Application value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma / 肿瘤研究与临床

Objective:To investigate the value of SHOX2 and RASSF1A gene promoter region methylation detection for screening and diagnosis of early-stage lung adenocarcinoma.Methods:The mRNA sequencing data of 471 lung adenocarcinoma patients and corresponding methylation data of 413 cases were downloaded from The Cancer Genome Atlas (TCGA) database, the methylation levels of SHOX2 and RASSF1A gene promoter regions were calculated, and the difference in methy lation level between normal lung tissues and tumor tissues was analyzed. The clinical data of 54 patients with early-stage lung adenocarcinoma and 31 patients with benign lung tumors who underwent surgery at Drum Tower Hospital Affiliated to Nanjing University Medical School from January 2018 to January 2019 were retrospectively analyzed. The methylation status of SHOX2 and RASSF1A in tumor tissues and normal lung tissues (>5 cm from the edge of the tumor foci) (called clinical samples) was detect, and a positive methylation in the promoter region of either gene was considered as a combination of two genes methylation positivity. Using pathological diagnosis as the gold standard, the efficacy of gene methylation positivity in diagnosing early-stage lung adenocarcinoma was analyzed by receiver operating characteristic (ROC) curve. Patients with >80% of tumor cells in paraffin samples were screened, and mRNA high-throughput sequencing was performed in their tumor tissues and normal lung tissues. The relationship between positive methylation of the two genes and clinicopathological features was analyzed, and the correlation between the promoter region methylation level of the two genes and mRNA expression levels in clinical samples and TCGA database samples was analyzed by Spearman method. Gene set variance analysis (GSVA) method was used to analyze the differences in Kyoto Encyclopedia of Genes and Genomes enrichment pathways between two-gene methylation-positive clinical lung adenocarcinoma samples and corresponding methylation-negative lung adenocarcinoma.Results:In TCGA database, the SHOX2 promoter region methylation island contained 6 sequenced methylation sites, of which sites cg04532033 and cg01557547 methylation levels were higher in lung adenocarcinoma tissues than in normal lung tissues (both P < 0.05); the RASSF1A gene promoter region methylation island contained 11 sequenced methylation sites, and the methylation levels of 6 of these sites in lung adenocarcinoma tissues were higher than those in normal lung tissues (all P < 0.05). Compared with normal lung tissues, the methylation level of SHOX2 promoter region was higher in stage Ⅰ and Ⅱ lung adenocarcinoma tissues (both P < 0.05); the methylation level of RASSF1A promoter region was higher in all stages of lung adenocarcinoma ( P < 0.001). Among 54 patients with early-stage lung adenocarcinoma, 28 were positive for SHOX2 promoter region methylation in tumor tissues, 21 were positive for RASSF1A promoter region methylation, and 40 were positive for combined methylation of both genes; 31 benign lung nodules were negative for SHOX2 and RASSF1A methylation. ROC curve analysis showed that the sensitivity of positive SHOX2 promoter region methylation for diagnosing early-stage lung adenocarcinoma was higher than that of RASSF1A promoter region methylation positivity (51.8% vs. 38.9%), and the area under the curve (AUC) for diagnosis by two-gene methylation positivity was larger than that for diagnosis by SHOX2 or RASSF1A gene methylation positivity alone (0.870 vs. 0.759 and 0.694). The circulating thresholds (Ct) of SHOX2 and RASSF1A methylation tested by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) in stage Ⅰ and Ⅱ lung adenocarcinoma were lower than those in normal lung tissues (all P < 0.05); patients with two-gene methylation positivity were characterized by older age, longer tumor longest diameter and more advanced pathological stage compared with patients with two-gene methylation negativity (all P < 0.05). In clinical stage Ⅰ-Ⅱ lung adenocarcinoma samples, the Ct of SHOX2 and RASSF1A promoter region methylation tested by qRT-PCR was negatively correlated with their mRNA relative expression levels ( r=-0.43, P = 0.003; r = -0.48, P = 0.001); in TCGA database stage Ⅰ-Ⅱ lung adenocarcinoma samples, the level of SHOX2 promoter region methylation was negatively correlated with its mRNA relative expression level ( r = -0.23, P < 0.001), and the level of RASSF1A promoter region methylation was also negatively correlated with its mRNA relative expression level, but without statistical difference ( r = -0.05, P = 0.310). In two-gene promoter methylation-positive lung adenocarcinoma samples, the pathways related to folate metabolism and DNA stability were upregulated, and the pathways related to vasoconstriction and cell growth and differentiation were downregulated. Conclusions:The combined detection of SHOX2 and RASSF1A promoter region methylation can be used as an indicator for screening and diagnosis of early-stage lung adenocarcinoma. Abnormal promoter region methylation of the two genes may affect multiple tumor-related pathways and promote the occurrence and progression of early-stage lung adenocarcinoma.

