Ras Association Domain Family 1A Gene Research Articles

Combined ultrasound endoscopy-guided fine-needle aspiration with DNA methylation of SHOX2 and RASSF1A genes to enhance the auxiliary diagnostic precision of pancreatic cancer

The purpose of this study was to assess the influence and the clinical effectiveness of the short stature homeobox 2 (SHOX2) and ras association domain family 1A (RASSF1A) genes by tissue sampling through ultrasound endoscopy-guided fine-needle aspiration (EUS-FNA) as auxiliary diagnostic tools for pancreatic cancer (PC). Methylation markers were detected in 96 patients using real-time fluorescence quantitative PCR (qPCR), and the performance of this diagnostic assay was compared with CA19-9, CEA, and puncture fluid-based exfoliative cytology using receiver operating characteristic curve (ROC) analysis. The PC group exhibited higher methylation rates for SHOX2, RASSF1A, and the combined assay of both genes compared to the control group (95.7 % vs. 54.0 %, 78.3 % vs. 36.0 %, and 73.9 % vs. 16.0 %, P < 0.05). The areas under the ROC curve (AUC) for CA19-9, CEA, liquid-based exfoliative cytology, SHOX2, RASSF1A, the combination of SHOX2 and RASSF1A, the combination assay with CEA, CA19-9, and liquid-based exfoliative cytology were 0.827, 0.692, 0.767, 0.770, 0.732, 0.870, 0.870, 0.933, and 0.900, respectively. Therefore, the methylation assay based on the combined SHOX2 and RASSF1A genes in EUS-FNA puncture fluid is more effective than using a single gene, liquid-based exfoliative cytology, or intravenous tumor markers for diagnosing PC. Combining the conventional marker CA19-9 enhances the diagnostic value, making it a promising approach to complement histology and cytology.

Ras Association Domain Family 1A Gene Promoter Methylation as a Biomarker for Chronic Viral Hepatitis C-related Hepatocellular Carcinoma.

Background One of the most prevalent aberrant epigenetic modifications found in hepatocellular carcinoma (HCC) is abnormal DNA methylation. Our study aimed to evaluate serum Ras association domain family 1A (RASSF1A) gene promoter methylation in patients with chronic viral hepatitis C (HCV)-associated liver cirrhosis with and without HCC as a potential new marker for the early detection of HCC. Methodology The 60 participants who participated in the trial were divided into the following three groups: 20 patients with newly diagnosed primary HCC on top of HCV-related liver cirrhosis, 20 patients with HCV-related liver cirrhosis, and 20 age- and sex-matched healthy individuals as a control group. All participants underwent methylation-specific polymerase chain reaction testing to detect the blood level of the RASSF1A gene's methylated promoter. Results Methylated RASSF1A was found in 30% of patients with liver cirrhosis caused by HCV and in 65% of patients with HCC, but not in any of the controls. It was discovered that the serum methylation RASSF1A had an accuracy of 82.50% and an area under the curve (AUC) of 0.825 for separating HCC patients from healthy controls. With an AUC of 0.675 and an accuracy of 67.50%, it was able to differentiate patients with HCC from those with HCV-related liver cirrhosis. Additionally, there was no statistically significant association between RASSF1A methylation status and HCC mass size (p = 0.449). Conclusions Serum RASSF1A promoter methylation status detection could be useful for detecting HCC early, especially in high-risk individuals such as those with HCV.

The effects of up-regulation of microRNA-148a on the expression of anti-oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC-1 cells

Objective To investigate the effect of up-regulation of microRNA (miRNA, miR)-148a on the methylation and protein expression of anti-oncogene preproenkephalin (ppENK), p16, ras association domain family 1A (RASSF1A) and the proliferation of pancreatic cancer AsPC-1 cells. Methods MiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group (transfected Lipofectamine), mimincs-NC group (transfected negative mimics) and mimincs-148a group (transfected miR-148a mimics). At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction (Real-time PCR). The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction (MSP). The expression of DNA methy transferas 1 (DNMT1), ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium (MTT) assay. Results The expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002), while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group (1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001). ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group (ppENK: 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16: 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A: 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002). The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group (P=0.026). Conclusion Up-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a. Key words: Pancreatic cancer; MicroRNA-148a; DNA methy transferas 1; Anti-oncogene; Methylation; Cell proliferation

The relationship between RASSF1A promoter methylation and thyroid carcinoma: A meta-analysis of 14 articles and a bioinformatics of 2 databases (PRISMA).

