A WNT4- and DKK3-driven canonical to noncanonical Wnt signaling switch controls multiciliogenesis.

Multiciliated cells contain hundreds of cilia whose directional movement powers the mucociliary clearance of the airways, a vital host defense mechanism. Multiciliated cell specification requires canonical Wnt signaling, which then must be turned off. Next, ciliogenesis and polarized ciliary orientation are regulated by noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling. The mechanistic relationship between the Wnt pathways is unknown. We show that DKK3, a secreted canonical Wnt regulator and WNT4, a noncanonical Wnt ligand act together to facilitate a canonical to noncanonical Wnt signaling switch during multiciliated cell formation. In primary human airway epithelial cells, DKK3 and WNT4 CRISPR knockout blocks, whereas ectopic expression promotes, multiciliated cell formation by inhibiting canonical Wnt signaling. Wnt4 and Dkk3 single-knockout mice also display defective ciliated cells. DKK3 and WNT4 are co-secreted from basal stem cells and act directly on multiciliated cells via KREMEN1 and FZD6, respectively. We provide a novel mechanism that links specification to cilium biogenesis and polarization for proper multiciliated cell formation.

ICBP90, an epigenetic regulator, induces DKK3 promoter methylation, promotes glioma progression, and reduces sensitivity to cis-platinum.

Glioma is the most common brain malignancy, characterized by high morbidity, high mortality, and treatment-resistance. Inverted CCAAT box Binding Protein of 90 kDa (ICBP90) has been reported to be involved in tumor progression and the maintenance of DNA methylation. Herein, we constructed ICBP90 over-expression and knockdown glioma cell lines, and found that ICBP90 knockdown inhibited glioma cell proliferation, migration, and invasion. ICBP90 silencing potentially enhanced cellular sensitivity to cis-platinum (DDP) and exacerbated DDP-induced pyroptosis, manifested by the elevated levels of gasdermin D-N-terminal and cleaved caspase 1; whereas, ICBP90 over-expression exhibited the opposite effects. Consistently, ICBP90 knockdown inhibited tumor growth in an in vivo mouse xenograft study using U251 cells stably expressing sh-ICBP90 and oe-ICBP90. Further experiments found that ICBP90 reduced the expression of Dickkopf 3 homolog (DKK3), a negative regulator of ß-catenin, by binding its promoter and inducing DNA methylation. ICBP90 knockdown prevented the nuclear translocation of ß-catenin and suppressed the expression of c-Myc and cyclin D1. Besides, DKK3 over-expression restored the effects of ICBP90 over-expression on cell proliferation, migration, invasion, and DDP sensitivity. Our findings suggest that ICBP90 inhibits the expression of DKK3 in glioma by maintaining DKK3 promoter methylation, thereby conducing to ICBP90-mediated carcinogenesis and drug insensitivity.

Deceased donor urinary DKK3 associates with future allograft function following kidney transplantation.

Predicting future kidney allograft function is challenging. Novel biomarkers, such as urinary Dickkopf-3 (uDKK3), may help guide donor selection and improve allograft outcomes. In this prospective multicenter pilot trial, we investigated whether donor uDKK3 reflects organ quality and is associated with future allograft function. We measured uDKK3/creatinine ratios (uDKK3/crea) from 95 deceased and 46 living kidney donors. Pre-nephrectomy uDKK3/crea levels were 100x higher in deceased than in living donors (9888 pg/mg versus 113 pg/mg, p<0.001). Among deceased donor transplantations, recipients were stratified by their corresponding uDKK3/crea donor levels ranging below (group A, n=68) or above (group B, n=65) median. The primary endpoint of best estimated glomerular filtration rate (eGFR) within the first 3 months after kidney transplantation was superior in group A (56.3 ml/min/1.73 m2) compared to group B (44.2 ml/min/1.73 m2, p=0.0139). Second, the composite clinical endpoint consisting of death, allograft failure or eGFR decline >50% occurred less frequent in group A. By mixed linear regression modelling, donor uDKK3/crea remained an independent predictor of eGFR after transplantation, with a slope of -4.282 ml/min/1.73 m2 per logarithmic increase in donor uDKK3/crea. In summary, urinary DKK3 may serve as a non-invasive, donor-dependent biomarker for assessing organ quality and future allograft function.

