Metastatic Suppressor Genes Inactivated By Aberrant Methylation In Gastric Cancer

Objective and significanceOver the past few years, the five—year survival rate of early gastric cancer has been enhanced. But it has been not improved for advanced gastric cancer. Postoperative recurrence and metastasis are the key factors that affect treatment outcome of patients with advanced gastric cancer. The metastasis of gastric cancer is a complicated course. Although many researches had been done, the mechanism is still poorly understood. Generally speaking, the metastasis of gastric cancer is because of some the mutation or deletion of the suppressor gene in the genetic level and is because of low expression of the suppressor gene in the epigenetic level. In these factor that cause the low expression of the suppressor gene, hyper-mythlation is an important mechanism. Aberrant methylation of CpG island has been shown recently to serve as a way of inactivating suppressor gene in many human caner. CpG islands are short sequences rich in the CpG dinucleotide and can be found in the 5' region of about one—half of all human genes. Methylation of cytosine within 5' CpG islands is associated with loss of gene expression and has been seen in physiological conditions such as X chromosome inactivation and genomic imprinting. Aberrant methylation also occurs during aging and carcinogenesis and is linked to transcriptional silencing of multiple genes, including known familial cancer genes. This has lead to the hypothesis that novel tumor suppressor genes could be isolated using aberrantly methylated CpG islands as a marker. In the past few years, several techniques were developed to de-tect aberrant methylation in cancer. Although these techniques are very powerful in detecting methylation differences, they are limited to known genes because they require sequence information for the design of PCR primers. More recently, to isolate differentially methylated CpG islands in cancer and normal tissues, a new technique (Methylated CpG Island Amplification) have been developed. MCA allows for the efficient PCR amplification of methylated CpG islands, which can detect methylation of many genes, or to clone CpG islands differentially methylated in cancer. By applying MCA coupled with RDA to gastric cancer, we can efficiently analyze whole genome methylation level, especially for unknown genes. In this research, the method was applied to analysis the methylated difference between gastric primary cancer and metastatic lymph nodes with gastric cancer in order to find novel genes which have relation to lymphatic metastasis of gastric cancer. Furthermore, these novel genes will be checked in gastric primary cancer tissues, metastatic lymph nodes and gastric cancer cell line by methylated special PCR (MSP) and RT — PCR.Samples and MethodsSamples and cell line: Samples of gastric cancer tissues and metastasis lymph nodes were obtained from Department of Surgical Oncology of the First Affiliated Hospital of China Medical University. The metastasis lymph nodes were verified by pathologic diagnosis. And the gastric cancer cell line SGC7901 was kindly provided by Oncology research institute of China Medical University.Methods:1. Methylated CpG island amplification (MCA): Genomic DNA was extracted from frozen tissues by the phenol—chloroform method. Digest 5 g of genomic DNA using 100 units of Smal (NewEngland Biolabs) over night. Add 20units of Xmal and incubate at 37t for 6 hours. RXMA and adaptors were prepared by incubation of the oligonu-cleotides RXMA24 at 65 °C for 2 min, followed by cooling to room temperature. DNA (0. 5 \i g) was ligated to 0. 5 nmol of RXMA adaptor using T4 DNA ligase (New England Biolabs). PCR was performed by using primer RXMA24.2. Representational difference analysis (RDA):The driver MCA amplicons using 100 Units Smal to remove the RXMA adaptor. Remove the adaptors using an Utracel —YM 100 centrifuge tube (Millpore). Change of adaptors on the tester amplicon Digest 5 g of tester MCA amplicons using 20 Units Xmal to remove the RXMA adaptor. Change of adaptors on the tester amplicon. The ratio of tester to driver from the first round to the third round hybridization is l:80, ls400 and l:800. After competitive hybridization, PCR was performed by using corresponding primer and then digest the single stranded amplified MCA products by using Mung bean nuclease (NEB). Then perform next round RDA.3. DNA clone, sequencing and analysis:After 3 round RDA, PCR