechanism restraining centriole amplification.