| Aberrant methylation of promoter CpG that causes silencing of tumor suppressor genes (TSGs) may play a key role in the carcinogenesis of many cancer types. RASSF1A, regarded as TSG has been extensively studied in lung cancers and other malignant tumors, whereas RASGRF2 and FBN2 have only been reported that they might play a role in the pathogenesis of pancreatic cancer cell lines. The aims of our study were (a) to determine the methylation profile of RASGRF2, (b) to compare the methylation profiles of RASGRF2 with RASSF1A in lung cancers and (c) to determine the methylation profile of FBN2. In this study, we examined methylation by methylation specific PCR (MSP) of RASSF1A, RASGRF2 and FBN2 respectively in lung cancer cell lines, and analyzed the methylation status of primary lung cancers and correlated them with the clinico-pathological features. We examined mRNA expression of RASGRF2 and FBN2 by reverse transcription PCR (RT-PCR) and treated those cell lines which showed loss of RASGRF2 and FBN2 mRNA expression with 5-Aza-CdR. Methods: 1. Methylation Assay. Genomic DNA was obtained from lung cancer cell lines, cultured nonmalignant cells, primary tumors and adjacent nonmalignant tissues by digestion with 20mg/ml proteinase K, followed by standard phenol/chloroform extraction and ethanol precipitation. One μg of genomic DNA was further subjected to bisulfite treatment. The modified DNA was used as a template for MSP. DNA methylation patterns in the CpG island of RASGRF2, RASGRF2 and FBN2 were determined by the method of MSP. Universal Methylated DNA which subjected to bisulfite treatment was used as a positive control for methylated alleles. Negative controls without DNA were included in each assay. Nine μl of each PCR product was loaded on 2% agarose gels stained with ethidium bromide. 2. Reverse Transcription-PCR for Gene Expression. mRNA expression was analyzed by RT-PCR. Total RNA was obtained from these cell lines, primary tumors and adjacent nonmalignant tissues by Single-step method. Reverse transcription reaction was performed on 5 μg of total RNA with the SuperScript II First-Strand Synthesis using oligo (dT) primer system, and aliquots of the reaction mixture were used for the subsequent PCR amplification. Expression of β-actin was used as an internal control to confirm the success of the reverse transcription reaction.These primer sequences were identical to the human target genes as was confirmed by BLAST searches. PCR products were analyzed on 2% agarose gels stained with ethidium bromide. NHBEC and normal trachea were used as normal controls for RT-PCR. Results: 1. Decreased RASGRF2 expression was presented in 36% (5/14) lung cancer cell lines while aberrant methylation of RASGRF2 was present in 29% (4/14) lung cancer cell lines. 2. The concordance between loss of expression and aberrant methylation of RASGRF2 was 86% (12/14) in cell lines. RASGRF2 expression was restored after treatment with 5-Aza-CdR in all four cell lines tested that downregulated RASGRF2 expression. 3. Expression of RASGRF2 was decreased in lung cancer tissues comparing to nonmalignant lung tissues. 4. Among primary NSCLC, RASGRF2 and RASSF1A methylation were observed in 34% (39/114) and 39% (44/114) cases respectively, while were observed only in 7% (4/57) and none of corresponding nonmalignant lung tissues. The difference was significant(p<0.0001). 5. Neither RASGRF2 nor RASSF1A methylation status associate with clinical characteristics, such as gender, age, smoking history (ever vs. never smoker), histology (adenocarcinoma vs. squamous cellcarcinoma) and survival. 6. There was no correlation between RASGRF2 and RASSF1A methylation status not only in cell lines but also in clinic samples. 7. Loss of FBN2 expression was observed in 50% (8/16) of lung cancer cell lines, respectively, in 64 % (7/11) of NSCLC cell lines, and 20% (1/5) of SCLC cell lines. Meanwhile, aberrant methylation of FBN2 was present in 55% (6 /11) of NSCLC cell lines, whereas it was absent in SCLC cell lines. 8. The concordan...
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