Ras Association Domain Research Articles

Promoter hypermethylation inactivates CDKN2A, CDKN2B and RASSF1A genes in sporadic parathyroid adenomas

Cyclin D1, a G1-S phase regulator, is upregulated in parathyroid adenomas. Since cyclin-dependent kinase (CDK) inhibitors, CDKN2A and CDKN2B, and RASSF1A (Ras-association domain family 1, isoform A) are involved in G1-S phase arrest and act as potential tumor suppressor genes, we aimed to study potential methylation-mediated inactivation of these genes in parathyroid adenomas. Gene expressions of cyclin D1 (CCND1) and regulatory molecules (CDKN2A, CDKN2B and RASSF1A) was analysed in parathyroid adenoma tissues (n = 30). DNA promoter methylation of cyclin D1 regulators were assessed and correlated with clinicopathological features of the patients. Gene expression analysis showed a relative fold reductions of 0.35 for CDKN2A (p = 0.01), 0.45 for CDKN2B (P = 0.02), and 0.39 for RASSF1A (p < 0.01) in adenomatous compared to normal parathyroid tissue. There was an inverse relationship between the expressions of CDKN2A and CDKN2B with CCND1. In addition, the promoter regions of CDKN2A, CDKN2B, and of RASSF1A were significantly hyper-methylated in 50% (n = 15), 47% (n = 14), and 90% (n = 27) of adenomas respectively. In contrast, no such aberrant methylation of these genes was observed in normal parathyroid tissue. So, promoter hypermethylation is associated with down-regulation of CCND1 regulatory genes in sporadic parathyroid adenomas. This dysregulated cell cycle mechanism may contribute to parathyroid tumorigenesis.

Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median (P(25)-P(75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group (P<0.001). DNA methylation levels of P16, RASSF1A and MGMT were 2.04±0.41, 2.19 (1.94-2.51), 2.22 (1.94-2.46)%5mC in exposure group, and 2.19±0.40, 2.41 (2.11-2.67), 2.44 (2.15-2.91)%5mC in control group. DNA methylation levels were lower in exposure group (P values were 0.005, 0.002 and 0.001, respectively). Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary εdA level (r values were -0.155, -0.137, and -0.198, respectively, P<0.05). No significant correlation was observed between the εdC level and any measured DNA methylation levels (P>0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95%CI) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95%CI) was -0.094 (-0.179--0.008), P=0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A and MGMT.

Short stature homeobox 2 methylation as a potential noninvasive biomarker in bronchial aspirates for lung cancer diagnosis

Gene methylation has been frequently observed in lung cancer. However, the use of methylated genes in bronchial aspirates of patients with lung cancer remains to be evaluated. The purpose of this study was to analyze whether the detection of genes with aberrant promoter methylation can be useful noninvasive biomarkers in bronchial aspirates from lung cancer. We found that the methylation status of the cyclin-dependent kinase inhibitor 2A (P16), Ras association domain family 1 isoform (RASSF1A), adenomatous polyposis coli (APC) and short stature homeobox 2 (SHOX2) genes was significantly correlated with lung cancer in bronchial aspirates. The P16, RASSF1A and APC methylation had a bad diagnostic effect in bronchial aspirates of patients with lung cancer compared with non-tumor controls (P16: sensitivity = 0.26, specificity = 0.99, area under the curve (AUC) = 0.67; RASSF1A: sensitivity = 0.40, specificity = 0.99, AUC = 0.66; APC: sensitivity = 0.17, specificity = 0.98, AUC = 0.65). The pooled sensitivity, specificity, and AUC of the SHOX2 methylation were 0.75, 0.94, and 0.94, respectively. Moreover, when squamous cell carcinoma (SCC) was compared to adenocarcinoma (AC), the SHOX2 gene had a significantly higher methylation rate in SCC than in AC (P < 0.001). Methylated P16, RASSF1A, APC and retinoic acid receptor beta2 (RARβ2) genes had similar frequencies in these two histotypes (P > 0.1). Our findings suggest that methylated SHOX2 gene could be a specific and potential noninvasive biomarker using bronchial aspirates for lung cancer diagnosis, especially for SCC.

