Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology. METHODS: In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre:Runx3f/f mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model. RESULTS: Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis. CONCLUSIONS: We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease.

Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3.

Application of dendritic cells (DCs) to prime responses to tumor Ags provides a promising approach to immunotherapy. However, only a limited number of DCs can be manufactured from adult precursors. In contrast, pluripotent embryonic stem (ES) cells represent an inexhaustible source for DC production, although it remains a major challenge to steer directional differentiation because ES cell-derived cells are typically immature with impaired functional capacity. Consistent with this notion, we found that mouse ES cell-derived DCs (ES-DCs) represented less mature cells compared with bone marrow-derived DCs. This finding prompted us to compare the gene expression profile of the ES cell- and adult progenitor-derived, GM-CSF-instructed, nonconventional DC subsets. We quantified the mRNA level of 17 DC-specific transcription factors and observed that 3 transcriptional regulators (Irf4, Spi-B, and Runx3) showed lower expression in ES-DCs than in bone marrow-derived DCs. In light of this altered gene expression, we probed the effects of these transcription factors in developing mouse ES-DCs with an isogenic expression screen. Our analysis revealed that forced expression of Irf4 repressed ES-DC development, whereas, in contrast, Runx3 improved the ES-DC maturation capacity. Moreover, LPS-treated and Runx3-activated ES-DCs exhibited enhanced T cell activation and migratory potential. In summary, we found that ex vivo-generated ES-DCs had a compromised maturation ability and immunogenicity. However, ectopic expression of Runx3 enhances cytokine-driven ES-DC development and acts as an instructive tool for the generation of mature DCs with enhanced immunogenicity from pluripotent stem cells.

EZH2 promotes cell proliferation by regulating the expression of RUNX3 in laryngeal carcinoma.

Enhancer of zeste homolog 2 (EZH2) is a highly conserved histone methyltransferase, which is overexpressed in different types of cancers such as breast and prostate cancer. It is reported that EZH2 can directly down-regulate RUNX3 by increasing histone H3 methylation. However, the role of EZH2 in the development and progression of laryngeal carcinoma has not yet been investigated, and the relationship between EZH2 and RUNX3 in laryngeal carcinoma is rarely reported. The current study aims to determine the role of EZH2 in the progression of laryngeal carcinoma, and investigate the interaction between EZH2 and the tumor suppressor RUNX3. Our study found that EZH2 is overexpressed in laryngeal carcinoma patients, and silencing EZH2 by EZH2 siRNA significantly inhibited the proliferation of laryngeal carcinoma cells. Besides, we also found that RUNX3 is repressed in laryngeal carcinoma patients. Moreover, RUNX3 as a downstream target protein of EZH2 is up-regulated by EZH2 siRNA accompanied by a decrease in the trimethylation modification pattern of H3K27. RUNX3 siRNA inhibits the decreased proliferation induced by EZH2 siRNA. Furthermore, ß-catenin protein expression is down-regulated by EZH2 siRNA and up-regulated by RUNX3 siRNA, and RUNX3 siRNA inhibits the down-regulation effect of EZH2 siRNA on ß-catenin protein expression. Additionally, the Wnt/ß-catenin activator BIO reverses the inhibitory effect of EZH2 siRNA on Hep-2 cell proliferation. Taken together, our results suggest that EZH2 regulates cell proliferation potentially by targeting RUNX3 through the Wnt/ß-catenin signaling pathway in laryngeal carcinoma.

Alteration in the expression of MGMT and RUNX3 due to non-CpG promoter methylation and their correlation with different risk factors in esophageal cancer patients.

