The Expressive Effect Of DNA Methylation On Human TIG2 Gene In Lung Cancer Cells

IntroductionPresently, the research focuses of molecular mechanisms of lung cancer are on molecular and cellular level, the tumor suppressors inactivation plays an important role in non - small cell lung cancer ( NSCLC ) progression. There are many ways of genes inactivation, among which anormal CpG islands methylation within promoter region is currently taken carefully into account.Tazarotene is a analogue of retinoic acid as a drug for therapeutics of skin diseases, TIG1 ,TIG2 and TIG3 genes are induced after endothelial cells are treated by Tazarotene. TIG2 gene is consistently expressed in human normal skin, but silenced or down - expressed in psoriatic patients. The current results indicated that TIG2 is natural ligand of orphan G protein — coupled receptor(GPCR) ChemR23 and its characteristics is able to recruitment of immature dendritic cells (DCs) and macrophages in vitro. The activation of TIG2 after proteolytic proteasea from serum and inflammation cleavage the C terminal of precursor TIG2. TIG2 gene is ubiquitously expressed in human normal tissues, but it was not reported to the expression and functions of TIG2 in human tumors.In the previous studies, we screened some differential expression genes from lung tumor tissue and its matching lung normal tissue based on suppressive subtractive hybridization (SSH) , among which TIG2 gene is expressed in normal lung cells but silenced in lung cancer cells, it is indicated that TIG2 gene may play an role in lung tumorigenesis. The thesis aims are to further detect TIG2 gene expression in lung cancer cells and normal lung cells, then observing the effect of demethylating agent on TIG2 expression in lung cancer cells. To identify methylation sites, we analyzed CpG islands distribution within TIG2 promoterand first extron region by MethPrime software, and tested by methylation specific PCR (MSP) in clinic specimens. Collective results may conduce to initial explain molecular mechanisms of TIG2 gene silenced in lung cancer cells, which is essential to further understand of TIG2 functions in lung cancer and to clinical early diagnosis for NSCLC.Materials and methodsGenomic DNA was extracted from lung cancer cell lines and clinical tissues using Phenol /CHCI, DNA samples were then treated with bisulfite to convert all unmethylated cytosines to uracil, while leaving methylated cytosines unaffected. Different primers could be designed using MethPrime software and could distinguish methylated/un - methylated status. Then PCR product was performed and observed in 2. 0% agarose gel. We also measure the effect of demethylating agent on TIG2 expression in lung cancer cells and the semi - quantitative RT -PCR method was used to assess the expression of TIG2 genes in three lung cell lines and clinical specimens.ResultsWe fond that TIG2 gene was expressed in normal lung cell WI - 38, but silenced in lung cancer cell lines by RT - PCR, as the same as clinical specimens. Typical CpG islands were discoveried within promoter and first extron region of TIG2 gene by MethPrime software. Interestingly, the TIG2 gene expression was restored in lung cancer cell A549 bb treatment with demethylating agent 5'- aza - CdR. The promoter methylation status of TIG2 gene was examined and fond that TIG2 gene was both unmethylated in normal lung cell WI -38 and normal lung tissues , but was frequently methylated in lung cancer cells and lung cancer tissues.ConclusionAberrant methylation of the promoter of TIG2 gene is frequently present in lung cancer cell lines and NSCLCs and methylation may be responsable to the decline of transcription of TIG2 gene.