WTAP and METTL14 regulate the m6A modification of DKK3 in renal tubular epithelial cells of diabetic nephropathy.

Diabetic nephropathy (DN) is an important cause of death in diabetes patients, which is mainly due to its complex pathogenesis. Here, we explored the role of N6-methyladenosine (m6A) RNA methylation in DN development. Renal tubular epithelial cells from DN patients and experimental DN mice treated with streptozotocin (STZ) exhibited a considerable increase in METTL14 and WTAP expression as well as overall m6A methylation. Knocking down the expression of METTL14 and WTAP inhibited the migration and proliferation of tubular epithelial cells. MeRIP-seq analysis of the renal tissues of DN patients revealed that the genes with elevated m6A methylation were concentrated in the Wnt/ß-Catenin signaling pathway. Dickkopf homolog 3 (DKK3) was screened out as the gene with the most significant increase in m6A methylation. In addition, the expression change pattern of DKK3 under DN circumstances is in line with those of METTL14 and WTAP. DKK3's m6A methylation sites were confirmed to be located in the 3'UTR region, which is how METTL14 and WTAP improved DKK3's mRNA stability. Finally, YTHDF1, a m6A reader, was demonstrated to recognize m6A-methylated DKK3 and promote DKK3 expression.

Peritoneal and renal DKK3 clearance in peritoneal dialysis.

BACKGROUND: Urinary Dickkopf 3 (DKK3) excretion is a recently established biomarker of renal functional development. Its excretion into the peritoneal cavity has not been reported. We here studied DKK3 in peritoneal dialysis. METHODS: DKK3 was assessed in serum, urine and dialysate in a prevalent adult peritoneal dialysis cohort and its concentration analyzed in relation to creatinine and clinical characteristics. RESULTS: Highest DKK3 concentrations were found in serum, followed by urine. Dialysate concentrations were significantly lower. Dialysate DKK3 correlated with both other compartments. Serum, dialysate and urine values were stable during three months of follow-up. Continuous ambulatory dialysis (CAPD) but not cycler-assisted peritoneal dialysis (CCPD) volume-dependently increased peritoneal DKK3 in relation to creatinine. RAAS blockade significantly decreased urinary, but not serum or peritoneal DKK3. CONCLUSION: Our data provide a detailed characterization of DKK3 in peritoneal dialysis. They support the notion that the RAAS system is essential for renal DKK3 handling.

DKK3 promotes renal fibrosis by increasing MFF-mediated mitochondrial dysfunction in Wnt/ß-catenin pathway-dependent manner.

BACKGROUND: Chronic kidney disease (CKD) lacks effective treatments and renal fibrosis (RF) is one of CKD's outcomes. Dickkopf 3 (DKK3) has been identified as an agonist in CKD. However, the underlying mechanisms of DKK3 in CKD are not fully understood. METHODS: H2O2-treated HK-2 cells and ureteric obstruction (UUO) mice were used as RF models. Biomarkers, Masson staining, PAS staining, and TUNEL were used to assess kidney function and apoptosis. Oxidative stress and mitochondria function were also evaluated. CCK-8 and flow cytometry were utilized to assess cell viability and apoptosis. Western blotting, IHC, and qRT-PCR were performed to detect molecular expression levels. Immunofluorescence was applied to determine the subcellular localization. Dual luciferase assay, MeRIP, RIP, and ChIP were used to validate the m6A level and the molecule interaction. RESULTS: DKK3 was upregulated in UUO mouse kidney tissue and H2O2-treated HK-2 cells. Knockdown of DKK3 inhibited oxidative stress, maintained mitochondrial homeostasis, and alleviated kidney damage and RF in UUO mice. Furthermore, DKK3 silencing suppressed HK-2 cell apoptosis, oxidative stress, and mitochondria fission. Mechanistically, DKK3 upregulation was related to the high m6A level regulated by METTL3. DKK3 activated TCF4/ß-catenin and enhanced MFF transcriptional expression by binding to its promoter. Overexpression of MFF reversed in the inhibitory effect of DKK3 knockdown on cell damage. CONCLUSION: Upregulation of DKK3 caused by m6A modification activated the Wnt/ß-catenin pathway to increase MFF transcriptional expression, leading to mitochondrial dysfunction and oxidative stress, thereby promoting RF progression.

