BACKGROUND: Autofluorescence imaging (AFI) is useful for diagnosing colon neoplasms, but what affects the AFI intensity remains unclear. This study investigated the association between AFI and the histological characteristics, aberrant methylation status, and aberrant expression in colon neoplasms.
METHODS: Fifty-three patients with colorectal neoplasms who underwent AFI were enrolled. The AFI intensity (F index) was compared with the pathological findings and gene alterations. The F index was calculated using an image analysis software program. The pathological findings were assessed by the tumor crypt density, cell densities, and N/C ratio. The aberrant methylation of
RESULTS: An increased N/C ratio, the aberrant expression of
CONCLUSIONS: AFI reflects the nuclear enlargement of tumor cells, the cell proliferation ability, and the altered status of cell proliferation-related genes, indicating that AFI is a useful and practical method for predicting the dysplastic grade of tumor cells and cell proliferation.

OBJECTIVE: Gastric cancer is more open related to genetic predisposition. In our RNA sequencing study on gastric cancer patients, Runt-related transcription factor-3 (RUNX3) expression was significantly down-regulated in gastric cancer. We showed that decreased levels of RUNX3 are significantly associated with c-MET (r = - 0.4216, P = 0.0130). In addition, c-MET expression is a candidate for targeted therapy in gastric cancer. Therefore, in the present study, the anti-cancer effects of the c-MET inhibitor on gastric cancer cells from positive or negative for c-MET amplification were evaluated.
RESULTS: INC280 treatment inhibits growth of a c-MET-amplified MKN45 (RUNX3-positive) and SNU620 (RUNX3-negative) diffuse type cells. Then, INC280 showed the highest inhibition and apoptotic rates with the lowest IC

Dysregulated microRNAs (miRNAs/miRs) directly modulate the biological functions of non‑small cell lung cancer (NSCLC) cells and contribute to the initiation and progression of NSCLC; however, the specific roles and underlying mechanisms of the dysregulated miRNAs in NSCLC require further investigation. The present study reported that miRNA‑629‑5p (miR‑629) was upregulated in NSCLC tissues and cell lines. High miR‑629 expression levels were significantly associated with tumour size, clinical stage and lymph node metastasis in patients with NSCLC. Functional experiments indicated that miR‑629 inhibition suppressed the viability and invasion NSCLC cells in vitro. Furthermore, bioinformatics prediction, luciferase reporter assay, reverse transcription‑quantitative polymerase chain reaction and western blot analysis demonstrated that runt‑related transcription factor 3 (RUNX3) was a direct target gene of miR‑629 in NSCLC. Restoration of RUNX3 expression suppressed the effects of miR‑629 inhibition in NSCLC cells. Rescue experiments revealed that RUNX3 knockdown partially abrogated the effects of miR‑629 inhibition on NSCLC cells. In summary, miR‑629 directly targeted RUNX3 to inhibit the progression of NSCLC, suggesting that this miRNA may be considered as a diagnostic and therapeutic target for patients with NSCLC.

AIM: Base excision repair (BER) gene polymorphisms are known to play an independent role in predisposition to developing different cancers as well as to be associated with clinicopathological traits of the disease modifying its clinical outcomes. One of the underlying mechanisms is presumed to include interplay between BER gene polymorphisms and key mutational, epigenetic and chromosomal events in tumor tissues. The present study was aimed at elucidating potential gene-gene interaction and assessing their mutual effects in bladder cancer (BC).
MATERIALS AND METHODS: The earlier obtained data on genotyping patients with verified diagnosis of BC for OGG1 rs1052133 (Ser326Cys) and XRCC1 rs25487 (Arg399Gln) polymorphisms were used for this study. The tumor tissue samples from the same patients were analyzed for mutations, epigenetic variations and losses of heterozygosity in some key genes involved in divergent pathogenic pathways of BC.
RESULTS: It was shown that the OGG1 (326 codon) heterozygous genotype as well as the minor 326Cys allele can intensify a mutational response of the RAS locus in urothelial carcinomas in the total cohort of patients simultaneously decreasing the mutation rates in the PIK3CA locus in smokers. The XRCC1 (399 codon) heterozygous genotype as well as the minor 399Gln allele reduced the frequency of LOH in the PTEN and TNKS genes, but did not affect the mutational variability in any locus tested. Both polymorphisms influenced the methylation status, carriers of OGG1 326Ser/Cys or Ser/Cys+Cys/Cys genotypes demonstrating increased frequency of methylated RUNX3 and ISL1 genes whereas the similar effect of XRCC1 polymorphism concerning methylation of p16 and TIMP3 genes. When dividing the total cohort into groups based on the extent of tumor spread, the observed associations were characteristic of non-muscle invasive BC.
CONCLUSION: The BER gene polymorphisms contributed to modification of key molecular events in urothelial carcinomas. Their mutual effects mainly manifested in non-muscle invasive BC. The underlying mechanisms as well as possible clinical outcomes need to be further explored to propose novel prognostic biomarkers for BC.

