Research Of Transcriptional Expression And Promoter Methylation Of Tumor Suppressor Gene In Lung Carcinoma

Lung cancer is a common malignant tumor; its increasing rate is in the first place among all the kind of carcinoma. Because of lacking effective early diagnosis methods, the majority of patients with lung cancer present advanced disease. So early finding is very important to the lung cancer.Recent study showed that DNA methylation was associated with the development of tumor. The methylation indicated that the-CH3 was carried on the cytosine by DNA methyltransferase and turned into 5-mC,which happened the 5'CpG island frequently. Promoter methylation could change the action protein and gene structure and suppressing the gene transcription through multiple ways. There were evidences showed that methylation before the tumor. Therefore, the detecting of 5'CpG island methylation was very useful to discover the tumor cell.In 1996, Otha discovered a new tumor suppressor gene -FHIT(Fragile Histidine Traid) by exon-capture technique. Abnormal FHIT has usually been found in various cancers. FHIT gene differs from the classical suppressor gene, abnormalities at transcriptional level were deletion of one or several exons ,sometimes were insertions, but point mutation has rarely been found .So, people has studied the methylation of FHIT Promoter in lung cancer speciments. Up to now, the research of the the relationship between the methylation of FHIT and lung cancer was few reported.The human Ras association domain family 1A gene (RASSF1A), was recently cloned from the lung tumor suppressor locus 3p21.3. Aberrant promoter hypermethylation of RASSF1A is a common epigenetic event in a wide variety of human cancers, and result in loss of RASSF1A expression. RASSF1A knockout mice were prone to spontaneous tumorigenesis and showed susceptibility to lung adenoma and hematologic malignancy in advanced age. Although the function of RASSF1A is undefined, it has been demonstrated that RASSF1A is involved in apoptotic signaling, microtubule stabilization, and mitotic progression.PTEN tumor suppressor is a 403-amino acids phosphoprotein/phospholipid dual-specificity phosphatase. Importantly, PTEN possesses lipid phosphatase activity, preferentially dephosphorylating phosphoinositides at the D3 position of the inositol ring. It is the only enzyme known to dephosphorylate the D3 position in inositol phospholipids, suggesting that PTEN may function as a direct antagonist of phosphatidylinositol 3-kinase (PI3-kinase) and phosphatidylinositol 3,4,5-trisphosphate (Ptdlns(3,4,5)P3)-dependent signaling. PTEN can downregulate the angiogenesis stimulators including the growth factors, vascular endothelial growth factor and basic fibroblast growth factor, and the induction of matrix remodeling via matrix metalloproteinases. Overexpression of PTEN inhibits cell migration, whereas antisense PTEN enhances migration. Integrin-mediated cell spreading and the formation of focal adhesions are down-regulated by wild-type PTEN but not by PTEN with an inactive phosphatase domain. PTEN interacts with the focal adhesion kinase FAK and reduces its tyrosine phosphorylation. The PTEN gene is mutated in high-grade gliomas as well as prostate, endometrial, breast, lung, and other tumors. In addition, PTEN is mutated in several rare autosomal dominant cancer predisposition syndromes, including Cowden disease, Lhermitte-Duclos disease and Bannayan-Zonana syndrome. But PTEN mutation is rare in some tumors with lose of PTEN expression. It seems that the promoter methylation be the main mechanism involved in.A member of the 14-3-3 superfamily, 14-3-3 is responsible for G2 cell cycle checkpoint control in response to DNA damage in human cells. 14-3-3 sequestered Cdc2/cyclin B1 complexes in the cytoplasm, thus arresting cells in G2 and preventing them from initiating mitosis before repair to their damaged DNA. The expression of 14-3-3 is reduced by p53 gene inactivation and by silencing of 14-3-3 gene via methylation of CpG islands. 14-3-3 hypermethylation have been reported in many tumors, including lung, gastric, ovarian, prostate, and hepatocellular carcinomas.CHFR gene, recently cloned from locus 12q24.3. Chromosomal instability (CIN) is a cytogenetic hallmark of human cancers. Increasing evidence suggests that impairment of mitotic checkpoint is causally associated with CIN. CHFR is one of the mitotic checkpoint regulators and it delays chromosome condensation in response to mitotic stress. Epigenetic inactivation of CHFR through promoter CpG hypermethylation may lead to CIN and has been reported in several human cancers. CHFR is responsible for a mitotic checkpoint function that delays chromosome condensation and entry into metaphase in response to microtubule-disrupting agents. Biochemically, CHFR is an ubiquitin ligase that targets other cell cycle regulatory proteins for degradation. While normal human cells expressing CHFR are delayed in entering mitosis in the presence of mitotic stress, cancer cell lines lacking CHFR enter metaphase prematurely. Ectopic expression of wild-type CHFR restores the checkpoint and enables the cells to survive mitotic stress. Interestingly, epigenetic inactivation of CHFR is frequently observed in human tumors. Methylation of CpG islands in promoter region seems to be correlated with CHFR silencing in several human cancer cell lines and primary tumors including lung, esophagea, colorectal, head and neck, gastric, and breast cancers.lung cancer tissues samples, surrounding normal and BALF, blood plasma were used in this study to detect FHIT methylation status with the method of MSP and colon in order to illustrate the relationship between the methylation of FHIT with the development of lung cancer. we analyzed the p16 methylation status in order to provide useful information for pre-warning of lung cancer.Method1. Five genes respectively mRNA expression level was measured by real-time reverse transcription polymerase chain reaction in 45 lung carcinoma , 23 lung carcinoma adjacent tissues and A549 cell lines. The promoter methylation status in lung carcinoma, lung carcinoma adjacent tissues and A549 cell lines were tested by methylation-specific PCR. 2. The data using chi-square test and t test was processed by meanes of SPSS 10.0 software .The significant level was set at p<0.05.Results The promoter methylation status of five genes including FHIT, RASSF1A, PTEN, 14-3-3σ, CHFR were examined. In 45 lung carcinoma cases, the frequencies of methylation for these 5 genes in lung cancer tissues, plasma, BALF and 23 free-cancer pulmonary diseases tissues. The frequencies of methylation of these five genes: FHIT:40%,31.11%,33.33%及8.70%;RASSF1A:53.33%,28.89%,42.22%及13.04%;PTEN:26.67%,15.56%,22.22%及0%;14-3-3σ:51.11%,31.11%,42.22%及17.39;CHFR:22.22%,13.33%,20%及0%. The frequencies of methylation in lung cancer tissues were higher than in free-cancer pulmonary diseases tissuesConclusions Frequency of promoter methylation of Promoter of TSGs is obviously higher in lung carcinoma tissue than lung tissue without cancer and expression level of TSGs is lost either lower in lung carcinoma than in lung tissues without cancer. It may contribute to the function in occurrence and development of lung carcinoma. More positive rate was found in lung cancer patients than the single gene through detecting of several TSGs methylation, indicating that the combined methylation incidence of many genes is very useful for prewarning of lung cancer.