Construction And Functional Estimation Of Human Interleukin-6 Gene Promoter Reporter Vector

ObjectiveOsteoporosis (OP) is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent in-creasee in bone fragility and susceptibility to fracture. OP is a common disease and a major health care problem. Bone remodeling is a dynamic process involving bone formation by osteoblasts and bone resorption by osteoclasts. Osteoporosis is associated with accelerated bone loss caused by increased bone resorption relative to bone formation. Estrogen is a critical monitor in bone development microenvironment, repressing the production of cytokines such as interleu-kin — 1 and interleukin - 6 from osteoblasts, decreasing the number of mature osteoclasts. Estrogen replace treatment (ERT) is the major therapy to post -menopausal osteoporosis resulting from lower level of estrogen.IL — 6 is thought to be the most important cytokine in initiating maturation of osteoclast and the key factor regulating bone resorption under pathology con-diction , playing a critical role in causation of PMOP. With binding to its receptor , IL — 6 promotes osteoclast maturation directly, increase the production of IL -6 and enhance the bone resorption. Estrogen deficiency can lead to elevated IL - 1 and TNF - α from mononuclear and macrophage, stimulating IL - 6 production and osteoclasts maturation, and inducing activity of bone resorption. Furthermore, estrogen receptor can act on IL - 6 promoter, repressing IL - 6 mRNA. In summary, IL -6 is a key target in estrogen pathway and can reflect the anti - OP effect of estrogen directly. Therefore, this study focued on the transrepression of estrogen to IL - 6 gene, and constructed a human IL - 6 promoter luciferase reporter construct, co - transfected with human estrogen recep-tor expression vector into mouse osteoblast - like cell, and estimated the effect of IL - 1 - induced IL - 6 promoter activity, to establish a molecular biologic evaluation protocol to the effect anti - OP effect of estrogen.Methods1. Construction of human IL — 6 gene promoter luciferase vector. Sequence analysis human IL - 6 gene promoter region, design the forwardand reverse primers and introduced restriction sites of Kpn I and Bgl II into them respectively. Amplified IL — 6 promoter using the method of polymerase chain reaction (PCR). After agarose gel electrophoresis and purification, PCR product was ligated with pMD18 -T simple Vector and transformed into JM109 competent cell in T — A cloning to create IL -6 promoter cloning vector (pMD18sim-HL6). PMD18sim - hIL6 and pGL3 -Basic were digested using double enzymes of Kpn I and Bgl II and two enzymic products were ligated to generate IL- 6 promoter luciferase reporter construct ( pGL3 - hIL6 ) . After confirming by DNA sequencing, pGL3 - hIL6 plasmid DNA of were prepared using according kit, as well as pRL - TK and human estrogen receptor expression vector (HEO).2. Cell culture, transient transfection and estrogen treatment.Mouse OB -like cells (MC3T3 -El) were cultivated in DMEM, buffered with bicarbonate and supplemented with 10% fetal bovine serum. Transient transfection of pGL3 - hIL6 plasmid was performed using Lipofectimine 2000, and cotransfected with pRL — TK vector as internal control as well as HEO. IL — 1 p (5ng) and 170 -E2(10"6, 10~\ andl0"10mol/l) were added 24 hours post - transfection, and undertook fluorescence measurement after another 8 hours.3. Analysis of luciferase activity.Promoter activity of pGL3 - hIL6 was evaluated by dual - luciferase reporter assay system according to the manufestors instructions.Results1. Structure Analysis of human IL - 6 promoter.The initiation codon of human IL - 6 gene was located 63 bp downstream of transcription initiation site. Transcription factors such as CREB, C/EBP and NFkB existed in its promoter, but no estrogen response element (ERE) was found.2. Construction of human IL - 6 promoter luciferase vectorPCR amplified of human IL - 6 promoter. PCR product was 222bp ( - 176 ~ +46). Establish the cloning vector of human IL -6 promoter (pMD18sim -hIL6) . DNA sequencing testified human IL - 6 nuclear promoter reporter construct (pGL3 -IL6).3. Repression of IL - 1 B - induced human IL - 6 promoter activity by estrogenIn the MC3T3 - El cell co - transfected with pGL3 - hIL6, pRL - TK and HEO, luciferase expression were induced by IL - IB greatly, and 17 B - E2 treatment significantly downregulated the IL - 1B - induced promoter activity of PGL3-IL6 (P<0.05).Conclusions1. Human IL - 6 gene promoter reporter luciferase was constructed.2. 17 B -E2 treatment represses promoter activity of human IL - 6 gene.