To explore the effect of miR-106a in breast cancer cell behavior and sensitivity to chemotherapeutic agents. Tumor tissue and adjacent normal tissue were derived from 40 breast cancer patients, and miR-106a expression was measured by reverse transcription-qPCR. Breast cancer cells, MDA-MB-231 and MCF-7, were treated with miRNA-106a mimic (MM) or miRNA-106a inhibitor (MI) and negative controls. Cell proliferation was measured by MTT assay. Clonogenicity was measured by colony-forming assay. Cell migration and invasion ability were measured by scratch test and transwell assay, respectively. Apoptosis was determined by flow cytometry, and chemosensitivity to cisplatin was measured by MTT assay. Finally, protein expression of p53, Bax, Bcl-2, RUNX3, and ABCG2 was quantified by western blot. miR-106a expression was significantly upregulated in human breast cancer tissue relative to adjacent normal tissue. Upregulation of miR-106a enhanced breast cancer cell proliferation, colony-forming capacity, migration, and invasion of cultured breast cancer cells. Additionally, miR-106a overexpression significantly decreased breast cancer cell apoptosis and sensitivity to cisplatin. Finally, we showed miR-106a overexpression upregulated the levels of Bcl-2 and ABCG2, and downregulated the expression of P53, Bax, and RUNX3. miR-106a promotes breast cancer cell proliferation and invasion through upregulation of Bcl-2, ABCG2, and P53, and downregulation of Bax and RUNX3.

Aberrant promoter methylation plays a vital role in colorectal carcinogenesis. However, its role in treatment responses is unclear, especially for metastatic disease. Here, we investigated the association between promoter methylation and treatment outcomes of irinotecan-based chemotherapy in 102 patients with metastatic colorectal cancer. Promoter methylation was examined by methylation-specific polymerase chain reaction for three loci (CHFR, WRN, and SULF2) associated with chemotherapy response and five CpG island methylator phenotype (CIMP)-specific markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1). Association between CHFR methylation and in vitro sensitivity to irinotecan was also evaluated. Promoter methylation of CHFR, WRN, and SULF2 was identified in 16 (15.7%), 24 (23.5%), and 33 (32.4%) patients, respectively. CIMP status was positive in 22 (21.6%) patients. CHFR methylation was associated with a significantly longer time to progression (TTP) (median: 8.77 vs. 4.43 months, P = .019), with trends favoring higher overall survival (OS) (median: 22.83 vs. 20.17 months, P = .300) and response rates (31.3% vs. 17.4%, P = .300). For patients with unmethylated CHFR, TTP (median: 5.60 vs. 3.53, P = .020) and OS (median: 20.57 vs. 9.23, P = .006) were significantly different according to CIMP status. Colorectal cancer cell lines with CHFR methylation demonstrated increased sensitivity to irinotecan. Both CHFR overexpression and combination with 5-aza-2'-deoxycytidine reversed irinotecan sensitivity in CHFR-methylated cell lines, whereas CHFR knockdown in unmethylated cells restored sensitivity to irinotecan. These data suggest that CHFR methylation may be associated with favorable treatment outcomes of irinotecan-based chemotherapy in patients with metastatic colorectal cancer.

BACKGROUND: We assessed the role of E-cadherin (CDH1), runt-related transcription factor 3, p21waf and p27 promoter methylation (PM) and protein expression in Helicobacter pylori (HP)-associated gastric carcinomas (GCs) and adjacent non-neoplastic tissues (ANNTs).
PATIENTS AND METHODS: 192 cases were assessed for PM and protein expression of CDH1, RUNX3, p21waf and p27 by methylation-specific PCR (MSP) and immunohistochemistry. The CagA gene was also assessed.
RESULTS: In GCs, 66 (34.4%) and 84 (43.8%) cases showed CDH1-PM and reduced expression. It is significantly affected in GCs rather than in non-neoplastic groups (p < 0.001). In ANNTs, 108 (56.3%) cases showed CDH1-PM and all cases revealed preserved protein expression. RUNX3-PM was detected in 78 GCs (40.6%) and 69 ANNTs (35.9%), whereas reduced protein expression was detected in 99 (51.65%) GC compared to ANNTs 90 (46.9%). p21WAF and p27 showed PM in (48.4% and 45.3%) GCs and ANNTs; respectively. p21waf protein was reduced in 90 (46.9%) cases and 91 ANNTs (47.4%). p27 was reduced in 86 (44.8%) cases and 87 ANNTs (45.3%). CDH1 aberrations correlated with HP in tumors and ANNTs and with diffuse/intestinal tumors (p = 0.014, p = 0.014 and p = 0.02). RUNX3 aberrations associated with HP (p = 0.04), high grade (p = 0.04), and advanced stage (p = 032). Tumor grade associated with RUNX3-PM, CDH, p21 and p27 protein (p < 0.05 for all). Tumor stage associated significantly with PM and reduced protein expression of all markers. Positive lymph nodes associated significantly with p27PM (p < 0.001).
CONCLUSIONS: HP plays an important role in the development and progression of GC through silencing of CDH1, RUNX3, p21WAF and p27 expression.