Study on the role of MYCN in retinoblastoma by inhibiting p53 and activating wnt/ßcatenin/Fra-1 signaling pathway by reducing DKK3.

Retinoblastoma (RB) is a pediatric malignancy, typically diagnosed at birth or during early childhood. The pathogenesis of RB is marked by the amplification of the Basic Helix-Loop-Helix (BHLH) Transcription Factor MYCN, which serves as a transcriptional regulator capable of binding to Dickkopf 3 (DKK3). However, the precise role of DKK3 in the malignant progression of RB cells caused by MYCN remains elusive. In the present study, the expression of MYCN was either overexpressed or interfered in RB cells. Subsequently, the expression level of DKK3 was assessed through quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was evaluated using the Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine staining, while cell cycle progression and apoptosis were analyzed by flow cytometry and western blot analysis, respectively. Additionally, the expression of proteins involved in the Wnt/ß-catenin/Fra-1/p53 signaling pathway was evaluated via western blot analysis. To gain further insights, Wnt agonists and the P53 inhibitor PFT-α were introduced into exploration. The current investigation revealed a negative correlation between the expression levels of MYCN and DKK3 in RB cells. Additionally, DKK3 overexpression inhibited cell proliferation, promoted cell apoptosis, and arrested cell cycle in RB cells with high expression of MYCN. Moreover, enhanced DKK3 expression inhibited proliferation, promoted cell cycle arrest and apoptosis of RB cells by modulating the wnt/ßcatenin/Fra-1/p53 signaling pathway. Furthermore, in vivo experiments revealed that overexpression of DKK3 inhibits the growth of RB tumors. Collectively, our findings elucidate that MYCN stimulates the Wnt/ß-catenin/Fra-1 pathway by suppressing DKK3 expression, ultimately suppressing p53 activity and contributing to malignant progression of RB.

DKK3 Expression in Glioblastoma: Correlations with Biomolecular Markers.

Glioblastoma is the most common malignant primary tumor of the CNS. The prognosis is dismal, with a median survival of 15 months. Surgical treatment followed by adjuvant therapies such as radiotherapy and chemotherapy characterize the classical strategy. The WNT pathway plays a key role in cellular proliferation, differentiation, and invasion. The DKK3 protein, capable of acting as a tumor suppressor, also appears to be able to modulate the WNT pathway. We performed, in a series of 40 patients, immunohistochemical and Western blot evaluations of DKK3 to better understand how the expression of this protein can influence clinical behavior. We used a statistical analysis, with correlations between the expression of DKK3 and overall survival, age, sex, Ki-67, p53, and MGMT and IDH status. We also correlated our data with information included in the cBioPortal database. In our analyses, DKK3 expression, in both immunohistochemistry and Western blot analyses, was reduced or absent in many cases, showing downregulation. To date, no clinical study exists in the literature that reports a potential correlation between IDH and MGMT status and the WNT pathway through the expression of DKK3. Modulation of this pathway through the expression of DKK3 could represent a new tailored therapeutic strategy in the treatment of glioblastoma.

NRF1 knockdown alleviates lipopolysaccharide-induced pulmonary inflammatory injury by upregulating DKK3 and inhibiting the GSK-3ß/ß-catenin pathway.

