To Investigate The Mechanism Of Expression Of Tumor - Associated Gene GIPC2

Background:Published data showed that mutations occur in GIPC2 exons in colorectal cancer, ovarian cancer and malignant melanoma.GIPC2 is upregulated in gastric cancer, whereas it isdownregulated in kidney cancer, acute lymphocytic leukemiaand adrenocortical carcinoma. Further study demonstrated thatthe GIPC2 promoter is hypermethylated in 29 of the 31 cases of acute lymphocytic leukemia. These data suggest GIPC2 is correlated with carcinogenesis.Method:A series of luciferase constructs comprising human or rat GIPC2sequences truncated at various lengths in the upstream of translation initiation site were prepared and inserted into Basic-pGL3 plasmid.And the core promoter sequence was confirmed by testing their transcription activity in HEK293T and PC12.1) Homologous sequence of GIPC2 was identified by comparing GIPC2 coding and non-coding sequence acrossdifferent species in Vista database; 2) A series of luciferase constructs comprising human or rat GIPC2sequences truncated at various lengths in the upstream of translation initiation site were tested in HEK293T and PC 12 to confirm the core promoter sequence; 3) Conserved GIPC2intronicsequences were inserted into the upstream of human core promoter to test for potential cis-acting regulatory activities; 4) A single nucleotide mutation in the highly conserved nucleotides within the sequences between human-40-human-30 and Rat-S8-Rat-S11 were carried out, and then transfect these mutants into HEK293T and PC12 cells to check the their effects on transcription activities; 5) we deliver plasmids harboring Rat-S8, Rat-S11and Rat-S14 into HEK293T and PC12 cells to examine the effect of a-KG on GIPC2 expression; 6) To explore the effect of 5’aza on GIPC2 expression, the levels of GIPC2 mRNA from 5’aza-treated HEK293T and PC 12 cells were detected by Q-PCR; 7) To check the effect of methylated human and rat exon 1 on the GIPC2 expression, the human and rat exonl was methylated, and then transfected PC12 cells.Result:Bioinformatics analysis revealed that GIPC2 was conserved across the vertebrate species, typically the homo logy of GIPC2 amino acid sequence is more than 80% among the mammalian species. The human and rat GIPC2 core promoter sequences spanning from +32 to +190 from upstream of translation initiation site were identified in our study. Additionally, a homologous sequence between human and rat in the upstream translation initiation sites represses GIPC2 promoter activity. Conversely, a homologous sequence derived from the first intron of human and rat could improve GIPC2 promoter activity; the human and rat exonl promotes GIPC2 transcription activity, but the methylated exonl failed to improve GIPC2 transcription activity. The target sequence of α-KG locates between Rat-S8 and Rat-S11, the inhibition activity of this sequence on GIPC2 transcription could be blocked by α-KG.GIPC2 level increased over 100 times in the HEK293T and PC12 treated with 5’aza for 96 hours.Conclusions:In the present study, we identified the human GIPC2 core promoter and multiple potential cis-acting elements in noncoding regions, which regulate the activity of GIPC2 promoter. Additionally, this study revealed that the exon 1 of GIPC2 plays a role in regulating the transcription of GIPC2.