DNA promoter methylation can suppresses gene expression and shows an important role in the biological functions of Ras association domain family 1A (RASSF1A). Many studies have performed to elucidate the role of RASSF1A promoter methylation in thyroid carcinoma, while the results were conflicting and heterogeneous. Here, we analyzed the data of databases to determine the relationship between RASSF1A promoter methylation and thyroid carcinoma. We used the data from 14 cancer-normal studies and Gene Expression Omnibus (GEO) database to analyze RASSF1A promoter methylation in thyroid carcinoma susceptibility. The data from the Cancer Genome Atlas project (TCGA) database was used to analyze the relationship between RASSF1A promoter methylation and thyroid carcinoma susceptibility, clinical characteristics, prognosis. Odds ratios were estimated for thyroid carcinoma susceptibility and hazard ratios were estimated for thyroid carcinoma prognosis. The heterogeneity between studies of meta-analysis was explored using H, I values, and meta-regression. We adopted quality criteria to classify the studies of meta-analysis. Subgroup analyses were done for thyroid carcinoma susceptibility according to ethnicity, methods, and primers. Result of meta-analysis indicated that RASSF1A promoter methylation is associated with higher susceptibility to thyroid carcinoma with small heterogeneity. Similarly, the result from GEO database also showed that a significant association between RASSF1A gene promoter methylation and thyroid carcinoma susceptibility. For the results of TCGA database, we found that RASSF1A promoter methylation is associated with susceptibility and poor disease-free survival (DFS) of thyroid carcinoma. In addition, we also found a close association between RASSF1A promoter methylation and patient tumor stage and age, but not in patients of different genders. The methylation status of RASSF1A promoter is strongly associated with thyroid carcinoma susceptibility and DFS. The RASSF1A promoter methylation test can be applied in the clinical diagnosis of thyroid carcinoma.

Value of the level of methylation of RASSF1A and WIF-1 in tissue and serum in neoadjuvant chemotherapeutic assessment for advanced breast cancer.

This study assessed the clinical efficacy of the neoadjuvant chemotherapy TAC scheme in treatment of patients with locally advanced breast cancer, and the value of the level of Ras association domain family 1A (RASSF1A) gene methylation and the Wnt inhibitory factor (WIF)-1 gene in tissue and serum of patients in clinical outcome prediction. In total, 126 patients were consecutively selected to receive TAC scheme (docetaxel, pirarubicin/epirubicin and cyclophosphamide) for at least four cycles with the total effective rate. The incidence of complications, progression-free survival and survival rate were recorded. Tumor tissues and peripheral blood samples collected in this study was used to detect methylation positive rate of RASSF1A and WIF-1 by methylation-specific PCR method and the relative level of expression of RASSF1A and WIF-1 mRNA by reverse transcription PCR method. Of the 126 patients, there were 18 cases with complete response (CR), 32 cases with partial response (PR), 50 cases with stable disease (SD), and 26 cases with disease progression (PD) with a total effective rate of 79.37%. Comparison on baseline data of effective group and ineffective group showed no difference (P>0.05), and comparison on adverse reactions occurrence showed no difference (P>0.05). Progression-free survival of the effective group was prolonged with a significant increase in survival rate (P<0.05). Positive rates of RASSF1A methylation and WIF-1 in tissue and serum of the patients in the effective group were significantly lower than those in the ineffective group, but the mRNA of RASSF1A and WIF-mRNA was significantly higher than the ineffective group (P<0.05). The sensitivity of clinical outcome prediction using tissue RASSF1A methylation was 67.0%, the specificity 15.4%, positive predictive value 69.0% and negative predictive value 31.0%. The above-mentioned indexes of tissue WIF-1 were 76.0, 31.4, 72.2 and 27.8, respectively. The indexes of serum RASSF1A were 85.0, 50.0, 76.2 and 23.8%, respectively, and the indexes of serum WIF-1 were 94.0, 75.0, 81.0 and 19.0%, respectively. The receiver operating characteristic curve analysis suggested that the accuracy of clinical outcome prediction using tissue RASSF1A mRNA level was 0.812. The sensitivity 85.2%, the specificity 76.3% and the critical value 0.4256. These indexes of tissue WIF-1 were 0.833, 86.7%, 75.4% and 0.3562 for CR, PR, SD and PD, respectively. These indexes of serum RASSF1A were 0.864, 88.3%, 77.4% and 0.2564, respectively, and for serum WIF-1 were 0.882, 89.4%, 73.5% and 0.1562, respectively. In conclusion, the detection of RASSF1A and WIF-1 gene methylation and level of mRNA expression in tissue and serum of patients with locally advanced breast cancer has an important application value in predicting clinical efficacy of neoadjuvant chemotherapy of the TAC scheme.