Impact of RASSF1A gene methylation on the metastatic axillary nodal status in breast cancer patients.

Hypermethylation of CpG islands is a hallmark of cancer and occurs at an early stage in breast tumorigenesis. To gain insight into the epigenetic switches that may promote and/or contribute to the initial neoplastic events during breast carcinogenesis, the present study focused on the DNA methylation profile of invasive breast carcinoma. The aim of the study was to evaluate the prognostic significance of Ras association domain family 1 isoform A (RASSF1A) promoter methylation status in operable breast cancer, and to analyze the utility of this biomarker regarding its association with metastatic and nonmetastatic axillary nodal status. For this purpose, formalin-fixed, paraffin-embedded tissue specimens from 116 breast cancer patients with known axillary nodal status were subjected to assessment of RASSF1A promoter methylation status by methylation-specific polymerase chain reaction (MSP) and methylation-sensitive high-resolution melting assay, and the results were subsequently validated by bisulfite sequencing. A multinomial logistic regression model was used to model the dependence of distinct levels of methylation status of the RASSF1A promoter on the nodal status. Promoter region CpG hypermethylation was identified by MSP in 97 (83.6%) of 116 primary breast tumors, while hypermethylation of RASSF1A was confirmed by MS-HRM in 107 (92.2%) of 116 cases of breast cancer. Based on the results of the multinomial logistic regression model, there was no significant difference between the frequency of RASSF1A promoter methylation and axillary lymph node status of patients in general. However, upon adjustment of pN stage, an association was identified between pN0 lymph node-negative status (without axillary metastases) and percentage of RASSF1A methylation in two groups of heterogeneous methylated alleles with ≤50% methylated (P<0.05) and >50% methylated alleles (P<0.0001). If a patients' nodal status changes from pN- to pN+ then the risk of having >50% methylated alleles increases by 7%. The present study revealed a specific phenomenon, suggesting that the presence of heterogeneous methylated alleles in the RASSF1A gene is significantly associated with lymph node-negative status in breast cancer patients. Furthermore, greater significance with negative axillary nodal status was observed with a higher level of heterogeneous methylated alleles in the RASSF1A gene.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Distinct DNA methylation alterations are associated with cribriform architecture and intraductal carcinoma in Gleason pattern 4 prostate tumors.

The aim of the present study was to explore DNA methylation aberrations in association with cribriform architecture and intraductal carcinoma (IDC) of the prostate, as there is robust evidence that these morphological features are associated with aggressive disease and have significant clinical implications. Herein, the associations of a panel of seven known prognostic DNA methylation biomarkers with cribriform and IDC features were examined in a series of 91 Gleason pattern (GP) 4 tumors derived from Gleason score 7 radical prostatectomies. Gene specific DNA methylation was compared between cribriform and/or IDC positive vs. negative cases, and in association with clinicopathological features, using Chi square and Mann-Whitney U tests. DNA methylation of the adenomatous polyposis coli, Ras association domain family member 1 and T-box 15 genes was significantly elevated in GP4 tumors with cribriform and/or IDC features compared with negative cases (P=0.045, P=0.007 and P=0.013, respectively). To the best of our knowledge, this provides the first evidence for an association between cribriform and/or IDC and methylation biomarkers, and warrants further investigation of additional DNA methylation events in association with various architectural patterns in prostate cancer.

