MicroRNA-10b's Function In Human Brain Tumors Associated Preliminary Study Of Gene Therapy

Research background:MicroRNAs are a class of small endogenous noncoding RNAs consist of 20-25 ribonucleotides, which modulate target gene expression by binding with target mRNA sequences in the 3'untranslated region (UTR) with full or partial complementarity way that cleavage of target mRNAs or inhibit mRNA translation. Many microRNAs have been reported to function as tumor oncogenes or anti-oncogenes. Recently, more and more microRNAs have been reported to contribute to tumor's invasive potential, more and more miRNAs have been found to be either activators or suppressors of tumor invasion and metastasis. Among these miRNAs, miR-10b was first reported to induce breast cancer cell invasion and metastasis, which was also overexpression and associated with tumor invasive potential in hepatic cancer, pancreatic cancer, glioma, esophageal cancer and neurofibromatosis. But the exact function of miR-10b in brain tumors, espically in pituitary adenoma and glioma are still unknown.Research objection:To measure the expression level of miR-10b in human pituitary adenoma and investigate that whether miR-10b is correlate with pituitary adenoma's invasive potential.To analyze the expression level of miR-10b in different grade of human glioma and investigate that whether miR-10b is associate with glioma's maligancy and invasive potential. To measure the expression level of miR-10b in different glioma cell lines and investigate the function of miR-10b in human glioma cells. Research Methods:Pituitary adenoma and glioma samples: Collect different pathological diagnosis of non-functional pituitary adenoma and malignant glioma, and normal human pituitary were collected by autopsy, as well as the surgical specimens of normal brain tissue from decompression of severe traumatic brain injury patients. Pituitary adenoma samples were divided into four groups according to the classified Hardy's method.Expression levels assay: TaqManMicroRNA assay-based real-time RT-PCR was used to analyze miR-10b levels in pituitaryadenoma and glioma samples as well as 4 well known glioma cell lines: U87, U251, LN229 and U373. Normal pituitary and brain tissues were considered as control. And further compared the relevance of the levels of miR-10b in different grade tumor groups.Function study: To investigate the biological function of miR-10b in the invasive progression of glioma, knock-down and regain of function experiments were employed. 2′-O-methyl (2′-O-Me) hsa-miR-10b inhibitors (miR-10b antisense oligonucleotides) were used to slience miR-10b expression in U87 and LN229 cells which expressed higer miR-10b levels and miR-10b mimics (miR-10b sense oligonucleotides) were used to elevate miR-10b expression level in U251 cells which expressed lower miR-10b level, synthesized by Shanghai GenePharma Company. In order to further study the mechamism of miR-10b induces glioma cell invasion, HOXD10 and miR-10b luciferase reporter vectors were constructed via technical support from Wuhan Genesil. And the expression of miR-10b in human glioma cells was detected by real-time quantitative RT-PCR. The effects of the invasion, apoptosis and cell cycle of infected glioma cells were evaluated via transwell assay and Flow cytometric method. Luciferase activity was analyzed by the Dual-Luciferase Reporter Assay System. Research Results:MiR-10b was higher expressed in human different-grade pituitary adenoma compared to normal pituitary tissue, and miR-10b levels of invasion group was higher than non-invasion group, showing an upward trend as pituitary adenoma invasive grand increased, miR-10b was also elevated in macroadenoma than microadenoma.MiR-10b was strongly up-regulated in all glioma samples and glioma cell lines, U87,LN229,U373 and U251 cells compared with normal brain tissue. MiR-10b expression levels showed upward trend with the increased malignancy grade of glioma, and its expression was positively correlated with tumor grade; miR-10b expression was highest in WHO- IV, and showed no statistical significance between WHO-II and WHO- III of glioma.Slience expression of miR-10b could effectively reduce U87 and LN229 glioma cells invasive potential. And overexpressing miR-10b in U251 cells could lead to an increased invasion cells. Additionally, both the miR-10b inhibitors and miR-10b mimics had no effect on cell apoptosis or cell cycle. Further research showed that HOXD10 was the direct target of miR-10b, and miR-10b induces glioma cells invasion by modulating MMP-14 and uPAR expression via HOXD10 gene.Research Conclusions: MiR-10b was elevated in human non-function pituitary adenoma, and showed positively correlated with tumor's invasive grade.MiR-10b showed different high level of expression in glioma samples and cell lines, and its expression positively correlates with tumor's maligancy grade in glioma. The aberrantly up-regulated miR-10b maybe function as tumor invasion activitor in human glioma.MiR-10b induced human glioma cell invasion by modulating MMP-14 and uPAR expression via HOXD10 gene, miR-10b/HOXD10/MMP-14, uPAR signaling pathway may contribute to glioma's invasion growth, miR-10b antisense inhibitors could be used for anti-invasion treatment.