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

Delineating the effect of demethylating agent 5-aza-2'-deoxycytidine on human Caco-2 colonic carcinoma cells.

Aberrant epigenetic changes are known to contribute to various phases of tumor development. The gene function loss caused by aberrant methylation is analogous to genetic mutations. Unlike genetic mutations, epigenetic alterations can be reversed. 5-Aza-2′-deoxycytidine (5-aza-CdR) has been approved by the Food and Drug Administration for the treatment of certain types of cancer, such as MDS and leukemia. The aim of the present study was to determine whether 5-aza-CdR has the potential to be used in the treatment of colon cancer using a human Caco-2 colonic carcinoma cell line. The effect of 5-aza-CdR on cell proliferation, cell cycle, apoptosis and reversal of aberrant methylation of the Ras association domain family 1A (RASSF1A) gene was also examined. The 5-aza-CdR was prepared at different concentrations in sterile tri-distilled water at 0.4, 1.6, 6.4, 25.6 and 102.4 µmol/l and employed to treat the human Caco-2 colonic carcinoma cells. An MTT assay was used to detect the effect of 5-aza-CdR on cell proliferation. Flow cytometry was used to examine the cell cycle and apoptosis. The RASSF1A mRNA transcript level was examined by reverse transcription-polymerase chain reaction. The results showed that 5-aza-CdR inhibited the proliferation of Caco-2 cells in a time- and concentration-dependent manner (p<0.01). The 5-aza-CdR treatment affected the cell cycle and caused accumulation of cells in the G0/G1 phase and this effect was concentration-dependent (p<0.05). 5-aza-CdR treatment caused an increase in the number of cells undergoing apoptosis and reactivated the RASSF1A tumor suppressor gene that was silenced by hypermethylation in Caco-2 cells. In conclusion, 5-aza-CdR inhibited growth and promoted apoptosis in Caco-2 cells by upregulating the epigenetically silenced tumor suppressor RASSF1A gene.