Excessive inflammatory injury is the main cause of the incidence of severe neonatal pneumonia (NP) and associated deaths. Although dickkopf-3 (DKK3) exhibits anti-inflammatory activity in numerous pathological processes, its role in NP is still unknown. In this study, human embryonic lung WI-38 and MRC-5 cells were treated with lipopolysaccharide (LPS) to induce inflammatory injury of NP in vitro. The expression of DKK3 was downregulated in LPS-stimulated WI-38 and MRC-5 cells. DKK3 overexpression decreased LPS-induced inhibition of cell viability, and reduced LPS-induced apoptosis of WI-38 and MRC-5 cells. DKK3 overexpression also reduced LPS-induced production of pro-inflammatory factors such as ROS, IL-6, MCP-1, and TNF-α. Nuclear respiratory factors 1 (NRF1) knockdown was found to upregulate DKK3 and inactivate the GSK-3ß/ß-catenin pathway in LPS-injured WI-38 and MRC-5 cells. NRF1 knockdown also suppressed LPS-induced inhibition on cell viability, repressed LPS-induced apoptosis, and inhibited the accumulation of ROS, IL-6, MCP-1, and TNF-α in LPS-injured WI-38 and MRC-5 cells. DKK3 knockdown or re-activation of the GSK-3ß/ß-catenin pathway reversed the inhibitory effects of NRF1 knockdown on LPS-induced inflammatory injury. In conclusion, NRF1 knockdown can alleviate LPS-triggered inflammatory injury by regulating DKK3 and the GSK-3ß/ß-catenin pathway.

DKK3 promotes oxidative stress injury and fibrosis in HK-2 cells by activating NOX4 via ß-catenin/TCF4 signaling.

Oxidative stress and fibrosis may accelerate the progression of chronic kidney disease (CKD). DKK3 is related to regulating renal fibrosis and CKD. However, the molecular mechanism of DKK3 in regulating oxidative stress and fibrosis during CKD development has not been clarified, which deserves to be investigated. Human proximal tubule epithelial cells (HK-2 cells) were treated with H2O2 to establish a cell model of renal fibrosis. The mRNA and protein expressions were analyzed using qRT-PCR and western blot, respectively. Cell viability and apoptosis were evaluated using MTT assay and flow cytometry, respectively. ROS production was estimated using DCFH-DA. The interactions among TCF4, ß-catenin and NOX4 were validated using luciferase activity assay, ChIP and Co-IP. Herein, our results revealed that DKK3 was highly expressed in HK-2 cells treated with H2O2. DKK3 depletion increased H2O2-treated HK-2 cell viability and reduced cell apoptosis, oxidative stress, and fibrosis. Mechanically, DKK3 promoted formation of the ß-catenin/TCF4 complex, and activated NOX4 transcription. Upregulation of NOX4 or TCF4 weakened the inhibitory effect of DKK3 knockdown on oxidative stress and fibrosis in H2O2-stimulated HK-2 cells. All our results suggested that DKK3 accelerated oxidative stress and fibrosis through promoting ß-catenin/TCF4 complex-mediated activation of NOX4 transcription, which could lead to novel molecules and therapeutic targets for CKD.

DKK3/CKAP4 axis is associated with advanced stage and poorer prognosis in oral cancer.

OBJECTIVE: We previously reported that dickkopf WNT signaling inhibitor 3 (DKK3) would modulate malignant potential of oral squamous cell carcinoma (OSCC) via activating Akt. Recently, cytoskeleton associated protein 4 (CKAP4) functions as receptor of DKK3, which activates Akt in esophageal squamous cell carcinoma, but its expression and function in OSCC were unclear. METHODS: We studied DKK3 and CKAP4 protein expression in OSCC tissue and investigated the correlation between protein expression and clinical data. We also investigated whether antibodies (Ab) for DKK3 or CKAP4 could suppress malignant potential of the cancer cells. RESULTS: DKK3/CKAP4 protein expression was observed in majority of OSCC cases and was associated with significantly higher T-stage and TNM stage. Multivariate analysis revealed that DKK3 and CKAP4 were independent prognostic biomarkers for overall survival (OS) and disease-free survival (DFS), respectively. Survival analyses revealed that DKK3-positive cases and CKAP4-positive cases showed significantly shorter OS and DFS, respectively, and that DKK3/CKAP4 double-negative cases showed significantly favorable prognosis. Both anti-DKK3Ab and anti-CKAP4Ab could suppress cancer cell proliferation, migration, and invasion. CONCLUSION: DKK3/CKAP4 axis is thought to be important in OSCC, and it would be a promising therapeutic target.