Promoter methylation reflects in the inactivation of different genes like O6-methylguanine-DNA methyltransferase DNA repair gene and runt-related transcription factor 3, a known tumor suppressor gene in various cancers such as esophageal cancer. The promoter methylation was evaluated for O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in CpG, CHH, and CHG context (where H is A, T, or C) by next-generation sequencing. The methylation status was correlated with quantitative messenger RNA expression. In addition, messenger RNA expression was correlated with different risk factors like tobacco, alcohol, betel nut consumption, and smoking habit. CpG methylation of O6-methylguanine-DNA methyltransferase promoter had a positive association in the development of esophageal cancer (p < 0.05), whereas runt-related transcription factor 3 promoter methylation showed no significant association (p = 1.0) to develop esophageal cancer. However, the non-CpG methylation, CHH, and CHG were significantly correlated with O6-methylguanine-DNA methyltransferase (p < 0.05) and runt-related transcription factor 3 (p < 0.05) promoters in the development of esophageal cancer. The number of cytosine converted to thymine (C→T) in O6-methylguanine-DNA methyltransferase promoter showed a significant correlation between cases and controls (p < 0.05), but in runt-related transcription factor 3 no such significant correlation was observed. Besides, messenger RNA expression was found to be significantly correlated with promoter hypermethylation of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in the context of CHG and CHH (p < 0.05). The CpG hypermethylation in O6-methylguanine-DNA methyltransferase showed positive (p < 0.05) association, whereas in runt-related transcription factor 3, it showed contrasting negative association (p = 0.23) with their messenger RNA expression. Tobacco, betel nut consumption, and smoking habits were associated with altered messenger RNA expression of O6-methylguanine-DNA methyltransferase (p < 0.05) and betel nut consumption and smoking habits were associated with runt-related transcription factor 3 (p < 0.05). There was no significant association between messenger RNA expression of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 with alcohol consumption (p = 0.32 and p = 0.15). In conclusion, our results suggest that an aberrant messenger RNA expression may be the outcome of CpG, CHG, and CHH methylation in O6-methylguanine-DNA methyltransferase, whereas outcome of CHG and CHH methylation in runt-related transcription factor 3 promoters along with risk factors such as consumption of tobacco, betel nut, and smoking habits in esophageal cancer from Northeast India.

13cRA regulates the differentiation of antler chondrocytes through targeting Runx3.

Although 13cRA is involved in the regulation of cellular proliferation and differentiation, its physiological roles in chondrocyte proliferation and differentiation still remain unknown. Here, we showed that 13cRA could induce the proliferation of sika deer antler chondrocytes and expression of Ccnd3 and Cdk6. Administration of 13cRA to antler chondrocytes resulted in an obvious increase in the expression of chondrocyte marker Col II and hypertrophic chondrocyte marker Col X. Silencing of Crabp2 expression by specific siRNA could prevent the 13cRA-induced up-regulation of Col X, whereas overexpression of Crabp2 showed the opposite effects. Further study found that Crabp2 mediated the regulation of 13cRA on the expression of Runx3 which was highly expressed in the antler cartilage and inhibited the differentiation of antler chondrocytes. Moreover, attenuation of Runx3 expression greatly raised 13cRA-induced chondrocyte differentiation. Simultaneously, 13cRA could stimulate the expression of Cyp26a1 and Cyp26b1 in the antler chondrocytes. Inhibition of Cyp26a1 and/or Cyp26b1 reinforced the effects of 13cRA on the expression of Col X and Runx3, while overexpression of Cyp26b1 rendered the antler chondrocytes hyposensitive to 13cRA. Collectively, 13cRA may play an important role in the differentiation of antler chondrocytes through targeting Runx3. Crabp2 enhances the effects of 13cRA on chondrocyte differentiation, while Cyp26a1 and Cyp26b1 weaken the sensitivity of antler chondrocytes to 13cRA.

Coreceptor gene imprinting governs thymocyte lineage fate.