The Clinical Significance Of Rassf1a And Cdh1 Hypermethylation In Breast Cancer Patients

Introduction:Aberrant DNAmethylation patterns in serum DNAmight be used as a biomarker for the early diagnosis and management of cancer patients. The aim of present study was to evaluate DNAmethylation of RASSF1Aand CDH1 in circulating cell free DNA(cfDNA) from serum and paired tissue DNAsamples of breast cancer patients. Material and methods: Methylation-specific PCR was used to assess the methylation status of the two genes in serum and paired tissue sample DNAof 50 breast cancer patients. Biochemical parameters were assessed using an electrochemiluminescence analyzer. Results: Significant correlation found between methylation status of RASSF1A and CDH1 in serum and paired tissue samples of patients. Among clinicopathological findings, CDH1 methylation showed significant association with advance staging and tumor and methylation of RASSF1A exhibited significant association with progesterone receptor and estrogen receptor status in both serum and paired tissue. Vitamins levels were significantly high in cases compared to control group. High folic acid levels were significantly associated with the RASSF1Amethylation. Conclusions: These findings suggest that methylation of cfDNAmay be important in the early detection of breast cancer.

Relationship between the overexpression of DNA methyltransferase 3B4 and clear cell renal cell carcinoma / 中国肿瘤临床

Objective:To determine the molecular role of DNA methyltransferase (DNMT) in kidney tumorigenesis. Methods:Tissue samples consisted of 15 cancer tissues and 15 matched adjacent tissues from clear cell renal cell carcinoma (ccRCC) patients who had undergone nephrectomy in 2012 at the First Affiliated Hospital of Baotou Medical College, Science and Technology University of Inner Mongolia were collected. Real-time PCR, Western blot, combined bisulfite restriction analysis (COBRA) and methylation specific PCR were used in this study. Real-time PCR was used to examine the mRNA expression levels of DNMT. The global methylation level, DNA methylation level, and the expression of the antioncogene RASSF1A in ccRCC tissues were concurrently detected. Results:Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were elevated in renal cell carcinoma tissue compared with those in con-troll tissue. Additionally, Alu was hypomethylated in ccRCC tissue (0.106±0.04) compared with control tissue (0.115±0.03) (P<0.05). Fur-thermore, the methylation of the promoter for RASSF1A, a tumor-suppressor gene, moderately increased in renal cell carcinoma tis-sue. By contrast, RASF1A expression decreased. Conclusion:DNMT3B4 overexpression may play an important role in human kidney tu-morigenesis via chromosomal instability and the decreased expression of RASSF1A.

Methylation status of RASSF1A gene promoter in upper tract urothelial carcinoma and its clinical significance / 北京大学学报(医学版)

Objective:To investigate the methylation status of the RASSF1A gene promoter in upper tract urothelial carcinoma (UTUC)tissues and its correlation with clinicopathologic characteristics and postoperative recurrence of primary UTUC.Methods:In a retrospective design,a total of 687 patients who underwent surgeries for primary UTUC in the urology department of Peking University First Hospital were enrolled.The methylation status of the RASSF1A gene promoter was analyzed using methylation-sen-sitive polymerase chain reaction on tumor specimens.Results:Aberrant methylation for the RASSF1A gene promoter was detected in 183 (26.6%) DNA samples in total.Aberrant methylation of the RASSF1A gene was strongly associated with tobacco consumption (P =0.044),ipsilateral hydronephrosis (P <0.001 ),tumor location (P <0.001 ),tumor stage (P =0.001 ),tumor grade (P =0.007), lymph node metastasis (P =0.001 )and growth pattern (P =0.013).The methylated RASSF1A gene promoter was an independent risk factor for bladder recurrence (P <0.001,HR =0.471)and contrala-teral recurrence (P =0.030,HR =0.269)of UTUC after surgery.Hypermethylated RASSF1A was pre-dictive for improved bladder recurrence-free survival (BRFS)(P <0.001)and contralateral recurrence-free survival (CRFS)(P =0.021)in the UTUC patients.Compared with the patients with unmethylated RASSF1A,the patients containing tumors with hypermethylated RASSF1A had tendency toward longer re-currence-free survival time [(114.4 ±3.9)months vs.(84.0 ±3.2)months for BRFS,(138.1 ±1.8) months vs.(132.9 ±1.9)months for CRFS]and higher estimated cumulative recurrence-free survive rates (five-year survival rate for example,79.8% ±3.4% vs.57.4% ±2.6% for BRFS,98.9% ± 0.8% vs.93.0% ±1.4% for CRFS).Additionally,tumor multifocality (P =0.002,HR =1.538), and ureteroscopy before surgery (P =0.001,HR =1.725)were independent risk factors for bladder re-currence in postoperative UTUC patients.Conclusion:The methylation status of the RASSF1A gene pro-moter appears to be a promising epigenomic biomarker for assessing the aggressiveness of UTUC and a predictor predicting the urinary tract recurrence after surgery.