Characterizing the SREB G protein-coupled receptor family in fish: Brain gene expression and genomic differences in upstream transcription factor binding sites.

The SREB (Super-conserved Receptors Expressed in Brain) family of orphan G protein-coupled receptors is highly conserved in vertebrates and consists of three members: SREB1 (orphan designation GPR27), SREB2 (GPR85), and SREB3 (GPR173). SREBs are associated with processes ranging from neuronal plasticity to reproductive control. Relatively little is known about similarities across the entire family, or how mammalian gene expression patterns compare to non-mammalian vertebrates. In fish, this system may be particularly complex, as some species have gained a fourth member (SREB3B) while others have lost genes. To better understand the system, the present study aimed to: 1) use qPCR to characterize sreb and related gene expression patterns in the brains of three fish species with different systems, and 2) identify possible differences in transcriptional regulation among the receptors, using upstream transcription factor binding sites across 70 ray-finned fish genomes. Overall, regional patterns of sreb expression were abundant in forebrain-related areas. However, some species-specific patterns were detected, such as abundant expression of receptors in zebrafish (Danio rerio) hypothalamic-containing sections, and divergence between sreb3a and sreb3b in pufferfish (Dichotomyctere nigroviridis). In addition, a gene possibly related to the system (dkk3a) was spatially correlated with the receptors in all three species. Genomic regions upstream of sreb2 and sreb3b, but largely not sreb1 or sreb3a, contained many highly conserved transcription factor binding sites. These results provide novel information about expression differences and transcriptional regulation across fish that may inform future research to better understand these receptors.

DKK3 ameliorates neuropathic pain via inhibiting ASK-1/JNK/p-38-mediated microglia polarization and neuroinflammation.

BACKGROUND: Neuropathic pain is a common and severely disabling state that affects millions of people worldwide. Microglial activation in the spinal cord plays a critical role in the pathogenesis of neuropathic pain. However, the mechanisms underlying spinal microglial activation during neuropathic pain remain incompletely understood. Here, we investigated the role of Dickkopf (DKK) 3 and its interplay with microglial activation in the spinal cord in neuropathic pain. METHODS: In this study, we investigated the effects of intrathecal injection of recombinant DKK3 (rDKK3) on mechanical allodynia and microglial activation in the spinal cord after spared nerve injury (SNI) in rats by western blot (WB), immunofluorescence (IF), quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that SNI induced a significant decrease in the levels of DKK3, Kremen-1 and Dishevelled-1 (DVL-1) and up-regulated the expression of phosphorylated apoptosis signal-regulating kinase 1 (p-ASK1), phosphorylated c-JUN N-terminal kinase (p-JNK), phosphorylated p38 (p-p38) in the spinal cord. Moreover, our results showed that exogenous intrathecal administration of rDKK3 inhibited expression of p-ASK1, p-JNK, p-p38, promoted the transformation of microglia from M1 type to M2 type, and decreased the production of pro-inflammatory cytokines compared to the rats of SNI + Vehicle. However, these effects were reversed by intrathecal administration of Kremen-1 siRNA or Dishevelled-1 (DVL-1) siRNA. CONCLUSIONS: These results suggest that DKK3 ameliorates neuropathic pain via inhibiting ASK-1/JNK/p-38-mediated microglia polarization and neuroinflammation, at least partly, by the Kremen-1 and DVL-1 pathways.

Establishment of anti-DKK3 peptide for the cancer control in head and neck squamous cell carcinoma (HNSCC).