Immature thymocytes are bipotential cells that are signalled during positive selection to become either helper- or cytotoxic-lineage T cells. By tracking expression of lineage determining transcription factors during positive selection, we now report that the Cd8 coreceptor gene locus co-opts any coreceptor protein encoded within it to induce thymocytes to express the cytotoxic-lineage factor Runx3 and to adopt the cytotoxic-lineage fate, findings we refer to as 'coreceptor gene imprinting'. Specifically, encoding CD4 proteins in the endogenous Cd8 gene locus caused major histocompatibility complex class II-specific thymocytes to express Runx3 during positive selection and to differentiate into CD4(+) cytotoxic-lineage T cells. Our findings further indicate that coreceptor gene imprinting derives from the dynamic regulation of specific cis Cd8 gene enhancer elements by positive selection signals in the thymus. Thus, for coreceptor-dependent thymocytes, lineage fate is determined by Cd4 and Cd8 coreceptor gene loci and not by the specificity of T-cell antigen receptor/coreceptor signalling. This study identifies coreceptor gene imprinting as a critical determinant of lineage fate determination in the thymus.

The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression.

OBJECTIVES: To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). METHODS: We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. RESULTS: The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10(-14)) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 'G' allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk 'A' allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). CONCLUSION: We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.

Hyperoxia-induced methylation decreases RUNX3 in a newborn rat model of bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary dysplasia (BPD) in premature infants is a predominantly secondary occurrence to intrauterine inflammation/infection and postpartum mechanical ventilation; in recent years, an association with epigenetics has also been found. DNA methylation, catalyzed by DNA methyl transferases (DNMTs), and tri-methylation of lysine 27 on histone H3 (H3K27me3), mediated by the methyltransferase, Enhancer of Zeste Homolog 2 (EZH2), are some of the most commonly found modifications in epigenetics. Runt-related transcription factor 3 (RUNX3) is associated with pulmonary epithelial and vascular development and regulates expression at the post-transcriptional level by DNA methylation through DNMT1 or DNMT3b. However, the involvements of these epigenetic factors in the occurrence of BPD are, as yet, unclear. METHODS: Newborn rats were randomly assigned to a model, hyperoxia (85 % O2) or control, normoxia group (21 % O2). Lung tissues and alveolar type 2 (AT2) epithelial cells were collected between 1-14 days. The expression of DNMTs, and EZH2 was detected by immunohistochemistry, Western blot and real-time PCR. The percentage of DNA methylation and H3K27me3 levels in the RUNX3 promoter region was measured by bisulfite sequencing PCR and chromatin immunoprecipitation assay. RUNX3 protein and mRNA expression in AT2 cells was also measured after inhibition using the DNA methylation inhibitor, 5-Aza-2'-deoxycytidine, the H3K27me3 inhibitor, JMJD3, and the EZH2 inhibitor, DZNep. RESULTS: Compared with the control group, RUNX3 protein was downregulated and DNMT3b and EZH2 were highly expressed in lung tissues and AT2 cells of the model group (P < 0.05), while high DNA methylation and H3K27me3 modifications were present in the RUNX3 promoter region, in lung tissues of the model group (P < 0.05). Following hyperoxia in the model group, JMJD3 and DZNep significantly reversed the hyperoxia-induced down-regulation of RUNX3 expression in AT2 cells (P < 0.05), more so than 5-Aza-2'-deoxycytidine (P < 0.05). CONCLUSIONS: 1) DNA methylation and H3K27 trimethylation are present in the BPD model; 2) RUNX3 down-regulation is attributed to both DNMT3b-catalyzed DNA methylation and EZH2-catalyzed histone methylation.

RUNX3 contributes to carboplatin resistance in epithelial ovarian cancer cells.