Noninvasive fetal RHD genotyping using cell-free fetal DNA incorporating fetal RASSF1A marker in RhD-negative pregnant women in Korea

PURPOSE: Conventional methods for the prenatal detection of fetal RhD status involve invasive procedures such as fetal blood sampling and amniocentesis. The identification of cell-free fetal DNA (cffDNA) in maternal plasma creates the possibility of determining fetal RhD status by analyzing maternal plasma DNA. However, some technical problems still exist, especially the lack of a positive control marker for the presence of fetal DNA. Therefore, we assessed the feasibility and accuracy of fetal RHD genotyping incorporating the RASSF1A epigenetic fetal DNA marker from cffDNA in the maternal plasma of RhD-negative pregnant women in Korea. MATERIALS AND METHODS: We analyzed maternal plasma from 41 pregnant women identified as RhD-negative by serological testing. Multiplex real-time PCR was performed by amplifying RHD exons 5 and 7 and the SRY gene, with RASSF1A being used as a gender-independent fetal epigenetic marker. The results were compared with those obtained by postnatal serological analysis of cord blood and gender identification. RESULTS: Among the 41 fetuses, 37 were RhD-positive and 4 were RhD-negative according to the serological analysis of cord blood. There was 100% concordance between fetal RHD genotyping and serological cord blood results. Detection of the RASSF1A gene verified the presence of cffDNA, and the fetal SRY status was correctly detected in all 41 cases. CONCLUSION: Noninvasive fetal RHD genotyping with cffDNA incorporating RASSF1A is a feasible, reliable, and accurate method of determining fetal RhD status. It is an alternative to amniocentesis for the management of RhD-negative women and reduces the need for unnecessary RhIG prophylaxis.

Association between the single nucleotide polymorphism of RASSF1A Ala133Ser and colorectal cancer in north Chinese Han population / 解放军医学杂志

Objective To evaluate the association between RASSF1A Ala133Ser single nucleotide polymorphism (SNP) and colorectal cancer (CRC) in patients of Han population in north China. Methods RASSF1A SNP was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 109 colon cancer (CC) patients, 67 rectal cancer (RC) patients and 189 age and sex matched healthy people (as controls). Unconditional logistic regression was used to analyze the gene-disease association and gene-sex interaction. Results The RASSF1A Ala133Ser SNP in CRC patients had no significant difference with that in normal control samples, but the interaction of genotype and sex existed in rectal cancer, Ala/Ser +Ser/Ser genotypes of RASSF1A gene increased the risk of rectal cancer in women (OR=3.78, 95%CI 1.31-10.89). Conclusion The RASSF1A 133Ser allele is associated with rectal cancer in women, but not colon cancer.

RASSF1A and RASSF1A methylation′s relationship with breast cancer / 实用肿瘤学杂志

Breast cancer is one of the most common malignant tumors in women.Early diagnosis plays a very important role in the treatment of breast cancer.Recently,with the development of epigenetics,it gives us a new orientation for early detection for cancers.The most understood mechanism of epigenetics is DNA methyla-tion.Ras associated domain family 1A gene(RASSFlA)promoter methylation occurres in tumor′s early stage,and have a statistically concern with tumor′s TNM stage,invasiveness and outcomes.Moreover,DNA methylation can be reversed,so RASSF1A can be a promising candidate for early breast cancer detection biomarker,and can be a potential orientation for breast cancer treatment.

Prognostic value of RAS association domain family 1A gene methylation in patients with hepatocellular carcinoma after surgery / 中华消化杂志