Methylation status of PCDH10 and RASSF1A gene promoters in colorectal cancer

To investigate the methylation status of promoters of protocadherin-10 (PCDH10) and Ras-association domain family 1A (RASSF1A) genes in colorectal cancer (CRC), and to study its relationship with development and progress of CRC. Tumor tissues were collected from 75 CRC patients who received surgical treatment in the Affiliated Hospital of Guizhou Medical University in the period from 2007 to 2010. Methylation-specific polymerase chain reaction (MSP) was applied to detect the methylation status of PCDH10 and RASSF1A gene promoters in CRC and adjacent normal colorectal mucosa. The relationship between methylation of PCDH10 and RASSF1A and clinicopathological features of CRC was analyzed using chi-squared test. The rate of PCDH10 methylation in CRC tissue was significantly higher than that in colorectal normal mucosa (58.7%(44/75)vs 22.7%(17/75), P<0.01). There was no significantly correlation between methylation of PCDH10 and patients' age, gender, tumor site, Dukes stage, and lymph node metastasis(all P>0.05). The rate of RASSF1A methylation in CRC tissue was significantly higher than that in colorectal normal mucosa (64.6%(42/65)vs 15.4%(10/65), P<0.01). There was no significantly correlation between methylation of RASSF1A and patients' age, gender, and tumor site (all P>0.05), but the patients in high Dukes stages and with lymph node metastasis had higher RASSF1A methylation rate(92.9%(26/28)vs 43.2%(16/37), 92.9%(26/28)vs 43.2%(16/37), both P<0.05). CRC tissues demonstrate high level of methylation of PCDH10 and RASSF1A genes, which may play a crucial role in the pathogenesis of CRC. The aberrant hypermethylation of RASSF1A gene is observed in more advanced CRC, suggesting that the RASSF1A gene methylation may be related to progression of CRC.

Meta-analysis of the Association between RASSF1A Gene Promoter Methylation and Non-small Cell Lung Cancer

背景与目的肿瘤发生、发展过程中，抑癌基因启动子区域CpG岛异常甲基化起着重要的作用。已有研究显示RAS相关区域家族1A(Ras association domain family 1A, RASSF1A)基因，作为一个抑癌基因其启动子区域甲基化与非小细胞肺癌(non-small cell lung cancer, NSCLC)的发生发展密切相关。在NSCLC患者癌组织中RASSF1A基因启动子往往表现为异常高甲基化。本研究采用meta分析的方法探讨RASSF1A基因启动子甲基化与NSCLC发生之间的关系。方法通过检索Medline、EMBASE、CNKI和万方数据库，按照已拟定的纳入与剔除标准筛选收集公开发表的关于RASSF1A基因启动子甲基化与NSCLC相关性的研究。以比值比(odds ratio, OR)和95%置信区间(confidence interval, CI)为效应指标，分析RASSF1A基因启动子甲基化与NSCLC的关系。结果共有23篇文献纳入本研究，RASSF1A基因启动子甲基化率在NSCLC患者肺部组织和对照组中分别为41.50%(95%CI: 34%-49%)和5.58%(95%CI: 2%-9%)，meta分析显示肿瘤组织中的甲基化率高于对照组(OR=8.72, 95%CI:4.88-15.58, P < 0.05)；亚组分析显示:肿瘤组织中的甲基化频率高于血浆(OR=10.99, 95%CI:2.48-48.68)和正常对照组织(OR=8.74, 95%CI: 4.39-17.41)。结论NSCLC患者肺癌组织中RASSF1A基因启动子甲基化率高于对照组，组织中RASSF1A基因启动子甲基化率对肺癌的发生更具影响，RASSSF1A甲基化可能与肺癌的发生存在相关并可作为肺癌诊断的潜在标志物。

EVALUATION OF HYPERMETHYLATION OF RAS ASSOCIATION DOMAIN FAMILY-1 A AND GLUTATHIONE S-TRANSFERASE PROTEIN-1 GENES AS DIAGNOSTIC MARKER FOR HEPATOCELLULAR CARCINOMA