OBJECTIVE: Resistance to platinum-based therapeutic agents represents a major hurdle in the treatment of epithelial ovarian cancer (EOC). There is an urgent need to better understand the underlying mechanisms. Here, we investigated the role of RUNX3 in carboplatin resistance in EOC cells. METHODS: Expression of RUNX3 was determined in human EOC cell line A2780s (cisplatin-sensitive) and A2780cp (cisplatin-resistant), human ovarian surface epithelium (OSE) and primary EOC cells. The effects of RUNX3 expression on sensitivity to carboplatin were determined in A2780s and A2780cp cells using neutral red uptake and clonogenic assays. Carboplatin-induced apoptosis was determined by measuring cleaved PARP using Western blotting. The expression of cellular inhibitor of apoptosis protein-2 (cIAP2) and its regulation by RUNX3 were assessed by quantitative RT-PCR and Western blotting. RESULTS: The expression of RUNX3 was elevated in A2780cp cells compared to A2780s cells and in EOC tissues from chemoresistant patients compared to those from chemosensitive patients. Overexpression of RUNX3 rendered A2780s cells more resistant to carboplatin, whereas inhibition of RUNX3 increased sensitivity to carboplatin in A2780cp cells. Inhibition of RUNX3 potentiated carboplatin-induced apoptosis in A2780cp cells as demonstrated by more pronounced PARP cleavage. Interestingly, the expression of cIAP2 was elevated in A2780cp cells compared to A2780s cells. Overexpression of RUNX3 increased cIAP2 expression in A2780s cells, whereas inhibition of RUNX3 decreased cIAP2 expression and potentiated carboplatin-induced decrease of cIAP2 in A2780cp cells. CONCLUSIONS: RUNX3 contributes to carboplatin resistance in EOC cells and may hold promise as a therapeutic target to treat EOC and/or a biomarker to predict chemoresistance.

Increased level of E protein activity during invariant NKT development promotes differentiation of invariant NKT2 and invariant NKT17 subsets.

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.

RUNX3 facilitates growth of Ewing sarcoma cells.

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma.

Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells.

RUNX3 is a tumor suppressor for a variety of cancers. RUNX3 suppresses the canonical Wnt signaling pathway by binding to the TCF4/ß-catenin complex, resulting in the inhibition of binding of the complex to the Wnt target gene promoter. Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells. Notably, RUNX3 expression increased the ratio of the Wnt signaling-high population in the KatoIII cells. although the maximum Wnt activation level of individual cells was similar to that in the control. As found previously, RUNX3 also binds to TCF4 and ß-catenin in KatoIII cells, suggesting that these molecules form a ternary complex. Moreover, the ChIP analyses revealed that TCF4, ß-catenin and RUNX3 bind the promoter region of the Wnt target genes, Axin2 and c-Myc, and the occupancy of TCF4 and ß-catenin in these promoter regions is increased by the RUNX3 expression. These results suggest that RUNX3 stabilizes the TCF4/ß-catenin complex on the Wnt target gene promoter in KatoIII cells, leading to activation of Wnt signaling. Although RUNX3 increased the Wnt signaling activity, its expression resulted in suppression of tumorigenesis of KatoIII cells, indicating that RUNX3 plays a tumor-suppressing role in KatoIII cells through a Wnt-independent mechanism. These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/ß-catenin complex by cell context-dependent mechanisms.

Expression of RUNX3 gene in pancreatic adenocarcinoma and its clinical significance.

We investigated the clinical significance of RUNX3 gene expression in human pancreatic carcinoma. Five samples of pancreatic tissues and 30 samples of pancreatic cancer tissues and paracancerous tissues were collected. RUNX3 expression was detected by real-time PCR and immunohistochemistry. The relationships between clinicopathological findings and the expression of RUNX3 were analyzed. The relative quantification level of RUNX3 mRNA expression in human pancreatic carcinoma tissues and paracancerous tissues was 2.60 (0.42-12.82) and 1.02 (0.19-3.58), respectively (P < 0.05). The percentage of positive cells expressing RUNX3 protein in human pancreatic tissues and paracancerous tissues was 45.5 ± 26.2 and 6.9 ± 6.0%, respectively (P < 0.01). The high RUNX3 group (N = 9) with 45.5% or more of the cancer cells staining for RUNX3 and the low RUNX3 group (N = 21) with less than 45.5% cancer cells staining for RUNX3. Low expression of RUNX3 correlated significantly with an advanced TNM stage (χ(2) = 6.897, P = 0.045), lymph node metastasis (χ(2) = 4.739, P = 0.029) and neural invasion (χ(2) = 5.44, P = 0.020). On the other hand, no association could be found between RUNX3 expression and clinicopathological variables including age, gender, tumor location, tumor size, tumor differentiation or the serum concentration of CEA and CA199. The expression of RUNX3 in pancreatic cancer tissues was obviously higher than that in the paracancerous tissues. Low expression of RUNX3 may have an important role in aggressiveness, lymph node metastasis and neural invasion in pancreatic cancer. In pancreatic carcinoma tissues, low expression of RUNX3 may indicate a poor prognosis.