Objective To evaluate the prognostic value of RAS association domain family 1A gene (RASSF1A) methylation in patients after hepatocellular carcinoma (HCC) hepatectomy.Methods A total of 260 patients with HCC who underwent hepatectomy were enrolled.HCC tissues and tumor adjacent tissues which were 2 cm away from the tumor edge of the patients were obtained.The clinicopathological data of patients were collected.The methylation of RASSF1A in HCC tissues and corresponding tumor adjacent tissues was determined by methylation specific polymerase chain reaction (PCR).The correlation between the expression rate of RASSF1A methylation and clinicopathological characteristics was analyzed by chi-square test.Log-rank test was performed to analyze the relation between RASSF1A methylation and overall survival rate.Univariate and multivariate Cox statistical techniques were used to identify the influence factors in the prognosis of HCC.Results Among 260 HCC tissues and corresponding tumor adjacent tissues,RASSF1A promoter hypermethylation was detected in 214 HCC tissues (82.3 ％) and 101 corresponding tumor adjacent tissues (38.8％),and the difference was statistically significant (x2 =102.824,P ＜ 0.01).There was no correlation between RASSF1A methylation and age,gender,liver cirrhosis,α-fetoprotein level,maximum diameter of tumor,Barcelona Clinic Liver Cancer (BCLC) stage,hepatitis B virus (HBV) infection,smoking and alcohol drinking (all P＞0.05).The 5-year overall survival rate of patients with negative RASSF1A methylation was 93％,while that of patients with positive RASSF1A methylation was 51 ％,and the difference in overall survival rate between the two groups was statistically significant (x2 =26.556,P ＜ 0.01).Univariate and multivariate Cox regression analysis indicated that liver cirrhosis,BCLC stage and RASSF1A methylation were the main influence factors in the death of patients with HCC after surgery (Wald=16.767,8.791,16.286; all P＜0.01).Conclusion RASSF1A methylation is not only one of the predictive factors of survival rate in patients with HCC after hepatectomy,but also an independent prognostic factor of HCC.

ImmunohistochemicaI Expression and CIinicaI ImpIication of RASSF1A Gene in LaryngeaI Squamous CeII Carcinoma and Keratosis of the Larynx / 听力学及言语疾病杂志

Objective To observe the RASSF1A expression in laryngeal keratosis and laryngeal squamous cell carcinoma and investigate the relationship between the gene and the occurrence and development of this disease. Methods The specimens of keratosis of larynx(43 cases)and laryngeal squamous cell carcinoma(31 cases)in Oto-laryngology-Head and Neck surgery of Anhui provincial hospital from Dec 2009 to Dec 2012 were collected.Nor-mal vocal fold mucosa from the cadaveric head as control group(8 cases)were collected.Immunohistochemical meth-ods were used to detect the expression of RASSF1A protein in these tissues.The diffuse distribution of brown gran-ules in the cell cytoplasm yielded positive results and the nucleus was not coloring.The percentage of positive cells of every section,and the average standard deviation(￣x±s)were calculated.SPSS17.0 statistical analysis software was used to analyze the data,P0.05).RASSF1A expression in the clinical stage from I to IV of laryngeal squamous cell carcinoma gradually de-clined.During the clinical stage,the difference had statistics significance (P0.05).ConcIusion The reduction of RASSF1A expression correlated with laryn-geal squamous cell carcinoma,which has certain significance for the different ion of carcinoma and laryngeal precan-cerous lesion.

Effects of 5-aza-2'-deoxycitydine on expression of RASSF1A gene in human ovarian cancer cell line / 国际肿瘤学杂志

Objective To investigate the effect of 5-aza-2'deoxycytidine (5-Aza-CdR) on proliferation and expression of RASSF1 A gene in human ovarian cancer cell line SKOV3 and 3AO.Methods SKOV3 and 3AO cells were treated with different concentrations (0.5,5,50 μmol/L) of DNA methyltransferase inhibitor 5-Aza-CdR.RT-PCR and Western Blot were adopted to detect expression of mRNA and protein of RASSF1A gene before and after treatment with 5-Aza-CdR respectively.Results Compared with control group,when the 5-Aza-CdR concentration was 0.5,5,50 μmol/L after drug treatment,human ovarian cancer cells could significantly inhibit tumor cell growth; SKOV3 and 3AO cells in control group were observed weaker expression of RASSF1A mRNA.After treated with 5-Aza-CdR,the expressions of RASSF1A mRNA were observed increased with the increase of the drug concentration.After treated with different concentration of 5-Aza-CdR,the expressions of RASSF1A mRNA treated with 0.5 μmol/L 5-Aza-cdR was lower than those treated with 5 and 50 μmol/L 5-Aza-cdR (t =-8.866,P =0.01 ; t =-12.256,P =0.000).However,expressions of RASSF1A mRNA treated with 5 and 50 μmol/L 5-Aza-cdR respectively showed no statistical significance (t =0.431,P =0.689).Expressions of RASSF1A protein treated with 0.5 μmol/L 5-Aza-cdR and 5 μmol/L 5-Aza-cdR didn't show statistically significant (t =-1.586,P =0.188).Conclusion Expressions of RASSF1A mRNA and protein in SKOV3 and 3AO cells were evidently enhanced.As one kind of methyltransferases inhibitors,5-Aza-CdR can inhibit ovarian cancer cell line SKOV3,3AO growth through the RASSF1A promoter methylation,and thus promote their apoptosis.