products were digested with Xmal. The adaptor was eliminated by Utracel —YM 100 centrifuge tube. The PCR products were then subcloned into pCAT<§)3—Control (promega). Transform it to JM109 bacterium. Extract plasmid DNA, digested with Xmal, electro-phorese and pick up the clones that can be cut into large than lOObp sequence. Sent these clones to United Gene Holdings LTD. to sequencing the DNA. Analyze the sequences by BLAST and RepeatMasker system.4. Dot blot:Use MCA—RDA products KL22 clone as a probe. Hybridize the probe to cancer tissues and metastasis lymph nodes MCA, and the first to the third RDA products on nylon membranes.5. Gastric cancer cell line culture and 5 — Aza —2' —deoxycytidine treatment:Divided cell line into two group and one of them was treated by 5—Aza — 2—deoxycytidine.6. Methylation—specific polymerase chain reaction (MSP): Genomic DNA was denatured by NaOH and modified by sodium bisulfite. DNA samples were then purfied using Wizard DNA Clean —up (Pro-mega) , and then performed PCR by using unmethylated primer and methylated primer.7. PTPRG gene RT-PCR:Extracted whole RNA by using TRIZOL agent and synthesized cDNA. PCR was performed by using PTPRG gene primers and p—actin RT—PCR was carried simultaneously.8. Statistics analysis:The x2 test was used to clarify the relationship between the rate of methylation of gastric primary cancer tissues and metastatic lymph nodes. A linear regression analysis was applied to illustrate the association between the quantity of metastatic lymph nodes and the methylation rate of metastatic lymph nodes.Result1. MCA:The MCA products of gastric cancer tissues and metastasis lymph nodes, after agarose gel electrophorese, can be seen relatively strong smears, ranging from 300 bp to 2 kb.2. RDA:After 3 rounds hybridization—amplification, 5 difference straps can be observed, ranging from lOObp to 500bp, which were gradually clear from the first to the third round hybridization.3. Clone, sequencing and DNA analysis:96 clones were obtained. Underwent restriction digestion, 27 sequences large than lOObp were obtained. 8 sequences were ALU repetitive sequence. All sequences rang from lOObp to 400bp,GC content> 50%. These sequences distribute each region in genome, including 5' region, exon, intron, and 3'region. KL59 was located in the first exon ofpl6. KL22 was located in PTPRG promoter.4. Dot blotThe products of 3 rounds RDA and tester hybridize with KL22 probe labeled by DIG all show positive signal. Driver was negative.5. PTPRG gene MSP and PTPRGmRNA expression of gastric primary cancer and metastatic lymph nodes:Positive rate of unmethylated PCR of gastric primary tumors was 77. 78% (28/36). Positive rate of unmethylated PCR of metastatic lymph nodes was 63. 89%(23/36), P>0. 05. Positive rate of methylated PCR of gastric primary tumors was 25. 0%(9/36). Positive rate of methylated PCR of metastatic lymph nodes was 52. 78% (19/36) , P<0. 05. A linear regression analysis revealed a significant association between the quantity of metastasis lymph nodes and the methylation rate of metastatic lymph nodes, r=0. 882, P<0. 05.Positive rate of RT—PCR of gastric primary tumors was 50. 0%(18/ 36). Positive rate of RT— PCR of metastatic lymph nodes was 25. 0%(9/ 36), P<0.05.6. Methylation of PTPRG gene and PTPRGmRNA expression before treatment by 5 — Aza— 2— deoxycytidin:158bp positive belts were detected in unmethylated PCR and 150bp positive belts were detected in methylated PCR of cell line before treatment by 5 — Aza—2—deoxycy—tidin.. PTPRGmRNA expression of cell line was negative before treatment by 5—Aza— 2— deoxycy tidin.7. Methylation of PTPRG gene and PTPRGmRNA expression after treatment by 5 — Aza— 2—deoxycytidin:158bp positive belts were detected in unmethylated PCR, but 150bp positive belts were not detected in methylated PCR of cell line after treatment by 5 — Aza — 2y— deoxy— cytidine. 177bp weak positive belts were detected in PTPRG gene RT—PCR of cell line after treatment by 5—Aza — 2— deoxycytidin.Conclusion1. There is a difference in DNA methylation between the tissues of primary cancer and lymph nodes with metastatic gastric cancer. MCA— RDA is an effective method to study the gene methylation. PTPRG gene may be a candidate gene for metastasis of gastric cancer.2. There was difference of PTPRG gene between gastric primary cancer and lymph nodes with metastatic gastric cancer. 5 — Aza—t—de-oxycytidin is an inhibitor of DNA methylation and can recovery the expression of PTPRG gene.