| PrefaceRASSF1A gene is a newly identified Tumor Suppressor Gene (TSG). According to previous research, RASSF1A participates in Ras signal transduction, DNA damage repairing and apoptosis induction, thus it is identified as a Tumor Suppressor Gene. Hypermethyla-tion of the promoter region s CpG island leads to silencing expression of the protein which results in occurrence of many types of tumors; such as lung carcinoma, gastric carcinoma, breast cancer, narsophan-ryngeal cancer, neuroblastoma, renal carcinoma, prostate carcinoma, etc. Because of CpG island hypermethylation in promoter region, RASSF1A's mRNA transcriptional levels were lowered and protein expression were decreased, the tumor suppressing function were inhibited , thus caused many types of cancers. Currently this mechanism is widely accepted and promoter region hypermethylation attracts more and more concerning in carcinogenesis process. According to previous research reports, this silencing mechanism in RASSF1A has not been investigated in Nephroblastoma. Therefore, we screened promoter methylation in 9 cases with nephroblastoma by methylation specific method.MethodSamples were collected from nine patients with nephroblastoma, including tumor tissue and the para - tumor tissue, then stored in -70t. DNA was collected through classical phenol - chloroform extraction method. DNA concentration and purity were tested by Ultraviolet spectrum, the sampled OD 260/OD280 value was larger than 1. 75. DNA was denatured by strong base and then treated with Sodium Bisulfate/benzenediol, and salt were eliminated through chromatogra-phy column. Nested Polymerase Chain Reaction was done with two forward primers and one reverse primer using RASSF1A promoter region as template. Primers sequences were as follows:Forward primers; ML 730;5 '-ACCCTCTTCCTCTAACACAATAAAACTAACC-3';Forward primers; ML 561;5'-CCCCACAATCCCTACACCCAAAT-3';Reverse primer; MU 379:5'-GTTITGGTAGTTTAATGAGTTTAGGTTTTTT-3'.The first reaction uses forward primer M730 and reverse primer MU379, the second reaction uses reverse primer MU379 and forward primer ML561 under the following condition;95 4min predenaturation95 Imin denaturation, 55T1 1 min annealing , and 74 2min extension, for 30 cycles,72 5min extension.PCR fragments were sequenced by Peking OKE Cooperation.ResultAmong the 9 cases, 2 PCR products showed positive results. In one case, the second, fourth, fifth, ninth, twelfth and fourteenth CpG sites had C to T conversion, which suggests that there was no methyla-tion in these sites. The other ten CpG have not C to T conversion, which suggests there was methylation in these sites. While in the coupling para - tumor regions DNA, all the 16 C in CpG island was intact and converted into T; This indicated that no methylation occurred here. In another case, the sixth, ninth, tenth, twelfth site had C to T conversion and the other twelve CpG island were methylated. Sequencing was failed in the para - tumor tissue in this case. Comparing the methylation site in the 2cases, 8 CpG island sites were same.DiscussionNephroblastoma is a kind of malignant and embryonic tumor commonly occurred in children. Because it has both embryonic genetic change and body cell mutation, it becomes a suitable model for studying genetic tumorigenesis. As previous study showed, this tumor had many suppressor genes, which related with deletion or abnormal expression of WT1, and also related with abnormal expression of WT2 ( candidate gene). Recent studies demonstrated that methylation which induced the elimination of printing gene and inactivation of suppressor gene had closely relationship with tumor genesis, such as imprinting deletion of IGF2/H19. PI 6 is a tumor suppressor gene in nephroblastoma, exon 1 and 2 s promoter region of it has methylation which lead to that the protein expression were inhibited and tumorigen-sis could not be inhibited. So it is necessary to study the methylation of RASSF1A.RASSF1A is a tumor suppressor gene. Previous rese... |
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