RASSF1A Suppresses Cell Migration through Inactivation of HDAC6 and Increase of Acetylated alpha-Tubulin / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The RAS association domain family protein 1 (RASSF1) has been implicated in a tumor-suppressive function through the induction of acetylated alpha-tubulin and modulation of cell migration. However, the mechanisms of how RASSF1A is associated with acetylation of alpha-tubulin for controlling cell migration have not yet been elucidated. In this study, we found that RASSF1A regulated cell migration through the regulation of histon deacetylase 6 (HDAC6), which functions as a tubulin deacetylase. MATERIALS AND METHODS: The cell migration was assessed using wound-healing and transwell assays. The role of RASSF1A on cell migration was examined by immunofluorescence staining, HDAC activity assay and western blot analysis. RESULTS: Cell migration was inhibited and cell morphology was changed in RASSF1A-transfected H1299 cells, compared with controls, whereas HDAC6 protein expression was not changed by RASSF1A transfection in these cells. However, RASSF1A inhibited deacetylating activity of HDAC6 protein and induced acetylated alpha-tubulin expression. Furthermore, acetylated alpha-tubulin and HDAC6 protein were co-localized in the cytoplasm in RASSF1A-transfected H1299 cells. Conversely, when the endogenous RASSF1A expression in HeLa cells was blocked with RASSF1A siRNA treatment, acetylated alpha-tubulin was co-localized with HDAC6 protein throughout the whole cells, including the nucleus, compared with scramble siRNA-treated HeLa cells. The restoration of RASSF1A by 5-Aza-dC treatment also induced acetylated alpha-tubulin through inhibition of HDAC6 activity that finally resulted in suppressing cell migration in H1299 cells. To further confirm the role of HDAC6 in RASSF1A-mediated cell migration, the HDAC6 expression in H1299 cells was suppressed by using HDAC6 siRNA, and cell motility was found to be decreased through enhanced acetylated alpha-tubulin. CONCLUSION: The results of this study suggest that the inactivation of HDAC6 by RASSF1A regulates cell migration through increased acetylated alpha-tubulin protein.

Study on methylation status of RASSFIA gene promoter and exon 1 in cervical cancer cell lines / 中国癌症杂志

Background and purpose:Loss or altered expression of Ras association domain family 1A gene (RASSF1A) through DNA methylation has been associated with the pathogenesis of a variety of cancers, which suggests the tumor suppressor function of this gene. This study aimed to explore the effect of DNA methyltransferase inhibitor 5-Aza-2’deoxycytidine (5-Aza-dc) on demethylation and expression of RASSF1A in cervical cancer cell lines. Methods:HPV positive cervical cancer cell lines HeLa and Caski, HPV negative cell lines HT-3 and C-33A were treated with two different concentration of 5-Aza-dc (5 μmol/L, 10 μmol/L). MSP (methylation-specific PCR) and Bisulfite genomic sequencing PCR (BGS) combined with TA clone were used to investigate methylation status of RASSF1A gene promoter and exon 1 before and after treatment of 5-Aza-dc. RASSF1A gene mRNA expression was detected by RT-PCR. Results:Two HPV positive cell lines showed hypomethylated RASSF1A promoters and expressed RASSF1A mRNA, and after treatment with 5-Aza-dc, the mRNA expression of RASSF1A did not change significantly (FHeLa=3.003, P=0.125; FCaski=0.045, P=0.956). Two HPV negative cervical cancer cell lines showed hypermethylation status of RASSF1A promoter and silenced RASSF1A. After treatment with 5-Aza-dc, demethylation occurred in the promoter region of RASSF1A gene, which subsequently induced re-expression of this gene in HT-3 and C-33A. The F test (FHT-3=18.002, P=0.03;FC-33A=17.179, P=0.03) and LSD-t test (P<0.05) demonstrated that significant difference in the expression of RASSF1A was found upon two different concentrations drug treatment.Conclusion:The methylation status of promoter and exon 1 of RASSFIA gene in HPV positive and HPV negative cervical cancer cell lines are different. The promoter hypermethylation is correlated with RASSF1A gene expression in HPV negative cervical cancer cell line HT-3 and C-33A, and plays a key role in RASSF1A silencing. 5-Aza-dc may effectively reverse the methylation status of RASSFIA gene promoter in cervical cancer HT-3 and C-33A cells and reactivate gene expression silenced by aberrant hypermethylation in a dose-dependent manner within certain extent.

