Regulation Of CYP2J2Via MicroRNA

Background:MicroRNAs (miRNAs) are small, noncoding RNA molecules of21to25nucleotides that regulate gene expression by binding to3’untranslated region (3’UTR) or5’UTR. It has become apparent that microRNAs play an important role in the process of tumor development by regulating the expression of proto-oncogenes and tumor suppressor genes.CYP2J2, a member of Human cytochrome P450epoxygenase family, seems to be primarily expressed in heart and vessel endothelial cells and in a variety of tissues (including lung, kidney and liver tissues). CYP2J2catalyzes the epoxidation of arachidonic acid into four regioisomers of cisepoxyeicosatrienoic acid (5,6-EET;8,9-EET;11,12-EET and14,15-EET). In the cardiovascular system, EETs (considered to be an endothelium-derived hyperpolarizing factor) could inhibit vascular smooth muscle cells proliferation, had anti-inflammatory effects in vascular system, and promoted endothelial cell proliferation, migration and angiogenesis. CYP2J2-derived EETs were found to play important roles in a host of processes related to tumor growth and metastasis. In our previous study, we found high expression of CYP2J2in human tumors, as well as in several human-derived carcinoma cell lines, but not in adjacent normal tissues and nontumoral human cell lines. Did miRNAs result in the differentially expression of CYP2J2in tumor tissue and adjacent tissues? Whether and how CYP2J2regulated by miRNA are not understood. Hence, the purpose of the present study was to investigate effects and mechanisms of miRNAs on tumor pathogenesis by targeting CYP2J2.Methods:1. Using bioinformatics analysis, we found potential target sites for miRNAs in3’UTR of human CYP2J2. To construct luciferase reporter plasmids, the3’UTR sequence of CYP2J2or mutants (carried mutated sequence in the complementary site for miRNAs seed region) were inserted into the pMIR-REPORT Luciferase Vector, respectively. MiRNAs were cotransfected with reporter plasmids, then luciferase activity was analyzed to investigate if miRNAs target the3’UTR of CYP2J2.2. We treated several tumor cell lines with miRNA, then western blot and ELISA were used to investigate the effects of exogenous let-7b on the protein level and enzymatic activity of endogenous CYP2J2proteins.3. We treated several tumor cell lines with miRNA to investigate the effects of miRNA on cell proliferation and apoptosis, and the expression of CYP2J2related signaling pathways.4. We investigated if miRNA affected tumor growth and metastasis by targeting cytochrome P450epoxygenase2J2in Xenograft Model.5. To investigate an association between let-7b level and the expression of CYP2J2in human carcinomas, the expression of CYP2J2protein and miRNA in cancer tissues and adjacent nontumor tissues was examined by western blot analysis and real-time RT-PCR, respectively.Results:1. Screening and validation miRNAs targeting3’UTR of CYP2J2We first applied luciferase reporter assay to screen miRNAs targeting3’UTR of CYP2J2. We found that transfection of pMIR/CYP2J2-3’UTR along with let-7b resulted in a significant reduction in reporter activity. However, miR-140transfection enhanced the luciferase reporter activity. Western blot analysis showed that overexpression of let-7b significantly downregulated the expression of CYP2J2in several cancer cell lines. ELISA showed that concentration of the metabolite of CYP2J2(14,15-EET) in cell culture media was significantly decreased by the transfection of let-7b.2. let-7b reduces cancer cell proliferation and induces apoptosis by downregulating CYP2J2We treated MDA-MB-435, Tca-8113, SK-MES-1and HeLa cells with let-7b. The EDU DNA Cell Proliferation analysis showed a significant inhibition of cell proliferation by let-7b. Apoptosis, measured by Annexin V and propidium iodide staining, significantly increased in cells treated with let-7b. The effects of let-7b expression on cell proliferation and apoptosis were enhanced by C26and abolished by14,15-EET. Furthermore, western blot analysis showed that let-7b regulating Cancer Cell proliferation and Apoptosis by regulating CYP2J2related signaling pathways (pAKT/AKT、pERK/ERK and Bax).3. let-7b-mediated downregulation of CYP2J2inhibits tumor growth and metastasisOur data showed that overexpression of let-7b in vivo significantly inhibit tumor growth and metastasis in a tumor xenograft model. Western blot analysis showed a markedly decreased expression level of CYP2J2protein and CYP2J2related signaling pathways (the PI3K, pAKT, pERK pathways) and a significantly increased expression level of proapoptotic protein Bax and antimetastatic protein nm-23. During the treatment period, we measured the14,15-DHET(the stable metabolite of CYP2J2) level in the urine of nude mice and found a significant reduction in14,15-DHET in the let-7b treatment group compared with the controls.4. Expression level of let-7b and CYP2J2protein in cancer and adjacent normal tissues are inversely correlatedWestern blot analysis showed that CYP2J2was highly expressed in the majority of human lung cancer tissue samples compared with their matched controls. Real-time RT-PCR was used to determine the mature let-7b levels. The results showed that levels of mature let-7b were significantly decreased in14lung tumors compared with adjacent normal tissues among18samples analyzed. And there is statistically significant inverse correlation between let-7b expression level and CYP2J2protein level in18sets of lung squamous tumors tissues and their matched adjacent nontumor tissues. Furthermore, we found that CYP2J2had inverse expression levels to let-7b in four paired human breast cancer tissues and adjacent Normal tissues.5. The regulation of CYP2J2by miR-140Luciferase reporter assay showed that miR-140significantly enhanced the luciferase reporter activity and the mRNA stability by targeting CYP2J2-3’UTR; western blot analysis showed that overexpression of miR-140significantly increased the expression level of CYP2J2protein in cancer cell line.Conclusion:1. In this study, we found that human CYP2J2is posttranscriptionally regulated by miRNAs:let-7b inhibited the protein level and enzymatic activity of CYP2J2by targeting the3’UTR of CYP2J2, however, western blot analysis showed that overexpression of miR-140increased the mRNA stability and protein level of CYP2J2.2. Applying molecular biology and cell biology, we investigated the effects and mechanisms of let-7b on cancer growth in vitro and in vivo, and we found that:(1) let-7b inhibited cancer cell proliferation and induces apoptosis by downregulating CYP2J2;(2) let-7b inhibits tumor growth and metastasis by downregulating CYP2J2;(3) levels of let-7b were significantly decreased in human cancer compared with adjacent normal tissues, expression Level of CYP2J2protein and let-7b were inversely correlated.