The molecular pathogenesis of HCC involves well-defined genetic and epigenetic alterations. The Ras association domain family 1A (RASSF1A) and the Glutathione S-transferase P 1 (GSTP1) genes are two tumour suppressor genes that are reported to be silenced by CpG island promoter hypermethylation which is a key to the tumourigenic process in HCC. The aim of this study was to analyze the methylation frequency of RASSF1A and GSTP1 genes in early stages of HCC , chronic hepatitis C and healthy subjects to evaluate its value as a diagnostic marker for early HCC. Patients and methods: Methylation-specific polymerase chain reaction (MSP) was used to detect RASSF1A and GSTP1 promotor methylation in DNA extracted from plasma samples of 25 patients with HCC, 25 patients with chronic hepatitis C and 25 healthy controls. Assessment of alpha fetoprotein (AFP) was performed in all groups by ELISA using commercially available kits. Results: Methylated RASSF1A was detected in 76 % of the HCC group (19/25), in 20% of the chronic hepatitis C patients (5/25) and in 16% of the healthy controls(4/25). The methylation frequencies were significantly higher in patients with HCC compared to the controls (P ≤ 0.001) and chronic hepatitis C patients ( P ≤ 0.001). While methylated GSTP1 was detected in 44% of the HCC group(11/25) ,in 12% of the chronic hepatitis C group (3/25) and in 8% of the controls (2/25). Although the sensitivity and specificity, for each gene as an epigenetic biomarker was moderate (76% and 44% for RASSF1A and GSTP1 respectively), the combination analysis of both genes resulted in an increased sensitivity and specificity to 88%,and 76% respectively) in discriminating HCC from normal control and chronic hepatitis C pateints. As regard AFP, Receiver operating characteristic curves were plotted and showed an optimal cutoff value of 9.5 ng/ml with sensitivity of 88 % and specificity of 58% when the area under the receiver operator characteristic (AUROC) curve was 0.87 with 95% Confidence Interval .Conclusion: The epigenetic changes observed in this study indicate that examination of methylation status of RASSF1A and GSTP1 could be of value for early diagnosis of HCC especially when using a combination of more than one epigenetic marker

Regulation of demethylation and re-expression of RASSF1A gene in hepatocellular carcinoma cell lines treated with NCTD in vitro.

Hepatocellular carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. Norcantharidin (NCTD), the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of cancer. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells. Human HepG2 cell lines were treated with NCTD at different concentrations (2.50, 5.00, 10.00, 20.00, 40.00 μg/mL) for 24 hours. Cell proliferation was evaluated by measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The methylation levels of RASSF1A (Ras-association domain family 1 A) in HepG2 cells were detected by methylation-specific PCR (MSP). The mRNA levels of RASSF1A in HepG2 cells were detected by real-time fluorescent quantitative PCR (RT-PCR). The levels of RASSF1A protein expression of HepG2 cells were detected by Western blotting assay. The inhibition of cell proliferation was observed when treated with NCTD at concentrations (2.5 μg/mL), and as concentration increased, the proliferation of HepG2 cells was markedly inhibited by NCTD in dose-dependent manners. The levels of methylation of RASSF1A decreased at the increasing concentration of 10, 20 and 40 μg/mL. The levels of RASSF1A mRNA and protein were decreased when treated with NCTD at the concentrations of 10, 20 and 40 μg/mL, which were also in a dose-dependent manner. NCTD can reverse the methylation state of RASSF1A gene and induce its re-expression, which will provide the theoretical basis for the clinical practice.

Cell proliferation and invasion in microRNA-143 and microRNA-29a transfected bladder cancer cells and its relationship of ras association domain family 1A methylation

Objective To evaluate the relationship among bladder cancer cell proliferation and invasion,microRNA-143 and 29a,and ras association domain family 1A (RASSF1A) methylation.Methods MiR-143 and miR-29a mimics were transfected to T24 cell lines.Transfection efficiency was confirmed by reverse transcription polymerase chain reaction (RT-PCR).All cell lines were divided into four groups:WT group (wild T24 cell lines without any treatment),NC group (transfected by negative control mimics),M1 group (transfected by miR-143 mimics) and M2 group (transfected by miR-29a mimics).Apoptosis rates,cell cycle,cell invasive inhibitor rates,RASSF1 A gene expression and methylation were detected.Results Transfection was successful confirmed by RT-PCR.Apoptosis rates of WT,NC,M1 and M2 group were 2.5％,3.4％,12.4％ and 13.3％ respectively.S-phase cells in M1 and M2 group increased significantly.Cell invasive inhibitor rates of NC,M1 and M2 group were 2.0％,16.5％ and 13.2％ respectively which showed the rates in M1 and M2 group were higher than NC group (P ＜0.05).RASSF1A gene expression was upgraded in M2 group and no changes were found in M1 group.What's more,MSP results showed that RASSF1 A gene was partial methylated in M2 group while it was completely methylated in other groups.Conclusion T24 bladder cancer cell proliferation and invasion decreased after miR-143 and miR-29a transfected.miR-29a transfection may inhibit RASSF1 A gene methylation and lead to gene expression uploaded. Key words: MicroRNA; Bladder cancer; Ras association domain family 1A; Methylation; Transfection

