Effects Of Estrogen On MMP-26 In Tumor Cell Lines And Cloning Of MMP-26 Promoter

Estrogen is an important hormone of steroid. It functions in the physiological and pathological processes. Estrogen maintains the features of female and the normal menstrual cycle while it can cause some diseases, especially the estrogen-dependent tumor. It is confirmed that estrogen correlates with breast cancer, endometrial cancer, ovarian cancer etc. The origination of cancer is a multi-stage process of complication. There is no clear mechanism of the estrogen causing cancer. Researchers have manifested that the process of estrogen causing cancer is manipulated by a lot of factors. Matrix metalloproteinase is a member of proteolytic enzyme family which can degrade the extracellular matrix. Extracellular matrix is a natural barrier of tumor formation and invasion, so matrix metalloproteinase functions in the tumor formation and invasion. Matrix metalloproteinase-26(MMP-26) is a new member of the matrix metalloproteinase family. Researches of MMP-26 are in the primary stage. Latest researches have shown that there is an estrogen receptor response element (ERE) in the promoter of MMP-26, we hypothesis that the transcription of MMP-26 is affected by estrogen.Our research aims at exploration of estrogen effect on the transcription of MMP-26. Clone the promoter of MMP-26 and construct the luciferase reporter vector which can be used as a tool for further research. We analyze the promoter of MMP-26 to get the elements in the promoter and get the tentative elements functioning in the transcription of MMP-26 and the process of origination of estrogen-dependent tumors. The clues and results are as follows:1. The expression of MMP-26 in tumor cell lines.We select the estrogen-dependent tumor cell lines, like breast cancer, ovarian cancer, hepatic cancer, osteosarcoma, etc. Apply Real time PCR to test the expression of ERα, ERβ, MMP-26 in the tumor cell lines. There are two groups, one is cells cultured in the dextran- carbon treated serum and H-DMEM without phenol red, the other is cells treated with 5×107 for 24 hours. The results show the expression of MMP-26 is down regulated in all cell lines except SGC-7901.The different expression may result from the cells origin and their malignance; and estrogen can down-regulate the expression of MMP-26 in most cell lines.2. Treatment with estrogen gradientsWe treat cell lines, MG-63, HO-8910 and MCF-7 with estrogen gradients. The results show expression of MMP-26 is estrogen-gradient dependant. Within a range, MMP-26 goes up with estrogen concentration, and then it gets the peak and goes down. The results suggest that the expression of MMP-26 maybe controlled by estrogen though ERα.3. Cloning of MMP-26 promoter and construction of luciferase reporter vector and the mutation of ERE elementWe take the genome of normal endometria as the template to amplify the promoter of MMP-26. We get the -506/+133 zone in the 5'flank of MMP-26 and insert the sequence into the pGL3-Enhancer luciferase reporter. Sequencing result shows we have constructed the luciferase reporter containing the promoter of MMP-26. Mutate the ERE element using PCR. By PCR, we mutate one base in the ERE element.4. Assessment of the function of MMP-26 PromoterTransfect the breast cancer cell line MCF-7 with the luciferase reporter containing the promoter of MMP-26. Examine the function of MMP-26 promoter by dual-luciferase reporter assay system. The result shows the promoter of MMP-26 can drive the expression of luciferase.