Relationship between methylation of Ras association domain family-1A and protein expression in ;endometriosis / 中国医师进修杂志

Objective To analyze the methylation status and protein expression of Ras association domain family- 1A (RASSF1A) in endometriosis (EMS). Methods The ectopic and corresponding eutopic endometrium tissues were collected from 45 women with EMS and normal endometrium tissues of 20 women without EMS. The methylation status of RASSF1A was examined by methylation specific PCR (MSP). Immunohistochemistry was performed to measure the level of RASSF1A in endometrium tissues. Results The RASSF1A protein expression rate in ectopic endometrium, eutopic endometrium, and normal endometrium was 37.78%(17/45), 60.00%(27/45) and 85.00%(17/20), and there was significant difference (χ2 = 13.136, P = 0.001). The frequency of aberrant methylation of RASSF1A was 55.56%(25/45), 33.33%(15/45) and 0 in ectopic endometrium , eutopic endometrium, and normal endometrium, and there was significant difference (χ2 =18.770, P = 0.000). The frequency of aberrant methylation of RASSF1A had no significant differnce throughout the menstrual cycle in ectopic endometrium and eutopic endometrium: 66.67%(14/21) vs. 45.83%(11/24), 38.10%(8/21) vs. 29.17%(7/24), P>0.05. In ectopic endometrium, the frequency of aberrant methylation of RASSF1A inⅢ-Ⅲstage was significantly higher than that in Ⅰ-Ⅱstage (χ2=5.940, P=0.015). In ectopic endometrium and eutopic endometrium, the RASSF1A protein expression had negative correlation with aberrant methylation of RASSF1A (r =- 0.594、- 0.577, P<0.01). Conclusions Epigenetic inactivation of RASSF1A through aberrant promoter methylation may be strongly correlated with the formation and progression of EMS, and assessment of RASSF1A methylation status in eutopic endometrium may be a potentially useful biomarker to enhance the early detection of EMS.

Effect of RASSF1A gene promoter methylation on its expression level in cervical cancer tissue and its clinical significance / 国际肿瘤学杂志

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.

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.

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.

RASSF1A expression mediated by lentivirus inhibits growth of small-cell lung cancer cell line H446 / 中国病理生理杂志

AIM: To explore the inhibitory effect of Ras-association domain family 1A ( RASSF1A) on the small-cell lung cancer cell growth .METHODS:The lentiviral expression vector containing RASSF1A gene was constructed and used to infect the small-cell lung cell line H446.The growth curve and cell cycle were detected by MTT assay and flow cytometry.The mRNA and protein levels of cell cycle-associated proteins were determined by real-time PCR and Western blotting.RESULTS:We obtained the H446 cells in which RASSF1A was stably expressed (named RASSF1A-H446). Compared with normal cell group and negative cell group , RASSF1A inhibited the proliferation of H446 cells, and arrested H446 cells in G1 phase.The expression of p21 and p27 was significantly increased , and E2F1 was significantly decreased in RASSF1A-H446 cells.CONCLUSION:RASSF1A inhibits the H446 cell growth by increasing the expressions of p 21 and p27, and decreasing the expression of E 2F1.

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.

Association of methylation status of RASSFIA and APC promoters with prostate cancer / 第二军医大学学报

Objective To investigate the promoter methylation status of Ras association domain family 1 isoform A (RASSFIA) and adenomatosis polyposis coli promoter (APC) and its association with prostate cancer (PCa), so as to pave a way for early diagnosis of Pea. Methods The prostatie tissues and clinical data were collected from 60 patients with prostate cancer and 40 patients with benign prostatie hyperplasia (BPH) tissues. Bisulphite Sequencing PCR was used to detect the promoter CpG methylation of RASSFIA and APC genes. Results The methylation rates of promoter CG sites of RASSFIA and APC genes in Pea group were significantly higher whan those in the BPH group (RASSFIA: 60. 8% vs 14%; APC: 48. 84% vs 1. 19%, P<0. 05). The methylation rates in RASSFIA and APC genes were significantly associated with PSA, Gleason score, pathologic stage and risk classification of PCa (P< 0.01). Combined detection of methylation status of RASSFIA and APC genes yielded a sensitivity of 95. 74% and a specificity of 82. 90% in differential diagnosis of PCa and BPH. Conclusion The promoter methylation of RASSFIA and APC genes are associated with the development and progression of PCa, and the changes of methylation rate is closely associated with risk classification of PCa. Combined detection of methylation in RASSFIA and APC genes may be a promising method for early diagnosis of prostate carcinoma.

Value of promoter methylation of RASSF1A, p16, and DAPK genes in induced sputum in diagnosing lung cancers / 中南大学学报(医学版)

Objective To determine the aberrant methylation status of RASSF1A,p16 and DAPK gene promoter region in induced sputum from lung cancer patients and the value of their combined detection in diagnosing lung cancers. Methods Methylation-specific PCR (MSP) was used to detect the promoter methylation status of RASSF1A,p16, and DAPK genes in induced sputum and pathological tissues from 82 patients with lung cancers and 25 patients with pulmonary benign lesion.We also analyzed the relation between methylation status and clinical pathological data.Results The positive rates of promoter methylation of RASSF1A,p16, and DAPK genes in pathological tissues from patients with lung cancers were 63.4%,59.8%, and 58.5%, respectively,and those in induced sputum were 54.9%,48.8%,and 51.2%, respectively. The promoter methylation of RASSF1A,p16, and DAPK genes were not detected in patients with pulmonary benign lesion.There was a significant difference between the lung cancer group and pulmonary benign lesion group (P＜0.05). The methylation rate of RASSF1A gene was significantly lower in the middle and high differentiation and non-metastastic lymph node of lung cancer tissues than that in the poor differentiation and the metastatic lymph node of lung cancer tissues(P＜0.05), and was not correlated with age, sex, smoking index, clinical stage, and pathological types.The methylation rate of p16, and DAPK genes was not significantly correlated with all the above mentioned factors (P＞0.05). The methylation rate of joint detecting RASSF1A, p16, and DAPK genes was 73.2%. Conclusion Joint detection for promoter hypermethylation of RASSF1A, p16, and DAPK genes in induced sputum may be used as a simple and effective index of the diagnosis and prognose of lung cancers, and can improve the positive rate.

Transcriptional expression and promoter hypermethylation of RAS association domain family 1A gene in glioma / 中华检验医学杂志

0. 05). In glioma, the expression level was higher in glioblastornathan in oligodendroglial tumors (P = 0. 011). While the frequency of promoter methylation of RASSFof RASSF1A gene was 39. 3% . It was not found the promoter methylation in normal brain tissues and U251 cell line. In 11 patients with the aberrant promoter methylation, five showed the gene inactivation (P = 0. 022). Conclusion Aberrant promoter methylation was found in glioma. Although the gene inactivation of RASSF1 A was not so common in glioma, its expression was lower in glioma than in normal brain tissues. Promoter methylation may contribute to the low level or loss of RASSFT A mRNA expression.