Detection efficiency of fetal epigenetic marker in maternal plasma

Objective To analyze the detection efficiency of non-sex dependent Ras association domain family 1A (RASSF1A) sequences in maternal plasma as a fetal epigenetic marker,and to explore the feasibility of replacing SRY gene in quantitative detection of fetal free DNA.Methods Maternal peripheral blood samples were collected in the first,second and third trimester from 50 normal pregnant women who had their prenatal care in the First and Fifth People's Hospital in Datong,Shanxi Province,between May 1,2011 and April 30,2012.Among them,pregnant women with single male fetus (n=28) were selected as study group,and another 20 normal healthy non-pregnant women in the same period were selected as control.The methylationsensitive restriction enzymes were used specifically to cut the maternal hypomethylated RASSF1A gene,the fetal hypermethylated sequences were determined by fluorescence quantitative polymerase chain reaction,and SRY sequences in maternal plasma were determined as a reference.Shapiro-wilk,Pearson or Spearman correlation analysis and Chi-square test were applied for statistical analysis.Results Among the 28 pregnant women with male fetus,the hypermethylated RASSF1A sequences in maternal plasma was tested 84 times and identified as early as at 8+2 weeks of gestation,giving the detection rate of 97.62％ (82/84).However,no RASSF1A or SRY gene was identified in control group.The mean concentrations of hypermethylated RASSF1A and SRY gene in the first,second and third trimester group were (60.19± 14.16) copies/ml vs (58.22± 15.13) copies/ml (r=0.933,P＜0.01),(99.18±28.44) copies/ml vs (95.43±32.33) copies/ml (r=0.931,P＜0.01)and (144.93±33.51) copies/ml vs (132.96±28.24) copies/ml (r=0.654,P＜0.01),respectively.The mean concentrations of hypermethylated RASSF1A and SRY gene showed positive correlation with gestational weeks (r=0.933 and 0.896,P＜0.01,respectively).Conclusions By using hypermethylated RASSF1A gene as an epigenetic marker,the detected free fetal DNA results is highly consistent with SRY gene without gender influence.Thus,it is helpful to expand its clinical application of noninvasive prenatal diagnosis. Key words: Tumor suppressor proteins; Methylation; Epigenomics; Genes, SRY; Prenatal diagnosis; Polymerase chain reaction

Quantitative detection of APC/RASSF1A promoter methylation in the plasma of patients with cervical diseases

To quantitatively detect adenomatous polyposis coli(APC) and Ras association domain family 1A( RASSF1A) promoter methylation levels in the plasma of patients with cervical disease and to determine the diagnostic value of the indicators of cervical disease. Preoperative blood samples were collected from 25 cases of healthy women and 118 cases of cervical disease, and tissue samples were also collected from 31 cases of them. The APC/RASSF1A promoter methylation levels of plasma and tissue were determined by duplex real-time quantitative methylation specific PCR (qMSP). Among 31 paired plasma and tissue samples, true negative rate of APC and RASSF1A genes were all 100%, and true positive rate of APC and RASSF1A genes were 3/5 and 7/9, respectively. In 143 cases of plasma samples, total positive rate of APC and (or) RASSF1A methylation was 3% (2/59) for control/low-grade lesions groups and 48% (40/84) for high-grade lesions/tumor groups (P < 0.01) . RASSF1A methylation rate was related to lymph node metastasis and International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.05). The plasma APC/RASSF1A methylation detection may be with some application prospect in the diagnosis of cervical diseases.