Circular RNAs (circRNAs), a novel class of non-coding RNAs, have emerged as indispensable modulators in human malignancies. Aberrant cellular senescence is a phenotype observed in various cancers. The association of circRNAs with cellular senescence in tumors is yet to determined. Here, we investigated the role of circLARP4 in cellular senescence and cell proliferation in hepatocellular carcinoma (HCC). Downregulated circLARP4 level was observed in HCC tissues and cell lines. Low expression level of circLARP4 independently predicted poor survival outcome. Gain-of-function and loss-of-function assays demonstrated that circLARP4 suppressed HCC cell proliferation, mediated cell cycle arrest and induced senescence in vitro. Levels of p53 and p21, 2 key regulatory molecules in cellular senescence, were increased in circLARP4-overexpressed HCC cells and decreased in circLARP4-silenced HCC cells. In vivo experiments further confirmed the tumor-suppressing activity of circLARP4. Further mechanistic studies showed that circLARP4 dampened HCC progression by sponging miR-761, thereby promoting the expression level of RUNX3 and activating the downstream p53/p21 signaling. Our study revealed the role of circLARP4/miR-761/RUNX3/p53/p21 signaling in HCC progression, providing a potential survival predictor and therapeutic candidate for HCC.

Up to 60% of untreated atypical hyperplastic endometrium will develop into endometrial carcinoma (EC), and for those who underwent a hysterectomy a coexisting EC is found in up to 50%. Gene promoter methylation might be related to the EC development. The aim of this study is to determine changes in gene promoter profiles in normal endometrium, atypical hyperplasia (AH) and EC in relation to K-Ras mutations. A retrospective study was conducted in patients diagnosed with endometrial hyperplasia with and without subsequent EC. Promoter methylation of APC, hMLh1, O6-MGMT, P14, P16, RASSF1, RUNX3 was analysed on pre-operative biopsies, and correlated to the final histological diagnosis, and related to the presence of K-Ras mutations. In the study cohort (n=98), differences in promoter methylation were observed for hMLH1, O6-MGMT, and P16. Promoter methylation of hMLH1 and O6-MGMT gradually increased from histologically normal endometrium to AH to EC; 27.3, 36.4% and 38.0% for hMLH1 and 8.3%, 18.2% and 31.4% for O6-MGMT, respectively. P16 promoter methylation was significantly different in AH (7.7%) compared to EC (38%). K-Ras mutations were observed in 12.1% of AH, and in 19.6% of EC cases. No association of K-Ras mutation with promoter methylation of any of the tested genes was found. In conclusion, hMLH1 and O6-MGMT promoter methylation are frequently present in AH, and thus considered to be early events in the carcinogenesis of EC, whereas P16 promoter methylation was mainly present in EC, and not in precursor lesions supporting a late event in the carcinogenesis.

Endogenous retroviruses (ERVs) are ancient viral elements that have accumulated in the genome through retrotransposition events. Although they have lost their ability to transpose, many of the long terminal repeats (LTRs) that originally flanked full-length ERVs maintain the ability to regulate transcription. While these elements are typically repressed in somatic cells, they can function as transcriptional enhancers and promoters when this repression is lost. Epstein-Barr virus (EBV), which transforms primary B cells into continuously proliferating cells, is a tumor virus associated with lymphomas. We report here that transformation of primary B cells by EBV leads to genome-wide activation of LTR enhancers and promoters. The activation of LTRs coincides with local DNA hypomethylation and binding by transcription factors such as RUNX3, EBF1, and EBNA2. The set of activated LTRs is unique to transformed B cells compared with other cell lines known to have activated LTRs. Furthermore, we found that LTR activation impacts the B cell transcriptome by up-regulating transcripts driven by cryptic LTR promoters. These transcripts include genes important to oncogenesis of Hodgkin lymphoma and other cancers, such as

Clear cell renal cell carcinoma (ccRCC) is the most frequent type of renal cell carcinoma (RCC). The present study aimed to examine prognostic markers and construct a prognostic prediction system for ccRCC. The mRNA sequencing data of ccRCC was downloaded from The Cancer Genome Atlas (TCGA) database, and the GSE40435 dataset was obtained from the Gene Expression Omnibus database. Using the Limma package, the differentially expressed genes (DEGs) in the TCGA dataset and GSE40435 dataset were obtained, respectively, and the overlapped DEGs were selected. Subsequently, Cox regression analysis was applied for screening prognosis‑associated genes. Following visualization of the co‑expression network using Cytoscape software, the network modules were examined using the GraphWeb tool. Functional annotation for genes in the network was performed using the clusterProfiler package. Finally, a prognostic prediction system was constructed through Bayes discriminant analysis and confirmed with the GSE29609 validation dataset. The results revealed a total of 263 overlapped DEGs and 161 prognosis‑associated genes. Following construction of the co‑expression network, 16 functional terms and three pathways were obtained for genes in the network. In addition, red, yellow (involving chemokine ligand 10 (CXCL10), CD27 molecule (CD27) and runt‑related transcription factor 3 (RUNX3)], green (involving angiopoietin‑like 4 (ANGPTL4), stanniocalcin 2 (STC2), and sperm associated antigen 4 (SPAG4)], and cyan modules were extracted from the co‑expression network. Additionally, the prognostic prediction system involving 44 signature genes, including ANGPTL4, STC2, CXCL10, SPAG4, CD27, matrix metalloproteinase (MMP9) and RUNX3, was identified and confirmed. In conclusion, the 44‑gene prognostic prediction system involving ANGPTL4, STC2, CXCL10, SPAG4, CD27, MMP9 and RUNX3 may be utilized for predicting the prognosis of patients with ccRCC.