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck. We identified cancer-specific genes in HNSCC and focused on DKK3 expression. DKK3 gene codes two isoforms of proteins (secreted and non-secreted) with two distinct cysteine rich domains (CRDs). It is reported that DKK3 functions as a negative regulator of oncogenic Wnt signaling and, is therefore, considered to be a tumor suppressor gene. However, our series of studies have demonstrated that DKK3 expression is specifically high in HNSCC tissues and cells, and that DKK3 might determine the malignant potentials of HNSCC cells via the activation of Akt. Further analyses strongly suggested that both secreted DKK3 and non-secreted DKK3 could activate Akt signaling in discrete ways, and consequently exert tumor promoting effects. We hypothesized that DKK3 might be a specific druggable target, and it is necessary to establish a DKK3 inhibitor that can inhibit both secreted and non-secreted isoforms of DKK3. METHODS: Using inverse polymerase chain reaction, we generated mutant expression plasmids that express DKK3 without CRD1, CRD2, or both CRD1 and CRD2 (DKK3ΔC1, DKK3ΔC2, and DKK3ΔC1ΔC2, respectively). These plasmids were then transfected into HNSCC-derived cells to determine the domain responsible for DKK3-mediated Akt activation. We designed antisense peptides using the MIMETEC program, targeting DKK3-specific amino acid sequences within CRD1 and CRD2. The structural models for peptides and DKK3 were generated using Raptor X, and then a docking simulation was performed using CluPro2. Afterward, the best set of the peptides was applied into HNSCC-derived cells, and the effects on Akt phosphorylation, cellular proliferation, invasion, and migration were assessed. We also investigated the therapeutic effects of the peptides in the xenograft models. RESULTS: Transfection of mutant expression plasmids and subsequent functional analyses revealed that it is necessary to delete both CRD1 and CRD2 to inhibit Akt activation and inhibition of proliferation, migration, and invasion. The inhibitory peptides for CRD1 and CRD2 of DKK3 significantly reduced the phosphorylation of Akt, and consequently suppressed cellular proliferation, migration, invasion and in vivo tumor growth at very low doses. CONCLUSIONS: This inhibitory peptide represents a promising new therapeutic strategy for HNSCC treatment.

Extracellular vesicles derived from endometrial epithelial cells deliver exogenous miR-92b-3p to affect the function of embryonic trophoblast cells via targeting TSC1 and DKK3.

BACKGROUND: Extracellular vesicles (EVs) could mediate embryo-maternal communication to affect embryo implantation by delivering biology information, including microRNA (miRNA), protein, lipid. Our previous research shows that miR-92b-3p was differentially expressed in EVs of uterine flushing fluids during the embryo implantation period. However, the role of miR-92b-3p from EVs in embryo implantation remains elusive. MATERIALS AND METHODS: EVs were isolated from porcine endometrial epithelial cells (EECs) by ultracentrifugation. MiR-92b-3p mimics and EVs were used to regulate the expression of miR-92b-3p in porcine trophoblast cells (PTr2 cells). Cell proliferation, migration and adhesion analyses were used to observe the phenotype. RT-qPCR, western blot and dual-luciferase reporter assay were used to assess the targets of miR-92b-3p. RESULTS: In this study, EVs derived from porcine EECs were identified and could be taken up by PTr2 cells. We found that the EVs derived from EECs transfected with miR-92b-3p mimic (EVs-miR-92b-3p) significantly promoted the proliferation, migration and adhesion of PTr2 cells. We verified that Tuberous sclerosis complex subunit (TSC1) and Dickkopf 3 (DKK3) were the target genes of miR-92b-3p. Moreover, our study showed that miR-92b-3p plays a vital role in PTr2 cells via targeting TSC1 and DKK3. Furthermore, the 3'UTR vectors of TSC1 and DKK3 can rescue the effect of miR-92b-3p on PTr2 cells. CONCLUSIONS: Taken together, this study reveals a novel mechanism that EVs derived from porcine EECs treated with miR-92b-3p crosstalk with trophoblasts by targeting TSC1 and DKK3, leading to an enhanced ability for implantation.

The role of Wnt pathway antagonists in early-stage lung adenocarcinoma.

