MicroRNAs Let-7b/i Suppress Human Glioma Cell Invasion And Migration By Targeting IKBKE Directly

Glioma is the most common and intractable central nervous system primary tumor, of which more than 2/3 for malignant glioma. Even with modern comprehensive treatment, the prognosis of malignant glioma has not been significantly improved, the median survival of patients for 15-17 months. Ability to destroy the highly invasive, nervous system tumors and surrounding normal brain tissue to diffuse infiltrative growth characteristics is the root cause of glioma recurrence. The invasion and migration characteristics of glioma has hindered the traditional treatment of glioma. So it is necessary to study its pathogenesis and develop new therapeutic approaches. However, recent advances in genetic research is exploring the occurrence, and glioma gene signaling pathways and molecular mechanisms related to the development of gene therapy has become a new treatment strategy.IKBKE also known as IKK e or IKK-i, is a member of the IKK family. IKBKE was found in breast cancer in the study of ovarian cancer and prostate cancer in activation and high expression. These studies also showed that IKBKE can induce cell survival, growth and chemoresistance and that the overexpression of IKBKE results in malignant transformation. Previous studies in our laboratory showed that high expression of IKBKE in human glioma cells, inhibition of IKBKE expression can significantly inhibit glioma cell proliferation and invasion by siRNA.The study shows that in recent years, some microRNA in human glioma showed abnormal expression, including let-7. Based on the study of miRNA provides a new idea for exploring glioma occurrence, development mechanism and treatment method. MiRNA is a length of about non coding single stranded small molecule RNA 22nt, and the target gene 3’untranslated regions (3’UTR) specific binding, the target gene degradation or inhibit translation, then the gene post transcriptional regulation of .MiRNA in cell growth, differentiation, apoptosis, proliferation play an important role inThe Let-7 family is highly conserved in function and sequence. The family of Let-7 as a tumor suppressor gene can inhibit key genes, oncogenes and some signal pathways such as:HMGA2, MYC, RAS[28]. Research shows that, through the Ras pathway, the let-7 family can reduce GBM cell growth and migration ability of[16,29, 30]. At present, some data indicate that there is indirect association between glioma prognosis and low expression of let-7, but no data showed low expression of let-7 in human glioma [31, 32]. However, bioinformatics analysis showed that, IKBKE is the target gene of microRNAs let-7b/i. Therefore, let-7b/i may be a new target for the treatment of glioma. In this study, we investigated that the effects of miR-let-7b and miR-let-7i on the invasion and migration of the glioma cell lines U251 and U87 occur through the direct targeting to IKBKE and explored the possible mechanisms responsible for this action.The present study was divided into two parts:The first part is the expression of let-7b/i in gliomas and targeted regulation of IKBKE. Application of real-time quantitative PCR (RT-PCR) method, we detected 3 cases of normal brain tissues and 2 human glioma cell line let-7b/i the expression level of TargetScan 6.2, microRNA-Targets and; using Expression to find the upstream regulatory gene IKBKE; 2 glioma cell lines using oligonucleotide let-7b/i mimics transfection, and through the expression effect of RT- PCR the detection of let-7b/i; expression of transfected let-7b/i mimics after application of Western target gene blot; upstream regulatory genes by luciferase reporter experiments IKBKE. The results showed that compared with normal tissue significantly decreased the expression of let-7b/i in glioma; TargetScan 6.2 analysis showed that the 99 miRNAs and IKBKE microRNA-Targets and Expression there is a direct correlation, analysis shows that there are 25 miRNAs and IKBKE there is a direct correlation. TargetScan 6.2 shows a stronger binding force between let-7b, let-7i and IKBKE genes, therefore IKBKE may be the target gene of let-7b/i. The tumor cells significantly increased the expression of let-7b/i let-7b/i mimics after transfection, IKBKE expression was decreased. Luciferase experiments showed that compared with control group, scramble group and pZEX-IKBKE-3’UTR-Mut group, mimics and pZEX-IKBKE-3’UTR-wild plasmids were transfected into let-7b/i when the fluorescence intensity increased significantly. It can be concluded that let-7b/i is the upstream regulatory gene IKBKE.The second part is let-7b/i through the invasion and migration of IKBKE/E-cadherin cell pathway. The scratch test, the invasion test after mimics was transfected into let-7b/i glioma cell migration and invasion in vitro; RNA interference mediated by oligofectamine siRNA targeting IKBKE was transfected into human glioblastoma U87 and U251 cells, expression of target protein was detected by Western blot. Results showed that the transfection of let-7b/i mimics, after the invasion and migration of glioma cells decreased; RNA interference mediated by oligofectamine targeting IKBKE siRNA after transfection, IKBKE expression, E-cadherin expression increased, and the first part of an experimental study found that tumor cells transfected with let-7b/i IKBKE expression decreased after mimics, consistent with the E-cadherin expression increased. These data suggest that let-7b/i is likely through the invasion and migration of IKBKE/E-cadherin cell pathway.Conclusion:low let-7b/i expression in glioma cell line, by bioinformatics analysis, Western blot and luciferase assays, we confirmed that let-7b/i is the upstream regulatory gene IKBKE. Through in vitro experiments, we found that over expression of let-7b/i IKBKE related pathways on the expression of E-cadherin was significantly increased. These data show that:let-7b/i probably through the regulation of IKBKE gene expression of IKBKE/E-cadherin pathway, regulates cell migration and invasion.