Background & Aims: Although nearly half of pancreatic ductal adenocarcinoma (PDAC) patients have diabetes mellitus with episodes of hyperglycemia, its tumor microenvironment is hypoglycemic. Thus, it is crucial for PDAC cells to develop adaptive mechanisms dealing with oscillating glucose levels. So far, the biological impact of such glycemic variability on PDAC biology remains unknown.
Methods: Murine PDAC cells were cultured in low- and high-glucose medium to investigate the molecular, biochemical, and metabolic influence of glycemic variability on tumor behavior. A set of in vivo functional assays including orthotopic implantation and portal and tail vein injection were used. Results were further confirmed on tissues from PDAC patients.
Results: Glycemic variability has no significant effect on PDAC cell proliferation. Hypoglycemia is associated with local invasion and angiogenesis, whereas hyperglycemia promotes metastatic colonization. Increased metastatic colonization under hyperglycemia is due to increased expression of runt related transcription factor 3 (Runx3), which further activates expression of collagen, type VI, alpha 1 (Col6a1), forming a glycemic pro-metastatic pathway. Through epigenetic machinery, retinoic acid receptor beta (Rarb) expression fluctuates according to glycemic variability, acting as a critical sensor relaying the glycemic signal to Runx3/Col6a1. Moreover, the signal axis of Rarb/Runx3/Col6a1 is pharmaceutically accessible to a widely used antidiabetic substance, metformin, and Rar modulator. Finally, PDAC tissues from patients with diabetes show an increased expression of COL6A1.
Conclusions: Glycemic variability promotes both local invasion and metastatic colonization of PDAC. A pro-metastatic signal axis Rarb/Runx3/Col6a1 whose activity is controlled by glycemic variability is identified. The therapeutic relevance of this pathway needs to be explored in PDAC patients, especially in those with diabetes.

The role of Runt-related transcription factor 3 ( RUNX3) gene in breast cancer remains not fully understood. We studied the correlation between RUNX3 gene promoter methylation and estrogen receptor (ER) expression status in breast cancer. Three breast cancer cell lines and 113 formalin-fixed, paraffin-embedded breast cancer tissue samples were analyzed for RUNX3 expression. Methylation-specific polymerase chain reaction was used to analyze RUNX3 methylation on the samples. Migration and invasion ability were evaluated in MCF7 cell line (RUNX3 methylated) treated with methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) to study the effect of RUNX3 methylation status. Our data showed that the expression of RUNX3 was high in MCF10A but not in MCF7 and SKBR3 cell lines, while the RUNX3 promoter showed hypermethylation in MCF7 but not in MCF10A and SKBR3. In tissues samples, Immunohistochemical (IHC) expression of RUNX3 protein was higher in ER-negative samples than in ER-positive cases, and it was negatively correlated with the methylation status of the RUNX3 gene promoter. Proliferation, migration, and invasion of MCF7 were suppressed when 5-Aza-CdR treated. Also, the hypermethylation status of RUNX3 gene promoter was reversed and RUNX3 expression was increased. In summary, our data suggest that hypermethylation of the RUNX3 gene promoter may play an important role in ER-positive breast tumor progression.

AIM: The prevalence and mortality rates of lung cancer in Xuanwei, Yunnan, China, are the highest in the world. The severe indoor air pollution caused by smoky coals with high benzo (a)pyrene (BaP) and quartz levels is the main environmental factor. The aim of this study was to investigate methylation profiles of promoters in eight genes in primary non-small cell lung cancers (NSCLC) exposed to smoky coals.
MATERIALS AND METHODS: Candidate genes including CDKN2A, DLEC1, CDH1, DAPK, RUNX3, APC, WIF1 and MGMT were determined for the promoter methylation status using Nested methylation-specific PCR (nMSP) in primary 23NSCLC tissues and in circulating tumor DNA (ctDNA) isolated from 42plasma samples (9matched to tissues) as well as 10healthy plasma samples, using Sanger sequencing to verify the results.
RESULTS: Seven of the 8genes, except MGMT, had relatively high methylation frequencies ranging from 39%-74% in tissues. Moreover, methylation frequencies in five genes identified in lung cancer plasma were 45% for CDKN2A, 48% for DLEC1, 76% for CDH1, 14% for DAPK, 29% for RUNX3, with a relatively good concordance of methylation among 9 tissues and paired plasma. However, the genes from all healthy plasma showed no methylation.
CONCLUSIONS: A panel of genes including CDKN2A, DLEC1, CDH1, DAPK and RUNX3 may be used as potential epigenetic biomarkers for early lung cancer detection. CDH1 promoter methylation was associated with lung cancer metastasis in areas of air pollution from buring of smoky coals. DLEC1 and CDH1 exhibited specific high methylation frequencies, different from previous reports.