Runt-related transcription factor 3 reverses epithelial-mesenchymal transition in hepatocellular carcinoma.

Loss or decreased expression of runt-related transcription factor 3 (RUNX3), a tumor suppressor gene involved in gastric and other cancers, has been frequently observed in hepatocellular carcinoma (HCC). The objective of this study was to identify the regulatory mechanism of the epithelial-mesenchymal transition (EMT) by RUNX3 in HCC. Human HCC cell lines, Hep3B, Huh7, HLF and SK-Hep1, were divided into low- and high-EMT lines, based on their expression of TWIST1 and SNAI2, and were used in this in vitro study. Ectopic RUNX3 expression had an anti-EMT effect in low-EMT HCC cell lines characterized by increased E-cadherin expression and decreased N-cadherin and vimentin expression. RUNX3 expression has previously been reported to reduce jagged-1 (JAG1) expression; therefore, JAG1 ligand peptide was used to reinduce EMT in RUNX3-expressing low-EMT HCC cells. Immunohistochemical analyses were performed for RUNX3, E-cadherin, N-cadherin and TWIST1 in 33 human HCC tissues, also divided into low- and high-EMT HCC, based on TWIST1 expression. E-cadherin expression was correlated positively and N-cadherin expression was correlated negatively with RUNX3 expression in low-EMT HCC tissues. Correlations between EMT markers and RUNX3 mRNA expression were analyzed using Oncomine datasets. Similarly, mRNA expression of E-cadherin was also significantly correlated with that of RUNX3 in low-EMT HCC, while mRNA expression of JAG1 was negatively correlated with that of RUNX3. These results suggest a novel mechanism by which loss or decreased expression of RUNX3 induces EMT via induction of JAG1 expression in low-EMT HCC.

Upregulated miR-130a increases drug resistance by regulating RUNX3 and Wnt signaling in cisplatin-treated HCC cell.

Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of patients with advanced liver cancer. However, acquisition of cisplatin resistance is common in patients with hepatocellular carcinoma (HCC), and the underlying mechanism of such resistance is not fully understood. In the study, we found that miR-130a levels were significantly increased in HCC patients treated with cisplatin-based chemotherapy. miR-130a levels were also higher in cisplatin-resistant Huh7 cells than in Huh7 cells. Overexpression of miR-130a contributed to cisplatin resistance in Huh7 cell, whereas knockdown of miR-130a overcame cisplatin resistance in cisplatin-resistant Huh7 cell. We further demonstrated that upregulated miR-130a directly inhibited expression of tumor suppressor gene RUNX3, which resulted in activation of Wnt/ß-catenin signaling and increased drug resistance. These data suggest that miR-130a/RUNX3/Wnt signaling represents a novel pathway regulating chemoresistance, thus offering a new target for chemotherapy of HCC.

Downregulation of runt-related transcription factor 3 associated with poor prognosis of adenoid cystic and mucoepidermoid carcinomas of the salivary gland.