Excess of a Rassf1-targeting microRNA, miR-193a-3p, perturbs cell division fidelity

Background:Several microRNA (miRNA) molecules have emerged as important post-transcriptional regulators of tumour suppressor and oncogene expression. Ras association domain family member 1 (RASSF1) is a critical tumour suppressor that controls multiple aspects of cell proliferation such as cell cycle, cell division and apoptosis. The expression of RASSF1 is lost in a variety of cancers due to the promoter hypermethylation.Methods:miR-193a-3p was identified as a RASSF1-targeting miRNA by a dual screening approach. In cultured human cancer cells, immunoblotting, qRT–PCR, luciferase reporter assays, time-lapse microscopy and immunofluorescence methods were used to study the effects of excess miR-193a-3p on RASSF1 expression and cell division.Results:Here, we report a new miRNA-mediated mechanism that regulates RASSF1 expression: miR-193a-3p binds directly to RASSF1-3′UTR and represses the mRNA and protein expression. In human cancer cells, excess of miR-193a-3p causes polyploidy through impairment of the Rassf1-Syntaxin 16 signalling pathway that is needed for completion of cytokinesis. In the next cell cycle the miR-193a-3p-overexpressing cells exhibit multipolar mitotic spindles, mitotic delay and elevated frequency of cell death.Conclusions:Our results suggest that besides epigenetic regulation, altered expression of specific miRNAs may contribute to the loss of Rassf1 in cancer cells and cause cell division errors.

MST50 is involved in multiple MAP kinase signaling pathways in Magnaporthe oryzae.

Appressorium formation plays a critical role in Magnaporthe oryzae. Mst50 is an adapter protein of the Mst11-Mst7-Pmk1 cascade that is essential for appressorium formation. To further characterize its functions, affinity purification was used to identify Mst50-interacting proteins (MIPs) in this study. Two of the MIPs are Mst11 and Mst7 that are known to interact with Mst50 for Pmk1 activation. Surprisingly, two other MIPs are Mck1 and Mkk2 that are the upstream kinases of the Mps1 pathway. Domain deletion analysis showed that the sterile alpha-motif of Mst50 but not the Ras-association domain was important for its interaction with Mck1 and responses to cell wall and oxidative stresses. The mst50 mutant was reduced in Mps1 activation under stress conditions. MIP11 encodes a RACK1 protein that also interacted with Mck1. Deletion of MIP11 resulted in defects in cell wall integrity, Mps1 phosphorylation and plant infection. Furthermore, Mst50 interacted with histidine kinase Hik1, and the mst50 mutant was reduced in Osm1 phosphorylation. These results indicated that Mst50 is involved in all three MAPK pathways in M. oryzae although its functions differ in each pathway. Several MIPs are conserved hypothetical proteins and may be involved in responses to various signals and crosstalk among signaling pathways.

Effect of RASSF1A gene promoter methylation on its expression level in cervical cancer tissue and its clinical significance

Objective To research the promoter methylation level of RAS association domain family 1A (RASSF1A) and RASSF1A gene mRNA expression level in cervical cancer tissue, and to analyze their relationships with clinicopathological parameters of cervical cancer and the clinical significance. Methods The RASSF1A gene promoter methylation and RASSF1A gene mRNA were detected respectively by methylation specific PCR and quantitative real-time PCR method in 40 cases of cervical cancer tissues and corresponding adjacent tissues. Results RASSF1A mRNA expression level in cervical cancer (0.26±0.05) was significantly lower than that in adjacent tissues (0.28±0.03), and the difference was statistically significant (t=2.27, P=0.026). The methylation rate of RASSF1A gene promoter region (0.71%±0.04%) was significantly higher than that in adjacent tissues (0.66%± 0.03%), and the difference was statistically significant (t=6.78, P=0.000). The expression of RASSF1A mRNA was significantly correlated with pathological differentiation (t=3.31, P=0.002), International Federation of Gynecology and Obstetrics (FIGO) stage (t=2.13, P=0.040), lymphatic metastasis (t=2.56, P=0.015). The promoter methylation level of RASSF1A gene was significantly correlated with pathological differentiation (t=2.08, P=0.045), FIGO stage (t=2.66, P=0.011), lymphatic metastasis (t=2.22, P=0.033), depth of invasion (t=2.12, P=0.041). Conclusion The RASSF1A gene promoter region methylation level and the RASSF1A gene mRNA expression level are associated with the malignant degree of cervical carcinoma. The RASSF1A gene promoter region methylation level may be used as a reference indicator for predicting the risk of metastasis of cervical cancer. Key words: Cervical neoplasms; Methylation; RAS association domain family 1A gene