Circular RNAs (circRNAs), a new class of endogenous non-coding RNAs, have recently been known to play critical roles in various cellular biological processes, including tumorigenesis, in which they act as an miRNA sponge that regulates gene expression. Thus, revealing the functions of circRNAs in carcinogenesis and cancer development has been of great interest. However, their expression and functions in gastric cancer (GC) development are still largely unknown. Therefore, the present study aimed to identify novel deregulated circRNAs in GC and reveal their biological functions and molecular mechanisms in GC. Quantitative real-time PCR (qRT-PCR) was performed to measure the expression levels of circRNAs in GC tissues, cell lines, and plasma. The MTT assay, colony formation assay, transwell assay, and tumor xenografts

INTRODUCTION: Runt-related transcription factor 3 (RUNX3) exerts a tumor suppressor gene associated with gastric and other cancers, including glioma. However, how its anti-tumor mechanism in functions glioma is unclear.
METHODS: We assayed expression of RUNX3 with a tissue microarray (TMA), frozen cancer tissues and malignant glioma cell lines using immunohistochemistry, qRT-PCR and Western bolt analysis. Cell proliferation, invasion, cell cycle distribution and apoptosis were also examined to confirm the effect of RUNX3 medicated malignant phenotype. TOP/FOP experiment was used to detect the β-catenin/Tcf-4 transcription activity by RUNX3.
RESULTS: Enforced RUNX3 expression inhibited proliferation and invasion, induced cell cycle arrest and promoted apoptosis in vitro and in vivo, Bim siRNA partically reversed the effect of RUNX3-induced apoptosis in LN229 and U87 cells, suggesting a dependent role of Bim-caspase pathway. Moreover, Mechanism investigations revealed that restoration of RUNX3 suppressed β-catenin/Tcf-4 transcription activity.
CONCLUSIONS: RUNX3 plays a pivotal role in glioma initiation and progression as a tumor suppressor via attenuation of Wnt signaling, highlighting it as a potential therapeutic target for glioma.

Increasing evidence showed that microRNAs (miRNAs) were abnormally expressed in cancers and made effects on the tumorigenesis. Aberrant expression of miR-20a-5p has been reported in human breast carcinoma. However, the functional mechanism of miR-20a-5p in human breast carcinoma, particularly in triple-negative breast cancer (TNBC), required further investigations. Here, firstly, we determined that miR-20a-5p was highly expressed in both TNBC tissues and cell lines. Then, we explored that the overexpression of miR-20a-5p promoted the migration and invasion of TNBC cells in vitro. The tendency was significantly reversed after the depletion of miR-20a-5p. Consistent result could be obtained with the in vivo nude mice tumorigenesis. Thirdly, the underlying molecular mechanism was investigated. The Runt-related transcription factor 3 (RUNX3) was identified as a target of miR-20a-5p in TNBC cells. High expression of miR-20a-5p significantly decreased both the mRNA and protein levels of RUNX3, as well as its direct downstream targets Bim and p21. These results verified the significance of miR-20a-5p and explored its functional mechanisms in TNBC, suggesting the potential clinical applications of miR-20a-5p in TNBC.

Emerging evidence has demonstrated that the deregulation of microRNAs (miRNAs) contributes to Wilms' tumour (WT) malignant progression. Therefore, identifying the essential miRNAs for WT onset and progression may be a promising therapeutic method for patients with this disease. Dysregulation of miRNA‑199b (miR‑199b) serves significant roles in various types of human cancer. However, its expression patterns, possible functions and associated mechanisms in WT are largely unknown. In the present study, the expression of miR‑199b in WT tissues was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis. The biological functions of miR‑199b overexpression in WT cells were determined using Cell counting kit‑8 and Transwell invasion assays. The mechanisms underlying the action of miR‑199b in WT cells were also investigated using bioinformatics analysis, a luciferase reporter assay, RT‑qPCR and western blot analysis. It was revealed that miR‑199b expression was upregulated in WT tissues. In addition, the downregulation of miR‑199b attenuated the proliferation and invasion of WT cells. Runt‑related transcription factor 3 (RUNX3) was mechanistically predicted as a potential target of miR‑199b. Subsequent experiments demonstrated that RUNX3 was a direct target gene of miR‑199b in WT. In addition, the downregulation of RUNX3 in the WT tissues was inversely correlated with the miR‑199b expression level. The recovered RUNX3 expression counteracted the oncogenic roles of miR‑199b in WT cells. Therefore miR‑199b may serve as an oncogene in WT progression by directly targeting RUNX3, thereby suggesting that the miR‑199b/RUNX3 axis may be a promising therapeutic target for patients with WT.