Association of RASSF1A genotype and haplotype with the progression of clear cell renal cell carcinoma in Japanese patients

What's known on the subject? and What does the study add? Ras association domain family 1A (RASSF1A) is a tumour suppressor and regulates cell cycle, apoptosis and microtubule stability. This is the first study to identify associations between RASSF1A polymorphisms and clinicopathological parameters and survival in patients with clear cell renal cell carcinoma (CCRCC). RASSF1A genotyping may be useful for predicting the prognosis of the clinical course of CCRCC, and this finding might provide a better understanding of the mechanism underlying the development and progression of CCRCC. However, functional and prospective studies with a larger number of patients are needed to confirm the results. To compare Ras association domain family 1A (RASSF1A) genotypes or haplotypes with clinicopathological characteristics and survival rates of patients with clear cell renal cell carcinoma (CCRCC). The study cohort comprised 224 Japanese patients who underwent radical nephrectomy and had CCRCC confirmed by histopathological analysis. • Three common polymorphisms in the RASSF1A gene, 133Ala/Ser (G/T), -710C/T and -392C/T, were genotyped using TaqMan assays and haplotypes were analysed using appropriate software. Patients with CCRCC with RASSF1A -710TT genotype exhibited a significantly higher tumour stage and higher stage grouping than those with -710CC or -710CT (P = 0.005 and P = 0.032, respectively). • There was no significant association between 133Ala/Ser or -392C/T genotype and clinicopathological characteristics. • RASSF1A 133Ala-710T-392T haplotype and -710TT genotype were significantly associated with poorer recurrence-free survival rates (P = 0.038 and P = 0.007, respectively). This is the first study to identify associations between RASSF1A polymorphisms and clinicopathological parameters and survival in patients with CCRCC. • RASSF1A genotyping may be useful in predicting the prognosis of the clinical course of CCRCC, and this finding might provide a better understanding of the mechanism underlying the development and progression of CCRCC. • Functional and prospective studies with a larger number of patients are needed to confirm the results.

Diagnosing Peripheral Lung Cancer: The Additional Value of the Ras-Association Domain Family 1A Gene Methylation and Kirsten Rat Sarcoma 2 Viral Oncogene Homolog Mutation Analyses in Washings in Nondiagnostic Bronchoscopy

The diagnostic yield of bronchoscopy in patients with endoscopically nonvisible (peripheral) tumors varies from 40% to 56%. Increasingly, molecular markers in bronchial washings are being investigated to improve the diagnostic yield. The aim of this study was to analyze the diagnostic value of the Ras association domain family 1A gene (RASSF1A) methylation analysis in washings in nondiagnostic bronchoscopy in the analysis of patients with suspected lung cancer who had peripheral tumors. Furthermore, the additional diagnostic value of Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations with RASSF1 methylation was analyzed. From a prospectively collected series, 129 patients with lung cancer and 28 control subjects were analyzed retrospectively regarding the methylation status of the promoter region of the RASSF1A gene by quantitative methylation-specific polymerase chain reaction and KRAS point mutations by using the sensitive Point-EXACCT method. A total of 40% of the lung cancer patients had peripheral tumors, and 17 patients had a nondiagnostic bronchoscopy. In these patients, RASSF1A methylation was detected in the washings of four patients (24%), and KRAS mutations were detected in the washings of two patients (12%). In total, 29% of the false-negative or doubtful cytology results were accompanied by RASSF1A methylation or KRAS mutation results that were highly suggestive of malignancy. The proportion of RASSF1A methylation was significantly higher in central and larger tumors. No relevant RASSF1A methylation was detected in control samples. Our data suggest that the molecular analysis of two biomarkers in nondiagnostic bronchial washings may better guide diagnostic procedures in patients with suspected lung cancer.