Diagnostic Capacity of RASSF1A Promoter Methylation as a Biomarker in Tissue, Brushing, and Blood Samples of Nasopharyngeal Carcinoma

Methylation of the RAS association domain family protein 1A (RASSF1A) promoter has been observed in nasopharyngeal carcinoma (NPC). This study investigated the correlation of RASSF1A promoter methylation with clinicopathological features and its utility as a diagnostic biomarker in NPC. A total of 926 patients with NPC and 495 non-tumor controls were analyzed in this study. RASSF1A promoter methylation was notably higher in NPC compared with non-tumor tissue, brushing and blood samples. RASSF1A promoter methylation was associated with clinical stage, lymph node status, distant metastasis, and T classification of patients with NPC, although it was not linked to age and sex. The pooled sensitivity, specificity, and AUC (area under the curve) of RASSF1A promoter methylation were determined in NPC samples vs. non-tumor samples (tissue: sensitivity=0.72, specificity=0.99, AUC=0.98; brushing: sensitivity=0.56, specificity=1.00, AUC=0.94; blood: sensitivity=0.11, specificity=0.98, AUC=0.97). Our findings show that RASSF1A promoter methylation may be correlated with the development, progression and metastasis of NPC. RASSF1A promoter methylation is a promising noninvasive biomarker for the diagnosis of NPC from tissue and brushing samples.

SHANK proteins limit integrin activation by directly interacting with Rap1 andR-Ras.

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN-domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN-domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN-domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1/RIAM/talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

Prognostic and predictive role of COX-2, XRCC1 and RASSF1 expression in patients with esophageal squamous cell carcinoma receiving radiotherapy.

Identification of biomarkers for predicting radiosensitivity would be useful for administering individualized radiotherapy (RT) to patients with esophageal cancer. The aim of the present study was to evaluate the association between cyclooxygenase-2 (COX-2), X-ray repair cross complementing group 1 (XRCC1), ras association domain family 1 (RASSF1) protein expression, clinicopathological characteristics, radiosensitivity and survival rate in 76 patients with esophageal squamous cell carcinoma (ESCC) who were treated with RT. Positive expression of COX-2, XRCC1 and RASSF1 was identified by immunohistochemistry in 81.6, 52.6 and 59.2% of ESCC cases, respectively. Negative COX-2 expression was associated with tumor (T) stage, node (N) stage, clinical stage and complete response (P<0.05), but not with gender, age, tumor location, differentiation degree, lesion length, progression-free survival (PFS) or overall survival (OS; P>0.05). XRCC1 expression was not associated with the clinicopathological features of ESCC, response to RT, PFS or OS. Positive RASSF1 expression was associated with the clinical stage, response to RT, PFS and OS (P<0.05), but not with gender, age, tumor location, T stage, N stage, differentiation degree or the lesion length (P>0.05). In the subgroup analysis, RASSF1 positive/XRCC1 negative expression was correlated with a longer median OS and PFS (P<0.05). Multivariate analyses revealed that the tumor response and RASSF1 expression were significant prognostic factors. Therefore, positive RASSF1 expression is associated with ESCC RT sensitivity, and may be a useful independent prognostic factor for ESCC.

PLCε1 regulates SDF-1α–induced lymphocyte adhesion and migration to sites of inflammation

Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.

Quantitative assessment of the relationship between RASSF1A gene promoter methylation and bladder cancer (PRISMA).

Methylation of the Ras-association domain family 1 isoform A (RASSF1A) gene promoter region is thought to participate in the initiation and development of many different cancers. However, in bladder cancer the role of RASSF1A methylation was unclear. To evaluate the relationship between RASSF1A methylation and bladder cancer, a quantitative assessment of an independent meta-analysis was performed. In addition, a DNA methylation microarray database from the cancer genome atlas (TCGA) project was used to validate the results of the meta-analysis. We searched published articles from computerized databases, and DNA methylation data were extracted from TCGA project. All data were analyzed by R software. The results of the meta-analysis indicated that the frequency of RASSF1A gene methylation in bladder cancer patients is significantly higher than in healthy controls. The hazard ratio (HR) was 2.24 (95% CI = [1.45; 3.48], P = 0.0003) for overall survival (OS), and the RASSF1A gene promoter methylation status was strongly associated with the TNM stage and differentiation grade of the tumor. The similar results were also found by the data from TCGA project. There was a significant relationship between the methylation of the RASSF1A gene promoter and bladder cancer. Therefore, RASSF1A gene promoter methylation will be a potential biomarker for the clinical diagnosis of bladder cancer.