Runt-related transcription factor 3 (RUNX3) is a transcription factor of the transforming growth factor (TGF)-ß superfamily and acts as a tumor suppressor gene, which is silenced by hypermethylation of the promoter region in various cancers. In this study, we examined the expression and methylation status of RUNX3 in the salivary gland cancers pleomorphic adenoma (PA), adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). The cytoplasmic expression rates of RUNX3 in PA, ACC and MEC were 65% (13/20), 22.2% (8/36) and 20.6% (7/34), respectively. Low expression or deletion of RUNX3 in ACC and MEC was significantly associated with tumor progression and poor prognosis. Using microdissected cDNA, we found that RUNX3 mRNA expression was lower in ACC and MEC than in PA and noncancerous salivary glands; furthermore, hypermethylation of RUNX3 was detected more frequently in PA (2/8, 25%), ACC (6/8, 75%) and MEC (7/8, 87.5%) than in noncancerous salivary glands (0/8, 0%). Our results suggest that low expression or deletion of RUNX3 in salivary gland tumors might play a pivotal role in tumorigenesis and tumor progression and poor prognosis in the case of salivary gland ACC and MEC. Recovery of the tumor suppressive function of RUNX3 might inhibit tumorigenesis and cancer progression in the human salivary gland.

Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia.

RUNX3/AML2 is a Runt domain transcription factor like RUNX1/AML1 and RUNX2/AML3. Regulated by 2 promoters P1 and P2, RUNX3 is frequently inactivated by P2 methylation in solid tumors. Growing evidence has suggested a role of this transcription factor in hematopoiesis. However, genetic alterations have not been reported in blood cancers. In this study on 73 acute myeloid leukemia (AML) patients (44 children and 29 adults), we first showed that high RUNX3 expression among childhood AML was associated with a shortened event-free survival, and RUNX3 was significantly underexpressed in the prognostically favorable subgroup of AML with the t(8;21) and inv(16) translocations. We further demonstrated that this RUNX3 repression was mediated not by P2 methylation, but RUNX1-ETO and CBFbeta-MYH11, the fusion products of t(8;21) and inv(16), via a novel transcriptional mechanism that acts directly or indirectly in collaboration with RUNX1, on 2 conserved RUNX binding sites in the P1 promoter. In in vitro studies, ectopically expressed RUNX1-ETO and CBFbeta-MYH11 also inhibited endogenous RUNX3 expression. Taken together, RUNX3 was the first transcriptional target found to be commonly repressed by the t(8;21) and inv(16) fusion proteins and might have an important role in core-binding factor AML.

Loss of RUNX3 expression correlates with differentiation, nodal metastasis, and poor prognosis of gastric cancer.

BACKGROUND: RUNX3 is a major growth regulator of gastric epithelial cells that is involved in gastric tumorigenesis in both humans and mice. In this study, we investigated the involvement of RUNX3 in tumor progression, and in the prognosis of human gastric cancer. METHODS: We analyzed the extent of RUNX3 protein expression by immunohistochemistry in 95 primary gastric adenocarcinomas, and correlated expression levels with clinicopathological parameters. We examined the effects of pFlag/RUNX3 on cell growth, apoptosis, and caspase-3 expression in AGS and SNU1 gastric cancer cell lines by colony-forming assay, terminal deoxynucleotidyl transferase (TdT)-mediate deoxyuridine triphosphatase (dUTP) nick-end labeling (TUNEL) assay, and Western blot analysis, respectively. The pFlag/RUNX3 effects on AGS invasion and migration potentials were also evaluated. RESULTS: RUNX3 expression was lost in 37 (39%) cases of gastric cancer. The expression of RUNX3 in diffuse- and mixed-type cancers was less frequent than expression in intestinal-type cancer (P < 0.001 and P = 0.001, respectively). In addition, the loss of RUNX3 expression was associated with lymph node metastasis (P = 0.02), and correlated with poor gastric cancer survival (P = 0.018). The growth of gastric cancer cells was suppressed after pFlag/RUNX3 transfection. The re-expression of RUNX3 resulted in the upregulation of caspase-3 and promoted apoptosis. Furthermore, Re-expression of RUNX3 induced significant inhibitions of AGS cell invasion and migration in vitro. CONCLUSIONS: This work shows that loss of RUNX3 expression is highly associated with lymph node metastasis and poor prognosis of gastric cancer. The re-expression of RUNX3 may induce apoptosis and inhibit the growth as well as invasion/migration of cancer cells. These results indicate that the targeting of the RUNX3 pathway could represent a potential modality for treating gastric cancer.