Methylation of E-cadherin, p16, RASSF1A, DAPK and MGMT in salivary gland tissues / 军事医学

Objective To investigate the methylation status of E-cadherin(E-cad), p16, RASSF1A, DAPK and MGMT in histologically normal salivary gland tissues and provide reference for determination of the methylation status of salivary gland tumors.Methods Methylation of E-cad, p16, RASSF1A,DAPK and MGMT was analyzed using methylation-specific polymerase chain reaction ( MSP) .The results were compared with the methylation status of these genes in salivary adenoid cystic carcinoma ( ACC) tumor tissues in our previous studies and the association between promoter methylation of E-cad, p16, RASSF1A, DAPK, and MGMT on one hand and the patients′gender, age, smoking and types of gland on the other hand was also analyzed .Results Promoter methylation was detected in 8 of the 60 (13%) salivary glands, E-cad in 4(7%), p16 in 2(4%), RASSF1A in 2(4%), DAPK in 2 (4%), and MGMT in 1(2%).Compared with our previous results, there was a significantly lower methylation ratio in promoter methylation of E-cad(P<0.01), p16 (P<0.01), RASSF1A (P<0.01),and DAPK (P<0.01) in salivary gland tissues than in ACC tumor tissues.Conclusion Promoter methylation of E-cad, p16 and RASSF1A is a rare event in histologically normal salivary gland tissues .

Promoter methylation of RASSF1A gene in hepatocellular carcinoma and its clinical significance / 中华普通外科杂志

Objective To explore the relationship between promoter methylation of RASSF1 A gene and clinico-pathological characteristics in hepatocellular carcinoma.Methods MS-PCR was used for analyzing the status of aberrant promoter methylation of RASSF1A in 100 primary HCC samples and adjacent noncancerous tissues,10 normal liver tissues,and six HCC cell lines.RT-PCR was used to assess reactivation of RASSF1A expression after HCC cell lines treated with demethylating agent 5'-aza-2' deoxycytideing.Results Abnormal promoter methylation of RASSF1A gene was found in 69(69％) cases of HCC,15 (15％) cases of adjacent normal tissues,and no abnormal promoter methylation of RASSF1A gene was found in normal liver tissues,and the difference was statistically significant (x2 =67.75,P ＜0.001).The methylation of RASSF1A gene was correlated to HBsAg (x2 =11.341,P ＜ 0.05) and histological differentiation(x2 =10.575,P ＜ 0.05).Four cell lines with abnormal CpG island methylation of RASSF1A gene were all re-expressed after treated with 5'-Aza-CdR.Conclusions RASSF1A gene promoter methylation was correlated to HBsAg and histological differentiation and this is one of the most important mechanism for low expression of RASSF1A in HCC.

Recrutamento do complexo repressivo polycomb 2 pelo RNA não codificador longo antissenso ANRASSF1 modula a expressão do gene RASSF1A e a proliferação celular / Recruitment of polycomb repressive complex 2 by intronic long noncoding RNA ANRASSF1 modulates RASSF1A expression and cell proliferation

O gene supressor tumoral RASSF1A tem sido associado com redução da proliferação celular em diversos tumores. Sua expressão é regulada por eventos epigenéticos que envolvem o complexo repressivo polycomb (PRC2), no entanto os mecanismos moleculares da modulação do recrutamento deste modificador epigenético para este locus ainda são desconhecidos. Neste trabalho identificamos e caracterizamos ANRASSF1, um RNA não codificador longo (lncRNA) intrônico unspliced, que é transcrito na fita oposta do gene RASSF1A, em várias linhagem celulares e tecidos, e se liga a PRC2. ANRASSF1 é transcrito pela RNAPII, possui cap-5´ e cauda poli-A, além de localizar-se no núcleo e possuir uma meia-vida em média quatro vezes menor comparada com outros lncRNAs ligados à PRC2. A super-expressão ectópica de ANRASSF1 reduziu os níveis de RASSF1A e aumentou a taxa de proliferação em células HeLa, enquanto seu silenciamento provocou efeito oposto. Essas mudanças nos níveis de ANRASSF1 não afetaram a abundância da isoforma RASSF1C em nenhuma das condições. A super-expressão de ANRASSF1 provocou um grande aumento tanto da ocupação de PRC2 como da marca de histona repressiva H3K27me3 especificamente na região promotora RASSF1A. Nenhum efeito da super-expressão de ANRASSF1 foi detectado na ocupação de PRC2 e na histona H3K27me3 nas regiões promotoras de RASSF1C e de outros quatro genes vizinhos, incluindo dois genes supressores tumorais bem caracterizados. Além disso, foi demonstrado que ANRASSF1 forma um híbrido de RNA/DNA e recruta SUZ12, um componente do PRC2, para o promotor de RASSF1A. Notavelmente, foi detectado pelo ensaio de RNase-ChIP que a degradação de ANRASSF1 diminui a ocupação de PRC2 neste promotor. Esses resultados demonstram um novo mecanismo de repressão epigenética do supressor tumoral RASSF1A, envolvendo um lncRNA unspliced antissenso, onde ANRASSF1 reprime seletivamente a expressão da isoforma de RASSF1 que sobrepõe o transcrito antissenso de modo local e específico...