Background: The objective of this study was to discover DNA methylation biomarkers for detecting non-small lung cancer (NSCLC) in bronchial washings and understanding the association between DNA methylation and smoking cessation.
Methods: DNA methylation was analyzed in bronchial washing samples from 70 NSCLCs and 53 hospital-based controls using Illumina HumanMethylation450K BeadChip. Methylation levels in these bronchial washings were compared to those in 897 primary lung tissues of The Cancer Genome Atlas (TCGA) data.
Results: Twenty-four CpGs (
Conclusions: The present study suggests that NSCLC may be detected by analyzing methylation changes of seven CpGs in bronchial washings. Furthermore, smoking cessation may lead to decreased DNA methylation in nonmalignant bronchial epithelial cells in a gene-specific manner.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females worldwide. Chemoresistance has been a major reason for the drug therapy failure. The present study performed a microarray analysis between MCF‑7 and MCF‑7/adriamycin (ADR) cells, and intended to identify long non‑coding (lnc)RNA expression character in drug resistant breast cancer cells. MCF‑7/ADR cells were induced from MCF‑7 cells via pulse‑selection with doxorubicin for 4 weeks, and the resistance to doxorubicin of ADR cells was confirmed by MTT assay. Microarray analysis was performed between MCF‑7 and MCF‑7/ADR cells. Total RNA was extracted from the two cell lines respectively and was transcribed into cDNA. The results of the microarray were verified by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Gene Ontology (GO) and pathways analysis were conducted to enrich the dysregulated lncRNAs presented in the microarray results. Compared to the MCF‑7 cells, 8,892 lncRNAs were differentially expressed in MCF/ADR cells (absolute fold‑change >2.0). A total of 32 lncRNAs were selected for RT‑qPCR by fold‑change filtering, standard Student's t‑test, and multiple hypothesis testing. Among the dysregulated lncRNAs, AX747207 was prominent because its associated gene RUNX3 was previously reported to be relative to malignant tumor chemoresistance. GO analysis results also indicated some biological processes and molecular functions linked to chemoresistance. The pathway enrichment results provided some potential pathways associated with chemoresistance. In the present study, the authors intended to identify lncRNA expression character in drug resistant cell line MCF‑7/ADR, corresponding to the parental MCF‑7 cell line. In addition, the study identified the lncRNA AX747207, and its potential targeted gene RUNX3, may be related to chemoresistance in breast cancer. These results may new insights into exploring the mechanisms of chemoresistance in breast cancer.

PURPOSE: Helicobacter pylori infection induces phenotype-stabilizing methylation and promotes gastric mucosal atrophy that can inhibit CpG-island methylation. Relationship between the progression of gastric mucosal atrophy and the initiation of CpG-island methylation was analyzed to delineate epigenetic period for neoplastic transformation.
Materials and Methods: Normal-appearing gastric mucosa was biopsied from 110 H. pylori-positive controls, 95 H. pylori-negative controls, 99 gastric cancer patients, and 118 gastric dysplasia patients. Gastric atrophy was assessed using endoscopic-atrophic-border score. Methylation-variable sites of eight CpG-island genes adjacent to Alu (CDH1, ARRDC4, PPARG, and TRAPPC2L) or LTR (MMP2, CDKN2A, RUNX2, and RUNX3) retroelements and stomach-specific TFF3 gene were analyzed using radioisotope-labeled methylation-specific polymerase chain reaction.
RESULTS: Mean ages of H. pylori-positive controls with mild, moderate, and severe atrophy were 51, 54, and 65 years and those of H. pylori-associated TFF3 overmethylation at the three atrophic levels (51, 58, and 63 years) tended to be periodic. Alu-adjacent overmethylation (50 years) was earlier than TFF3 overmethylation (58 years) in H. pylori-positive controls with moderate atrophy. Cancer patients with moderate atrophy showed late Alu-adjacent (58 years) overmethylation and frequent LTR-adjacent overmethylation. LTR-adjacent overmethylation was frequent in cancer (66 years) and dysplasia (68 years) patients with severe atrophy.
CONCLUSION: Atrophic progression is associated with gastric cancer at moderate level by impeding the initiation of Alu-adjacent methylation. LTR-adjacent methylation is increased in cancer patients and subsequently in dysplasia patients.