Quantitative detection of the hypermethylated RASSF1A gene in maternal plasma of pre-eclampsia

To investigate the level of hypermethylated ras association domain family 1A (RASSF1A) gene in maternal plasma of pre-eclampsia and its clinical value. Sixty pre-eclampsia women including 30 mild and 30 severe cases were selected, 60 women with normal pregnancy were studied as control. Free DNA from plasma samples was extracted, fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the concentrations of RASSF1A gene before and after methylation-sensitive restriction digestion. Meanwhile, beta-actin gene was detected as a control to confirm complete enzyme digestion. The median concentration of hypermethylated RASSF1A gene was 3.31-fold higher in samples from pre-eclamptic pregnancies than that in controls. There was significant difference between the mild and severe pre-eclamptic subjects (P<0.05), with the median concentrations of 1659 copies/mL and 2036.50 copies/mL, respectively. Hypermethylated RASSF1A gene in pre-eclampsia plasma was significantly increased and the concentrations were related to the severity of pre-eclampsia.

Genetic Polymorphisms of MTHFR and Aberrant Promoter Hypermethylation of the RASSF1A Gene in Bladder Cancer Risk in a Chinese Population

Epidemiological studies have shown that folate deficiency increases the risk of cancer by affecting DNA repair and methylation. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. In this study, it was hypothesized that MTHFR (C677T and A1298C) polymorphisms would be associated with bladder cancer and also with hypermethylation of the promoter of the Ras association domain family 1A (RASSF1A) gene. This hospital-based, case-control study of 312 bladder cancer patients and 325 cancer-free controls found that individuals carrying the MTHFR 677TT genotype had a 2.00-fold increased risk of bladder cancer compared with those carrying the 677CC genotype. None of the MTHFR A1298C polymorphisms alone were associated with bladder cancer, but the combined haplotype 677TT/1298AA was associated with a 2.27-fold increased risk compared with haplotype 677CC/1298AA. There was no association between MTHFR gene variants and methylation status of the RASSF1A gene in the 45 bladder cancer patients in whom this was studied. It is concluded that the MTHFR 677TT genotype and the TTAA haplotype may increase the risk of bladder cancer.

Inactivation of RASSF1A, RARβ2 and DAP-kinase by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma

Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor β2 (RARβ2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and nine normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RARβ2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RARβ2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RARβ2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RARβ2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis.To assess the functional significance of the epigenetic silencing of RARβ2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RARβ2 through aberrant methylation (p = 0.003) in NPC biopsies.We concluded that the inactivation of RASSF1A, RARβ2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.

Aberrant CpG Island Hypermethylation of RASSF1A in Gastric Cardia Adenocarcinoma

Ras-association domain family 1A (RASSF1A) gene, a candidate tumor suppressor gene, is inactivated in several human tumors, usually by hypermethylation of its promoter region. RASSF1A induces cell cycle arrest through inhibition of cyclin D1 accumulation. In this work, the promoter methylation status of the RASSF1A in 92 gastric cardia adenocarcinoma (GCA) and corresponding normal tissues were investigated using Methylation-specific PCR (MSP) approach, immunohistochemistry method and RT-PCR were used respectively to examine the protein expression and mRNA expression of RASSF1A in tumors and corresponding normal tissues. Cyclin D1 expression was examined by immunohistochemistry. RASSF1A was methylated in 54/92 (58.7%) tumor specimens, which was significantly higher than that in corresponding normal tissues (p <.001). Methylation frequencies of stage III and IV tumor tissues were significantly higher than that in stage I and II tumor tissues (p <.05). By immunostaining, 43/92 (46.7%) tumor tissues demonstrated heterogeneous, positive immunostaining of tumor tissues was significantly reduced with comparison to matched normal tissues (p <.001). mRNA expressions of RASSF1A in GCA tumor tissues were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2376 ± 0.2315 vs 0.6874 ± 0.2668, p <.001). RASSF1A mRNA expression in methylation group of GCA was significantly different from that in unmethylation group (p <.001). Cyclin D1 hyper-expression was found in 72/92 (78.3%) cases and correlated with RASSF1A methylation (p <.05). Our data suggested that epigenetic silencing of RASSF1A gene expression by promoter hypermethylation may play an important role in GCA.