Tumor-suppressor RASSF1A gene down-regulation has been implicated in increasing cell proliferation in several tumors. Its expression is regulated by epigenetic events involving polycomb repressive complex 2 (PRC2), however the molecular mechanisms modulating recruitment of this epigenetic modifier to the locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand of RASSF1 gene in several cell lines and tissues, and binds to PRC2. ANRASSF1 is transcribed by RNA Polymerase II, 5'-capped, polyadenylated, displays nuclear localization, and has on average a four-fold shorter half-life compared to other lncRNAs that bind PRC2. ANRASSF1 ectopic overexpression decreases RASSF1A abundance and increases the proliferation rate of HeLa cells, whereas its silencing causes opposite effects. These changes in NRASSF1 levels do not affect RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase both in PRC2 occupancy and in histone H3K27me3 repressive mark specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression is detected on PRC2 occupancy and on histone H3K27me3 at the promoter regions of RASSF1C and of four other neighbor genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrate that ANRASSF1 forms an RNA/DNA hybrid, and recruits SUZ12, a PRC2 component, to the RASSF1A promoter. Notably, depletion of ANRASSF1 disrupts SUZ12 occupancy on RASSF1A promoter as measured by RNAse-ChIP assay. Together, these results show a new mechanism of epigenetic repression of RASSF1A tumor suppressor gene involving an antisense unspliced lncRNA, in which ANRASSF1 selectively represses expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the...

Non-invasive Prenatal Diagnosis of Trisomy 21 by Dosage Ratio of Fetal Chromosome-specific Epigenetic Markers in Maternal Plasma / 华中科技大学学报（医学）（英德文版）

This study examined the methylation difference in AIRE and RASSF1A between maternal and placental DNA,and the implication of this difference in the identification of free fetal DNA in maternal plasma and in prenatal diagnosis of trisomy 21.Matemal plasma samples were collected from 388 singleton pregnancies,and placental or chorionic villus tissues from 112 of them.Methylation-specific PCR (MSP) and methylation-sensitive restriction enzyme digestion followed by fluorescent quantitative PCR (MSRE + PCR) were employed to detect the maternal-fetal methylation difference in AIRE and RASSF1A.Diagnosis of trisomy 21 was established according to the ratio of fetal-specific AIRE to RASSF1A in matemal plasma.Both methods confirmed that AIRE and RASSF1A were hypomethylated in maternal blood cells but hypermethylated in placental or chorionic villus tissues.Moreover,the differential methylation for each locus could be seen during the whole pregnant period.The positive rates of fetal AIRE and RASSF1A in maternal plasma were found to be 78.1％ and 82.1％ by MSP and 94.8％ and 96.9％ by MSRE + PCR.MSRE + PCR was superior to MSP in the identification of fetal-specific hypermethylated sequences (P＜0.05).Based on the data from 266 euploidy pregnancies,the 95％ reference interval of the fetal AIRE/RASSF1A ratio in maternal plasma was 0.33-1.77,which was taken as the reference value for determining the numbers of fetal chromosome 21 in 102 pregnancies.The accuracy rate in 98 euploidy pregnancies was 96.9％ (95/98).Three of the four trisomy 21 pregnancies were confirmed with this method.It was concluded that hypermethylated AIRE and,RASSF1A may serve as fetal-specific markers for the identification of fetal DNA in maternal plasma and may be used for noninvasive prenatal diagnosis of trisomy 21.

Noninvasive Prenatal Diagnosis using Cell-Free Fetal DNA in Maternal Plasma: Clinical Applications

Owing to the risk of fetal loss associated with prenatal diagnostic procedures (amniocentesis, chorionic villus sampling), noninvasive prenatal diagnosis (NIPD) is ultimate goal of prenatal diagnosis. The discovery of circulating cell-free fetal DNA (cffDNA) in maternal plasma in 1997 has opened up new probabilities for NIPD by Dr. Lo et al. The last decade has seen great development in NIPD. Fetal sex and fetal RhD status determination by cffDNA analysis is already in clinical use in certain countries. For routine use, this test is limited by the amount of cell-free maternal DNA in blood sample, the lack of universal fetal markers, and appropriate reference materials. To improve the accuracy of detection of fetal specific sequences in maternal plasma, internal positive controls to confirm to presence of fetal DNA should be analyzed. We have developed strategies for noninvasive determination of fetal gender, and fetal RhD genotyping using cffDNA in maternal plasma, using real-time quantitative polymerase chain reaction (RT-PCR) including RASSF1A epigenetic fetal DNA marker (gender-independent) as internal positive controls, which is to be first successful study of this kind in Korea. In our study, accurate detection of fetal gender through gestational age, and fetal RhD genotyping in RhD-negative pregnant women was achieved. In this assay, we show that the assay is sensitive, easy, fast, and reliable. These developments improve the reliability of the applications of circulating fetal DNA when used in clinical practice to manage sex-linked disorders (e.g., hemophilia, Duchenne muscular dystrophy), congenital adrenal hyperplasia (CAH), RhD incompatibility, and the other noninvasive pregnant diagnostic tests on the coming soon. The study was the first successful case in Korea using cffDNA in maternal plasma, which has created a new avenue for clinical applications of NIPD.