Abstract PR02: Epigenetic and molecular drivers of inflammation-driven colorectal cancer

Abstract Colorectal tumors arise from intestinal epithelial cells in a multistep process that extends over several years resulting in progression from a normal mucosa to invasive carcinoma. Approximately 5% of all CRC are due to inherited genetic mutations (sporadic CRC) while 95% of the cases originate from a state of chronic inflammation, such as observed in CRC arising in patients with inflammatory bowel diseases (IBD-CRC) or with primary sclerosing cholangitis (PSC-CRC). About 70-75% of PSC patients have ulcerative colitis (UC, a form of IBD) and thus PSC-IBD is very similar to IBD-CRC. It is now established that the persistent pro-inflammatory environment that exists in patients with IBD or PSC can trigger molecular changes leading to tumor development. In this study, we have explored the origins of abnormal chronic intestinal inflammation, what sustains it and how it promotes CRC if uncontrolled. Furthermore, we can eliminate intestinal inflammation and prevent invasive carcinoma in a mouse model of chronic inflammation driven malignancy using the azoxymethane [AOM]/dextran sodium sulphate [DSS] model. In this model, DSS injures the colonic mucosa and in repeated chronic cycles promotes inflammation-induced carcinogenesis, which is accelerated by an initial exposure to the pro-carcinogen AOM. The tumor-suppressor protein, Ras association domain family 1A, (RASSF1A or 1A) is epigenetically silenced in numerous cancers, including CRC. 1A can negatively regulate NFκB and intestinal inflammation and its loss triggered acute colitis in a rodent model for IBD. In this study, we utilized mouse models, cell biology, patient samples and 3D intestinal organoids to demonstrate epigenetic silencing of 1A in IBD, PSC and CRC patients and detrimental effects of the expression loss of 1A during chronic inflammation-injury in the AOM/DSS model of IBD-CRC. Loss of 1A resulted in the appearance of colonic hyperplasia and invasive carcinoma. The timing of expression loss of 1A resulted in the hyperactivation of the nucleotide-binding oligomerization domain-containing protein2 (NOD2)/receptor interacting protein kinase 2 (RIPK2) pathway and Yes associated protein (YAP) dependent transcriptional activity. Abnormal phosphotyrosine (pY) activities of both RIPK2 and YAP contributed to the appearance of invasive carcinoma. YAP, when tyrosine phosphorylated, is a transcriptional co-activator of p73-driven cell death and TEAD/RUNX2/SMAD2 driven proliferation, whereas tyrosine phosphorylated RIPK2 can promote the activation of NFkB, the autophagic response and contribute to enhance growth and metastasis. Both RIPK2 and YAP maintain the chronic inflammation state and can drive colonic malignancy and the appearance of CRC. Furthermore, we validated the importance of both pY-YAP and pY-RIPK2 in IBD patient FFPE sections as well as in 3 case study IBD-CRC patients, utilized a small molecule anti-inflammatory to inhibit in vivo chronic inflammation and prevent invasive carcinoma. Both YAP and RIPK2 are novel biomarkers of inflammation-driven CRC and expression loss of RASSF1A may be an early marker for molecular changes during chronic inflammation that may predict the progression to CRC. This abstract is also being presented as Poster A01. Citation Format: Marilyn Gordon, Mohamed Salla, Ahmed Said, Volodko Natalia, Levinus Dieleman, Shariaz Baksh. Epigenetic and molecular drivers of inflammation-driven colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR02.