RUNX3 protein is overexpressed in human epithelial ovarian cancer.

OBJECTIVE: RUNX family genes, including RUNX3, are developmental regulators that are important in human cancers. The purpose of this study was to evaluate expression and oncogenic potential of RUNX3 in ovarian carcinoma. METHODS: Immunohistochemical staining was performed on 60 malignant, 14 borderline, and 5 normal ovarian specimens. Correlation between RUNX3 expression with tumor histology was performed. RUNX3 expression was evaluated by quantitative real-time polymerase chain reaction (QRT-PCR) in microdissected normal and malignant epithelial ovarian tissues. Cell proliferation and viability studies were performed on cells expressing RUNX3 by lentiviral infection and cells with silenced RUNX3 expression by siRNA. RESULTS: RUNX3 expression by immunohistochemistry was higher in serous ovarian carcinomas versus normal ovarian epithelium (P<0.001). Immunofluorescent staining confirmed upregulation of cytoplasmic RUNX3 in ovarian cancer cell lines and tissues. QRT-PCR showed higher RUNX3 mRNA expression in microdissected borderline and malignant ovarian tumor tissues compared with the normal ovarian surface epithelial cells (HOSE) (P=0.006 and P=0.023). Forced RUNX3 expression by lentiviral gene delivery in ovarian cancer cells, SKOV3, that initially showed undetectable RUNX3 expression, resulted in increased cell viability (P=0.043). Silencing RUNX3 expression by siRNA transfection into ovarian cancer cells, OVCAR429, initially expressing high levels of endogenous RUNX3 resulted in a decrease in proliferation (P=0.021). CONCLUSION: These results suggest that RUNX3 has a role in cell proliferation and viability in ovarian cancer.

Hsa_circRNA_33287 promotes the osteogenic differentiation of maxillary sinus membrane stem cells via miR-214-3p/Runx3.

BACKGROUND: Circular RNAs (circRNAs) comprise a novel class of noncoding RNAs that play important roles in a variety of diseases. However, the mechanism by which circRNAs regulate the osteogenic differentiation of maxillary sinus membrane stem cells (MSMSCs) remains largely unclear. METHODS: Microarray analysis was used to explore the expression profiles of circRNAs during the osteogenic differentiation of normal and BMP2 induced-MSMSCs. CircRNA_33287 was identified by agarose electrophoresis, quantitative real-time PCR (qRT-PCR), and western blotting. The function of circRNA_33287 was assessed by loss- and gain-of-function techniques and Alizarin red staining. Potential miRNA binding sites for circRNA_33287, and the target genes of miR-214-3p, were predicted by using online bioinformatics analysis tools. The relationships among the regulatory roles played by circRNA_33287, miR-214-3p, and Runt-related transcription factor 3 (Runx3), during the osteogenic differentiation of MSMSCs were verified by use of the dual luciferase reporter assay, qRT-PCR, and western blotting techniques, respectively. In addition, the molecular sponge potential of circRNA_33287 for miRNA was assessed via in vivo ectopic bone formation and a histological analysis performed after hematoxylin and eosin staining. RESULTS: Expression of circRNA_33287 was confirmed to be up-regulated during the osteogenic differentiation of MSMSCS. Overexpression and silencing of circRNA_33287 increased and decreased the expression levels of key markers of osteogenesis, respectively, including Runx2, OSX, and ALP. Furthermore, circRNA_33287 acted as a molecular sponge for miR-214-3p, which regulated Runx3 expression by targeting its 3'UTR. Moreover, circRNA_33287 protected Runx3 from miR-214-3p-mediated suppression. In addition, circRNA_33287 was shown to increase ectopic bone formation in vivo and displayed the strongest ability to stimulate bone formation when co-transfected with a miR-214-3p inhibitor. CONCLUSION: The novel pathway circRNA_33287/miR-214-3p/Runx3 was found to play a role in regulating the osteoblastic differentiation of MSMSCs in the posterior maxilla.