BACKGROUND/AIMS: Emerging evidence points towards an important role of long noncoding RNAs (lncRNAs) in the tumorigenesis and progression of gastric cancer (GC). MT1JP has recently been reported to be differentially expressed and act as a tumor suppressor in different tumors, with its mechanisms not fully understood in GC.
METHODS: RT-qPCR was used to detect the expression of MT1JP, miR-214-3p and RUNX3 in tumor tissues and cell lines of GC. The CCK-8 assay, colony formation, Transwell assay and wound healing assay were used to evaluate the proliferation, invasion and migration of GC cells, respectively. Bioinformatics analysis and luciferase reporter assay were performed to disclose the interaction between MT1JP, miR-214-3p and RUNX3. Western blot and immunofluorescence were applied to assess the downstream signaling of RUNX3.
RESULTS: MT1JP was found downregulated in GC tissues and cells. Low expression of MT1JP was significantly correlated with advanced TNM stage and lymphatic metastasis. The expression of plasma MT1JP was also found decreased in GC patients compared to healthy controls, with an area under the ROC curve (AUC) of 0.649 for diagnosis of GC. Gain- and loss-of-function of MT1JP revealed that MT1JP functioned as a ceRNA for miR-214-3p to facilitate RUNX3 expression and then upregulated p21 and Bim levels suppressing GC cell proliferation, invasion and migration, and promoting apoptosis. Furthermore, MT1JP overexpression suppressed tumor growth and inhibited the expression of miR-214-3p and proliferation antigen Ki-67, but increased the expression of RUNX3, p21 and Bim in vivo.
CONCLUSIONS: Our results suggest a potential ceRNA regulatory network involving MT1JP regulates RUNX3 expression by competitively binding endogenous miR-214-3p in tumorigenesis and progression of GC. This mechanism may contribute to a better understanding of GC pathogenesis and provide potential therapeutic strategy for GC.

Loss of runt‑related transcription factor 3 (RUNX3) has been reported in various cancers, and one of the mechanisms mediating loss of RUNX3 expression is DNA methylation. However, the role of RUNX3 expression and its DNA methylation status as prognostic factors in endometrial cancer remain unclear. In the present study, the expression and promoter methylation of RUNX3 was examined in endometrial cancer tissues and cell lines, as well as their association with endometrial cancer prognosis. Fifty‑five endometrial cancer tissues and two endometrial cancer cell lines (HEC1‑α and Ishikawa) were studied. RUNX3 expression and promoter methylation were examined using reverse transcription‑polymerase chain reaction (RT‑PCR), methylation specific PCR (MS‑PCR), and immunohistochemical staining. The demethylating agent 5‑aza‑2'‑deoxycytidine (ADC) was used to reverse the methylation of the RUNX3 promoter. Loss of RUNX3 expression was observed in 50.9% (27/53) of endometrial cancer tissues and in the HEC1‑α cell line by immunohistochemistry and RT‑PCR, respectively. Methylation of the RUNX3 promoter was observed in 62.2% (33/53) of endometrial cancer tissues, 12.5% (1/8) of normal endometrial tissues, and the HEC1‑α cell line by MS‑PCR. Tumor grade and stage were significantly correlated with loss of RUNX3 expression. The expression of RUNX3 was restored by treatment with ADC and resulted in growth inhibition in HEC1‑α cells. The present results suggested that methylation may serve a critical role in the silencing of RUNX3 and loss of RUNX3 expression may serve as a prognostic marker in endometrial cancer.

KDM6A, an X chromosome-encoded histone demethylase and member of the COMPASS-like complex, is frequently mutated in a broad spectrum of malignancies and contributes to oncogenesis with poorly characterized mechanisms. We found that KDM6A loss induced squamous-like, metastatic pancreatic cancer selectively in females through deregulation of the COMPASS-like complex and aberrant activation of super-enhancers regulating ΔNp63, MYC, and RUNX3 oncogenes. This subtype of tumor developed in males had concomitant loss of UTY and KDM6A, suggesting overlapping roles, and points to largely demethylase independent tumor suppressor functions. We also demonstrate that KDM6A-deficient pancreatic cancer is selectively sensitive to BET inhibitors, which reversed squamous differentiation and restrained tumor growth in vivo, highlighting a therapeutic niche for patient tailored therapies.

A number of genes have been therapeutically targeted to relieve cancer, but cancer relapse is still a growing issue. The concept that the surrounding tumor environment is critical for the progression of cancer may foster an answer to the issue of cancer malignancy. Runt domain transcription factors (RUNX1, 2, and 3) are evolutionarily conserved and have been intensively studied for their roles in normal development and pathological conditions. During tumor growth, a hypoxic microenvironment and infiltration of the tumor by immune cells are common phenomena. In this review, we briefly introduce the consequences of hypoxia and immune cell infiltration into the tumor microenvironment with a focus on RUNX3 as a critical regulator. Furthermore, based on our current knowledge of the functional role of RUNX3 in hypoxia and immune cell maintenance, a probable therapeutic intervention is suggested for the effective management of tumor growth and malignancy. [BMB Reports 2018; 51(4): 174-181].