Domain analysis of Ras‐association domain family member 6 upon interaction with MDM2

The tumor suppressor Ras-association domain family member 6 (RASSF6) has Ras-association domain (RA) and Salvador/RASSF/Hippo domain (SARAH). RASSF6 antagonizes MDM2, stabilizes p53, and induces apoptosis and cell cycle arrest. We previously demonstrated the interaction between RASSF6 and MDM2, but did not determine how both proteins interact with each other. We have shown here that N-terminal, RA, and SARAH domains of RASSF6 interact with MDM2 at distinct regions. RA binds to the RING-finger region of MDM2 and stabilizes p53. SARAH binds RA and blocks the interaction between RA and MDM2. RA overexpression induces p53-dependent apoptosis and senescence. In the presence of active KRas, the interaction between RA and MDM2 is recovered. In this way, RA and SARAH play an important role in Ras-mediated regulation of p53.

MiR-214 regulates oral cancer KB cell apoptosis through targeting RASSF5.

Ras association domain family member 5 (RASSF5), a member of the Ras association domain family, induces cell apoptosis by phosphorylating FOXO3a, which triggers target gene BIM (pro-apoptotic factor) activation. MiR-214 is overexpressed in oral cancer tissue, indicating its possible involvement in oral cancer pathogenesis. Bioinformatics analysis has revealed a complimentary sequence between miR-214 and the 3'-UTR of RASSF5 mRNA. However, whether miR-124 regulates RASSF5 in oral cancer remains poorly understood. We aimed to investigate the role of miR-214 in RASSF5 expression regulation in oral cancer. Tumor and paracarcinoma tissues were obtained from 48 oral cancer patients to examine miR-214 and RASSF5 expression. The relationship between miR-214 and RASSF5 was investigated by dual luciferase reporter gene assay. Oral cancer KB cells were cultured in vitro and divided into inhibitor NC, miR-214 inhibitor, Scramble-pMD18, RASSF5-pMD18, and miR-214 inhibitor + RASSF5-pMD18 groups. Caspase 3 activity, cell apoptosis, and total protein expression were measured by spectrophotometry, flow cytometry, and western blot, respectively. MiR-214 expression was significantly increased, while that of RASSF5 decreased in oral cancer tumor tissues compared to paracarcinoma tissues. Luciferase assay showed that miR-214 suppressed RASSF5 expression by targeting its 3'-UTR. Down-regulation of miR-214 and/or enhancement of RASSF5 expression markedly increased FOXO3a phosphorylation, BIM expression, caspase 3 activity, and apoptosis. In conclusion, miR-214 expression was elevated and RASSF5 was down-regulated in oral cancer. Moreover, miR-214 regulated KB cell apoptosis through targeted inhibition of RASSF5 expression, FOXO3a phosphorylation, and BIM expression, suggesting its possible application as a novel therapeutic oral cancer target.

Establishment of two data mining models of lung cancer screening based on three gene promoter methylations combined with telomere damage.

To identify the significance of a support vector machine (SVM) model and a decision tree (DT) model for the diagnosis of lung cancer combined with the detection of fragile histidine triad (FHIT), RAS association domain family 1 (RASSF1A) and cyclin-dependent kinase inhibitor 2A (p16) promoter methylation levels and relative telomere length (RTL) of white blood cells from peripheral blood DNA. The levels of p16, RASSF1A and FHIT promoter methylation and the RTL of white blood cells in peripheral blood DNA of 200 healthy individuals and 200 lung cancer patients were analyzed by SYBR Green-based quantitative methylation-specific PCR and quantitative PCR. Based on the 4 biomarkers, SVM and DT models were developed. The levels of FHIT, RASSF1A and p16 promoter methylation were 3.33 (1.86-6.40) and 2.85 (1.39-5.44) (p = 0.002); 27.62 (9.09-52.86) and 17.17 (3.86-50.87) (p = 0.038); and 0.59 (0.16-4.50) and 0.36 (0.06-4.00) (p = 0.008) in cases and controls, respectively. RTL was 0.93 ± 0.32 and 1.16 ± 0.57 (p<0.001). The areas under the receiver operating characteristic (ROC) curves of the Fisher discriminant analysis, SVM and DT models were 0.670 (0.569-0.761), 0.810 (0.719-0.882) and 0.810 (0.719-0.882), respectively. The SVM and DT models for diagnosing lung cancer were successfully developed through the combined detection of p16, RASSF1A and FHIT promoter methylation and RTL, which provided useful tools for screening lung cancer.