PURPOSE: We aimed to examine the expression levels of the genes encoding adenomatous polyposis coli (APC) 1, APC-2, Dickkopf related protein (DKK)-1, DKK-3, secreted frizzled-related protein (SFRP)-2, SFRP-4, and SFRP-5, which play roles in the Wnt signaling pathway, in lung adenocarcinoma and adjacent normal lung tissues and to evaluate their relationships with clinicopathologic factors. MATERIALS AND METHODS: The expression levels of genes in formalin-fixed paraffin-embedded samples of tumor tissue and adjacent intact lung tissue from 57 patients who underwent surgery for lung adenocarcinoma between 2011 and 2018 were determined by real-time PCR analysis. RESULTS: The expression levels of the DKK-1 in tumor tissue, especially in stage I-II tumor tissue, were significantly suppressed compared to those in normal tissue (p < 0.025). Whereas DKK-1 expression was suppressed in the tumor tissue of patients with early-stage lung adenocarcinoma, expression of the SFRP-5 in these patients was significantly higher in tumor tissue than in normal tissue (p < 0.039). CONCLUSION: In our study, opposing regulation was found between the SFRP-5 and DKK-1, which are known to be extracellular antagonists of the Wnt signaling pathway. The SFRP-5 was found to have an oncogenic role in adenocarcinoma development. Studies of the opposing regulation between these genes in early-stage lung adenocarcinoma may shed light on the mechanisms associated with the development of carcinogenesis. The relationships or interactions of these genes may serve as potential therapeutic targets.

MiR-363-3p promotes prostate cancer tumor progression by targeting Dickkopf 3.

BACKGROUND: Prostate cancer (PCa) is a frequent malignant tumor worldwide with high morbidity along with mortality. MicroRNAs (miRNAs) have been identified as key posttranscriptional modulators in diverse cancers. Herein, we purposed to explore the impacts of miR-363-3p on PCa growth, migration, infiltration along with apoptosis. METHODS: The expressions of miR-363-3p along with Dickkopf 3 (DKK3) were assessed in clinical PCa specimens. We adopted the PCa cell line PC3 and transfected it using miR-363-3p repressors or mimic. The relationship of miR-363-3p with DKK3 was verified by a luciferase enzyme reporter assay. Cell viability along with apoptosis were determined by MTT assay coupled with flow cytometry analysis. Cell migration along infiltration were detected via wound healing, as well as Transwell assays. The contents of DKK3, E-cadherin, vimentin along with N-cadherin were analyzed via Western blotting accompanied with qRT-PCR. RESULTS: MiR-363-3p was found to be inversely associated with the content of DKK3 in clinical PCa specimens. Further investigations revealed that DKK3 was miR-363-3p's direct target. Besides, overexpression of miR-363-3p decreased the contents of DKK3, promoted cell viability, migration coupled with infiltration, and reduced cell apoptosis, while silencing of miR-363-3p resulted in opposite influence. Upregulation of miR-363-3p diminished E-cadherin contents but increased vimentin along with N-cadherin protein contents in PC3 cells; in contrast, miR-363-3p downregulation produced the opposite result. CONCLUSION: Our study indicates that miR-363-3p promotes PCa growth, migration coupled with invasion while dampening apoptosis by targeting DKK3.

Hsa_circ_0006404 and hsa_circ_0000735 Regulated Ovarian Cancer Response to Docetaxel Treatment via Regulating p-GP Expression.