BACKGROUND: Long non-coding RNAs (LncRNAs) exert their functions mainly by binding to their corresponding proteins. Runt-related transcription factor 3 (Runx3) is an important transcription factor that functions as a tumor suppressor in gastric cancer. Whether there is an interplay between LncRNAs and Runx3 remains unclear.
METHODS: RPISeq was applied to screen the LncRNAs that potentially bind to Runx3. The interaction between LncRNA HOX antisense intergenic RNA (HOTAIR) and Runx3 was validated by RNA Immunoprecipitation and RNA pull-down assays. The role of Mex3b in the ubiquitination of Runx3 induced by HOTAIR was assessed by immunoprecipitation. Pearson's correlation between HOTAIR mRNA expression and Runx3 protein expression was analyzed. Cell migration and invasion were explored by transwell assays.
RESULTS: We found that HOTAIR was bound to Runx3 protein and identified the fragment of HOTAIR spanning 1951-2100 bp as the specific binding site. In addition, mex-3 RNA binding family member B (Mex3b) was an E3 ligase involved in HOTAIR-induced ubiquitous degradation of Runx3. Silencing the expression of HOTAIR or Mex3b attenuated the degradation of Runx3. In human gastric cancer tissues, HOTAIR was negatively associated with the expression level of Runx3 protein (Pearson coefficient - 0.501, p = 0.025). Inhibition of HOTAIR significantly suppressed gastric cancer cell migration and invasion through upregulating claudin1, which could be reversed by co-deficiency of Runx3.
CONCLUSIONS: These results uncovered the novel interaction between HOTAIR and Runx3, and provided potential therapeutic targets on the metastasis of gastric cancer.

Cholangiocarcinoma (CCA) is the most common primary liver cancer in Northeastern Thailand where liver fluke infection is highly endemic. Although aberrant DNA methylation in CCA has been reported by several investigators, little is known regarding the associations between them. In the present study, the results obtained from our previously published methylation array were analyzed and 10 candidate genes involved in DNA repair [protein phosphatase 4 catalytic subunit (PPP4C)], apoptosis [runt related transcription factor 3 (RUNX3), interferon regulatory factor 4 (IRF4), ubiquitin C‑terminal hydrolase L1 (UCHL1) and tumor protein p53 inducible protein 3 (TP53I3)], cell proliferation [cyclin D2 (CCND2) and Ras association domain family member 1 (RASSF1)], drug metabolism [aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and solute carrier family 29 member 1 (SLC29A1)] and angiogenesis [human immunodeficiency virus‑1 tat interactive protein 2 (HTATIP2)] were selected for quantification of their methylation levels in 54 CCA and 19 adjacent normal tissues using methylation‑sensitive high‑resolution melting. The associations between the methylation status of the individual genes and clinical parameters were statistically analyzed. High methylation levels were observed in UCHL1, IRF4, CCND2, HTATIP2 and TP53I3. The median methylation level of UCHL1 was 57.3% (range, 3.15 to 88.7%) and HTATIP2 was 13.6% (range, 7.5 to 36.7%). By contrast, low methylation of HTATIP2 and UCHL1 was identified in adjacent normal tissues. The methylation status of HTATIP2 and UCHL1 was associated with patients' overall survival. CCA patients with high methylation of HTATIP2 and low methylation of UCHL1 exhibited longer overall survival. In addition, multivariate Cox regression analysis demonstrated that UCHL1 methylation was an independent factor for CCA with hazard ratio of 1.81 (95% confidence interval, 1.01‑3.25) in high methylation group. The combination of HTATIP2 and UCHL1 methylation status strongly supported their potential predictive biomarker in which patients with CCA who had high methylation of HTATIP2 and low methylation of UCHL1 showed longer overall survival than those with low HTATIP2 methylation and high UCHL1 methylation. In conclusion, the present study revealed the value of aberrant DNA methylation of HTATIP2 and UCHL1, which may serve as a potential predictive biomarker for CCA.

(1) Background: Epithelial ovarian cancer (EOC) is the most lethal cancer of the female reproductive system. In an earlier study, we identified multiple genes as hypermethylated in tumors of patients with poor prognosis. The most promising combination of markers to predict a patient's outcome was

Progression of solid cancers, colorectal carcinomas among them, from their primaries to metastatic lesions traditionally is thought to proceed by a stepwise acquisition of and selection for genomic aberrations. To test if patterns of genomic aberrations would be consistent with this model, we studied 10 colorectal carcinoma primary-metastasis pairs, 9 with 1 liver metastasis each and 1 with 2 metastases. Next-generation targeted sequencing (50-gene panel) with samples obtained from different regions of the primaries and their metastases demonstrated 1-11 gene mutations per lesion. But only in 2 tumors were there seen mutations in all samples from the metastasis and not any of the primaries (BRAF

Breast cancer is one of complex diseases that are influenced by environment. Various genetic and epigenetic alterations are provoking causes of breast carcinogenesis. Dynamic epigenetic regulation including DNA methylation and histone modification induces dysregulation of genes related to proliferation, apoptosis, and metastasis in breast cancer. DNA methylation is strongly associated with the repression of transcription through adding to the methyl group by DNA methyltransferases (DNMTs), and tumor suppressor genes such as CCND2 and RUNX3 have been investigated to undergo hypermethylation at promoter region in breast cancer. In addition, histone deacetylases (HDACs) contribute to transcriptional repression by removing acetyl group at lysine residues leading to tumorigenesis. Since epigenetic changes are reversible, therapeutic approaches have been applied with epigenetic modification drugs such as DNMT inhibitors and HDAC inhibitors. In this chapter, we will summarize the feature of epigenetic markers in breast cancer cells and the effect of single or combination of epigenetic reagents for breast cancer therapy.