RASSF1A promoter methylation is associated with increased risk of thyroid cancer: a meta-analysis.

ObjectivePrevious studies have reported that Ras-associated domain family 1A (RASSF1A), the most commonly silenced tumor suppressor via promoter methylation, played vital roles in the development of carcinogenesis. The purpose of this meta-analysis was to determine whether RASSF1A promoter methylation increased the risk of thyroid cancer.MethodsPubMed, Embase, ISI Web of Knowledge, and Chinese National Knowledge Infrastructure databases were searched to obtain eligible studies. The pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of the associations, using Stata 12.0 software. The methodological quality of included studies was evaluated using Newcastle–Ottawa scale table. Egger’s test and Begg’s test were applied to detect publication biases. TSA 0.9 software was used to calculate the required information size and whether the result was conclusive.ResultsA total of 10 articles with 12 studies that included 422 thyroid cancer patients, identifying the association of RASSF1A promoter methylation with thyroid cancer risk, were collected in this meta-analysis. Overall, RASSF1A promoter methylation significantly increased the risk of thyroid cancer (total, OR=8.27, CI=4.38–15.62, P<0.05; Caucasian, OR=9.25, CI=3.97–21.56, P<0.05; Asian, OR=7.01, CI=2.68–18.38, P<0.05). In the subgroup analysis based on sample type, a significant association between thyroid cancer group and control group was found (normal tissue, OR=9.55, CI=4.21–21.67, P<0.05; adjacent tissue, OR=6.80, CI=2.49–18.56, P<0.05). The frequency of RASSF1A promoter methylation in follicular thyroid carcinoma was higher than in control group (OR=11.88, CI=5.80–24.32, P<0.05). In addition, the results indicated that the RASSF1A promoter methylation was correlated with papillary thyroid carcinoma in Caucasians and Asians (total, OR=8.07, CI=3.54–18.41, P<0.05; Caucasian, OR=11.35, CI=2.39–53.98, P<0.05; Asian, OR=6.67, CI=2.53–17.64, P<0.05). On the basis of the trial sequential analysis, the significant association of RASSF1A promoter methylation with thyroid cancer risk was found, and there was no need to perform further studies.ConclusionThis meta-analysis confirms that RASSF1A promoter methylation is a risk factor for thyroid tumor.

MiR-181a-5p promotes the progression of gastric cancer via RASSF6-mediated MAPK signalling activation

We previously discovered that Ras association domain family member 6 (RASSF6) was downregulated and predicted poor prognosis in GC patients. However, the mechanisms of the down regulation of RASSF6 in GC remained unclear. Increasing evidence indicates that dysregulation of microRNAs promotes the progression of cancer through the repression of tumour suppressors. Here, we identified miR-181a-5p as a novel regulator of RASSF6 in GC. Functionally, ectopic expression or silencing of miR-181a-5p, respectively, promoted or inhibited GC cell proliferation, colony formation and cell cycle transition, as well as enhanced or prevented the invasion, metastasis of GC cells and epithelial to mesenchymal transition of GC cells in vitro and in vivo. Molecularly, miR-181a-5p functioned as an onco-miRNA by activating the RASSF6-regulated MAKP pathway. Overexpression or silencing of RASSF6 could partially reverse the effects of the overexpression or repression of miR-181a-5p on GC progress caused by activation of the MAKP pathway in vitro and in vivo. Clinically, high miR-181a-5p expression predicted poor survival in GC patients, especially combined with low RASSF6 expression. Collectively, we identified miR-181a-5p as an onco-miRNA, which acts by directly repressing RASSF6 in GC.