Isolation And Identification Of The Promoter Of The Human ITLN-1 Gene Promoter

Background ITLN-1 is a novel fat factor which mainly expressed in omental adipose tissue,also known as omentin.It is also expressed in other tissues such as endothelial cells,human epicardial fat,thymus,small intestine,colons,reticulocytes,ovary,lungs and placentas.However,which is rarely expressed in subcutaneous fat.ITLN-1 seems to be downregulated in obesity or diabetes,and which has a negative correlation with waist circumference,body mass index and leptin.Recent studies have found that the plasma levels of ITLN-1 in patients with coronary heart disease are significantly lower than those in normal and metabolic syndrome patients.Plasma ITLN-1 levels in patients with inflammatory bowel disease were significantly lower than those in normal controls,while the ITLN-1 levels in prostate cancer patients were significantly higher.The high expression of ITLN-1 in gastric cancer cells and intestinal metaplasia cells is negatively correlated with the degree of gastric cancer invasion,which is an independent factor for predicting the survival time of gastric cancer patients.In addition,ITLN-1 can promote the proliferation of bone marrow derived mesenchymal stem cells and inhibit the apoptosis.It has also been reported that ITLN-1 can significantly reduce cerebral infarction volume,promote angiogenesis and reduce apoptosis in rats.This suggests that ITLN-1 has a protective effect on the nerves after focal cerebral ischemia,suggesting that ITLN-1 may be a new therapeutic target and a candidate gene for the treatment of cerebral ischemia.In view of the fact that human ITLN-1 is involved in many physiological and pathological activities,it may be a new biological marker and a potential target for the treatment of diseases.However,the mechanism of differential expression of human ITLN-1 gene in different states is not clear.The study of transcriptional mechanism of ITLN-1 provides theoretical support for further study of gene physiological function and intervention of disease treatment.Objective To Clone and analyze of the promoter of Human ITLN-1 Gene.To isolate and to identify the activity of the human ITLN-1 promoter.Methods To have truncation analysis in the region of Human ITLN-1 Promoter.The DNA fragment of the human ITLN-1 promoter region from 5’-flanking region was amplified from human peripheral blood leucocyte genomic DNA by PCR.The length of DNA sequence are 0.3 kb,0.7 kb,1 kb,1.3 kb,1.7kb,2 kb,3 kb,and then they were inserted into basic reporter vector p GL 4.10[luc2].The human ITLN-1 promoter reporter vector was correctly constructed,and named as p GL4.10(0/+296),p GL4.10(-350/+296),p GL4.10(-705/+296),p GL4.10(-1003/+296),p GL4.10(-1385/+296),p GL4.10(-1751/+296),p GL4.10(-2035/+296),p GL4.10(-3012/+296),The recombinant vectors were correctly identified by enzyme digestion and gene sequencing.Both of the luciferase reporter vectors and p GL4.74 [h Rluc/TK] were cotransfected into HEK 293.And after 48 hour measured the activity of Luciferase.To predict the transcriptional factors of human ITLN-1 gene promoter in the(-705/+296)region.The transcriptional factor "ZNF 35,Sox-6,KLF14,Foxc1" was preliminarily verified in HEK 293 cells by RT-PCR technique.Results The recombinant vectors which containing the promoter DNA fragment of human ITLN-1 gene with different lengths were successfully constructed.The activity of all recombinant plasmids was higher than the base plasmid p GL4.10(P<0.05).The activity of p GL4.10(0/+296)was 6 times of p GL4.10.The p GL4.10(0/+296)had the highest activity.Conclusion The human ITLN-1 gene 5’-flanking region(0/+296)had the highest promoter activity.The expression of transcription factor "ZNF 35,Sox-6,KLF14,Foxc1" were found in HEK 293 cells.It may bind to human ITLN-1 promoter(0/+296)and play a regulatory role in the transcription of the human ITLN-1.This study provided an important basis for illuminate the regulation mechanism of human ITLN-1 gene.