Polymorphism of exon G435T in RASSF 1A gene associated with the susceptibility of breast cancer / 肿瘤

Objective: To explore the relationship between the polymorphism of the third exon G435T in Ras association domain family 1A (RASSF 1A ) gene and the susceptibility of breast cancer. Methods: The polymorphism of G435T in RASSF 1A gene was determined in 212 female patients with breast cancer, 218 female patients with breast benign disease and 220 female healthy controls by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, and the correlation of RASSF 1A polymorphism with the susceptibility of breast cancer was evaluated. Results: The frequencies of GT+TT and T allele at G435T site in RASSF 1A gene were significantly higher in patients with breast cancer than those with benign breast disease and healthy controls. Conclusion: The polymorphism of the third exon G435T in RASSF 1A gene may be associated with the susceptibility of breast cancer. RASSF 1A 435T allele can be a potential predictive factor for the risk of breast cancer. Copyright© 2011 by the Editorial Board of Tumor.

Abnormal methylation of RASSF1A and BLU genes in renal carcinoma / 第二军医大学学报

Objective: To investigate the methylation status of the promoters of RASSF1A and BLU genes in the renal carcinoma tissues and to assess their roles in the tumorigenesis of renal carcinoma. Methods: Methylation specific PCR (MSP) method was used to examine the methylation status of the promoters of RASSF1A and BLU genes in the renal carcinoma tissues of 26 patients with renal carcinoma and their corresponding adjacent normal tissues; and the results were analyzed. Results: We found that 17 (65.4%) of the renal carcinoma tissues had abnormal methylation of RASSF1A gene, and there was no abnormal methylation of RASSF1A in the adjacent normal tissues. Eleven(42.3%) of 26 renal cancer tissues had hypermethylation of BLU gene, and there was no hypermethylation of BLU gene in the corresponding adjacent tissues. Conclusion: Hypermethylation of RASSF1A and BLU genes is present in the renal carcinoma tissues, suggesting the hypermethylation of RASSF1A and BLU might be associated with the tumorigenesis of renal carcinoma.

Effects of 5-aza-2′-deoxycitydine on proliferation of prostate cancer cell line PC3 and transcriptional regulation of tumor suppressor gene GSTP1 and RASSF1A / 第二军医大学学报

Objective: To observe the effects of 5-aza-2′-deoxycitydine (5-aza CdR) on the proliferation and transcription of tumor suppressor gene GSTP1 and RASSF1A in prostate cancer cell line PC3. Methods: The status of 5′CpG island methylation of RASSF1A and GSTP1 genes in PC3 was analyzed by methylation specific PCR (MSP) before treatment with 5-aza CdR. RASSF1A and GSTP1 mRNA were quantified by real time PCR during the demethylation process by 5-aza-CdR. MTT assay and flow cytometry were used to examine the proliferative activity of PC3 cells before and after 5-aza-CdR treatment. Results: The 5′ CpG island methylation of RASSF1A and GSTP1 genes were detected in human prostate cancer cell line PC3. Compared with control group, RASSF1A and GSTP1 mRNA expression had no significant change 24 h after culture with 5-aza-CdR; their expression was up-regulated 48 h after cultured with 5-aza-CdR, with significant difference found between 5 μmol/L and 10 μmol/L 5-aza-CdR groups. Compared with control group, the expression of RASSF1A and GSTP1 mRNA was significantly increased 72 h after cultured with all concentrations of 5-aza-CdR. MTT assay and cell cycle examination indicated that exposure to 5-aza-CdR for 24 h and 48 h resulted in no obvious growth inhibition and cell cycle change; exposure to 5-aza-CdR for 72 h induced significant growth inhibition (P<0.05) and cell cycle change (P<0.05); and cells were arrested at G0/ G1, phase. Conclusion: The 5′CpG island methylation of RASSF1A and GSTP1 genes is probably responsible for RASSF1A and GSTP1 silencing in PC3 cells. 5-aza-CdR can inhibit the proliferation of PC3 cells, disturb the cell cycle, and elevate transcription of GSTP1 and RASSF1A.