| Backgroud:Glioblastoma (hereafter referred to as glioma) which is the most common originates from neural ectoderm intracranial malignant tumor, account for about half of intracranial tumor. It possesses some characteristics that difficult to completely removed by surgical removal, radiation or chemotherapy effect is not obvious and also easy recurrence and poor prognosis. MicroRNA (miRNA) is a kind of non-coding small RNA molecules that translational repression the expression of its target gene. A growing body of research suggests miRNA widely modulate many kinds of biological characteristics of malignant tumors. However, little is known about the role of miRNA in the development of glioma. Research the roles and mechanisms of miRNA in glioma have important clinical significance for further explain the pathogenesis, and for diagnosis and treatment of glioma.Objective:(1) The main purpose of this study was to screen difference expression of miRNAs in glioma.(2) Find out several miRNAs that possible regulation biological characteristics and drug resistance of glioma.(3) Seek for the target genes of miRNA through bioinformatics software and conduct related research for molecular mechanism.Methods:(1) According to the results of microarray assay that had been reported, we firstly find out a variety of miRNA that was downregulated in glioma compared with the normal control group. Then choose the miRNAs that could inhibit the activity of U87 and U251 glioma cell lines by MTT. Further to find the genes that was potentially regulated by miRNA through bioinformatics software which called miRanda and DIANA microT v3.0.(2) 16 cases with high-grade glioma and 8 cases with head injury by surgical removal were collected from people's hospital of wuhan university from 2011 to 2014. Detect the expression of miR-129 and miR-873 in normal brain tissues, glioma tissues, U87 and U251 glioma cell lines by qPCR.(3) Detecting the proliferation of U87 and U251 cells after overexpression of miR-129 by MTT. Detecting cell apoptosis by flow cytometry. Detecting autophagic activity affected by miR-129 through qPCR, Western blot, confocal, transmission electron microscopy and also implantation experiment in nude mouse.(4) To verify whether Notch-1 was a target gene of miR-129 through luciferase assay, qPCR and Western blot experiments. To test the influence of miR-129 on mTOR phosphorylation and expression of E2F7 and Beclin-1 by qPCR and Western blot. To detect the influence of Notch-1 on E2F7 and Beclin-1 and the influence of E2F7 on Beclin-1 by Western blot.(5) Build U87 and U251 cisplatin resistance cell lines, which named U87/DDP and U251/DDP respectively. Identified miRNAs that differential expression in cisplatin resistant cells and wild-type cells by qPCR. Determine the influence of miR-873 on proliferation of cisplatin resistant cells by MTT. Determine the effect of miR-873 on cell viability and proliferation under cisplatin by MTT and clone formation assay. Detecting the influence of miR-873 on apoptosis of cisplatin resistant cells under cisplation by flow cytometry and caspase-3/7 activity assay.(6) To verify whether Bcl-2 is a target of miR-873 through luciferase assay, qPCR and Western blot. Detecting the function of Bcl-2 which regulated by miR-873 on cisplatin sensibilization by MTT assay, clone formation or flow cytometry and caspase-3/7 activity assay.Results:(1) According to the results of microarray assay that had been reported, we find 8 miRNAs which were downregulated in glioma samples. They are miR-128?miR-129? miR-133a?miR-139?miR-153?miR-323?miR-488 and miR-873. MTT results showed that miR-129 and miR-873 could significantly inhibit U87 and U251 cell activity. miR-129 could target Notch-1, Bcl-2 and other related genes predicted by miRanda and DIANA microT v3.0, while miR-873 could target Bcl-2 and other tumor related genes.(2) QPCR detection results show that, miR-129 and miR-873 expression significantly decreased in glioma tissues and cell lines compared with normal brain tissues.(3) Overexpression of miR-129 not only inhibit proliferation and promote apoptosis, but also trigger autophagy in U87 and U251 cells. Inhibit endogenous miR-129 can inhibit rapamycin-induced autophagy effectively. The result of implantation experiment showed that the tumor volume in Lv-miR-129 injected group was smaller that Lv-NC or PBS injected groups, and the expression of LC3 and beclin-1 was upregulated in tumors.(4) Luciferase assay, qPCR and Western blot have established that miR-129 could regulate the expression of Notch-1. In addition, miR-129 could promote autophagy of glioma cells through Notch-1/mTOR or Notch-1/E2F7/Beclin-1 pathways.(5) miR-873 was reduced significantly in U87/DDP and U251/DDP cells compared to wild-type cells. Overexpression of miR-873 could inhibit proliferation and promote apoptosis of U87/DDP and U251/DDP cells. Moreover, miR-873 could increase the killing effect of cisplatin on U87/DDP and U251/DDP cells.(6) Luciferase assay, qPCR and Western blot have established that miR-873 could regulate the expression of Bcl-2. In addition, miR-873 could increase the killing effect of cisplatin on U87/DDP and U251/DDP cells by target Bcl-2.Conclusions:In summary, this study found that miR-129 and miR-873 were downregulated in glioma tissues and cell lines. Overexpression of miR-129 and miR-873 not only inhibit cell proliferation but also promote apoptosis in U87 and U251 cells. Moreover, miR-129 could promote autophagy of glioma cells through Notch-1/mTOR or Notch-1/E2F7/Beclin-1 pathways. Bcl-2 mediated the function of miR-873 sensitize glioma cells to cisplatin. miR-873 could increase the killing effect of cisplatin on cisplatin-resistant glioma cells by target Bcl-2. |
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