Several microRNAs (miRNAs) and circular RNAs (circRNAs) were reported to be involved in the Docetaxel (DTX) chemoresistance of cancer treatment, but the underlying mechanisms remain to be explored. In this study, we established cellular and animal models respectively to study the effect and underlying molecular mechanisms of the dysregulation of circRNA_0006404 and circRNA_0000735 in tumor response to DTX treatment. Quantitative real-time PCR was performed to measure the expression of circRNA_0006404, miR-346, circRNA_0000735, miR-526b, Dickkopf-related protein 3 (DKK3), and Dickkopf-related protein 4 (DKK4) mRNA. The expression of circRNA_0006404 and circRNA_0000735 was remarkably suppressed and activated in DTX-treated SKOV3-R cell lines, respectively. As revealed by luciferase assays, circRNA_0006404 and circRNA_0000735 was found to be respectively targeted by miR-346 and miR-526b, while DKK3 and DKK4 were respectively targeted by miR-346 and miR-526b. Moreover, the expression of DKK3 and DKK4, which were targets of miR-346 and miR-526b, respectively, was significantly altered along with the expression of p-GP. Furthermore, circ_0006404 shRNA and circRNA_0000735 shRNA showed remarkable efficiency in stimulating the expression of circRNA_0006404, miR-346, DKK3, circRNA_0000735, miR-526b, DKK4, and p-GP in cellular and animal models. Accordingly, the cell apoptosis and proliferation were apparently changed by circ_0006404 shRNA and circRNA_0000735 shRNA in both cellular and animal models. In summary, our study found the involvement of the circRNA_0006404/miR-346/DKK3/p-GP and circRNA_0000735/miR-546b/DKK4/p-GP axis in the tumor response to DTX. Both the up-regulation of circRNA_0006404 and down-regulation of circRNA_0000735 could inhibit the expression of p-GP in vivo and ex vivo, leading to the suppressed tumor response to DTX treatment.

The long noncoding RNA TUNAR modulates Wnt signaling and regulates human ß-cell proliferation.

Many long noncoding RNAs (lncRNAs) are enriched in pancreatic islets and several lncRNAs are linked to type 2 diabetes (T2D). Although they have emerged as potential players in ß-cell biology and T2D, little is known about their functions and mechanisms in human ß-cells. We identified an islet-enriched lncRNA, TUNAR (TCL1 upstream neural differentiation-associated RNA), which was upregulated in ß-cells of patients with T2D and promoted human ß-cell proliferation via fine-tuning of the Wnt pathway. TUNAR was upregulated following Wnt agonism by a glycogen synthase kinase-3 (GSK3) inhibitor in human ß-cells. Reciprocally, TUNAR repressed a Wnt antagonist Dickkopf-related protein 3 (DKK3) and stimulated Wnt pathway signaling. DKK3 was aberrantly expressed in ß-cells of patients with T2D and displayed a synchronized regulatory pattern with TUNAR at the single cell level. Mechanistically, DKK3 expression was suppressed by the repressive histone modifier enhancer of zeste homolog 2 (EZH2). TUNAR interacted with EZH2 in ß-cells and facilitated EZH2-mediated suppression of DKK3. These findings reveal a novel cell-specific epigenetic mechanism via islet-enriched lncRNA that fine-tunes the Wnt pathway and subsequently human ß-cell proliferation.NEW & NOTEWORTHY The discovery that long noncoding RNA TUNAR regulates ß-cell proliferation may be important in designing new treatments for diabetes.

Histone deacetylase inhibitor chidamide regulates the Wnt/ß-catenin pathway by MYCN/DKK3 in B-ALL.

Our previous studies revealed that MYCN downregulates the expression of DKK3, activates the Wnt/ß-catenin signalling pathway at the transcriptional level, and thereby promotes the development of B cell acute lymphocytic leukaemia (B-ALL) but does not affect the methylation of the DKK3 promoter. Some studies have shown that MYCN is associated with histone acetylation. We speculate that histone deacetylase inhibitors (HDACis) can inhibit the Wnt/ß-catenin signalling pathway by inhibiting MYCN and increasing the expression of DKK3. Based on previous experiments, we tested this hypothesis by analysing the changes in MYCN, DKK3 and the Wnt/ß-catenin signalling pathways in B-ALL cells after treatment with the selective HDACi chidamide. The in vitro and in vivo experiments confirmed that chidamide inhibited the expression of MYCN and increased the expression of DKK3 by inhibiting the activity of histone deacetylase, and these effects resulted in inhibition of the Wnt/ß-catenin signalling pathway and the proliferation of B-ALL cells. These findings indicate that chidamide might be used alone or in combination with other chemotherapy regimens for patients with B-ALL and thus provide a new approach to the treatment of B-ALL.