To Study The Regulation Mechanism Of MiRNA-21 And LncRNA HOTIR In Human Glioma

Objectives: 1. To establish lentivirus containing mi R-21 inhibitor and luciferase reporter plasmid system for PPARA and VHL. To confirm that PPARA and VHL were direct targets for mi R-21 and try to elucidate the regulatory mechanisms of mi R-21 in regulating EGFR signaling. Combination of mi R-21 inhibitor and nimotuzumab in treating glioma both in vitro and in vivo, and providing new ideas for optimizing glioma therapy. 2. To find signal pathways regulated by lnc RNA HOTAIR through bioinformatics. To clarify the molecular mechanisms of lnc RNA HOTAIR in modulating glioma cell cycle progression by introducing small molecule inhibitors and expression plasmids.Methods: 1. Infecting U87, LN229 and U251 glioma cells with lentivirus containing mi R-21 inhibitor, and detecting the expression changes of EGFR, p-EGFR, AKT and p-AKT by Western blot. Cell colony formation, flow cytometry analysis and transwell were used to examine the effects of mi R-21 on cell proliferation, cell cycle progression, cell invasion and apoptosis.2. Finding putative targets for mi R-21 and confirming that PPARA and VHL are direct targets for mi R-21 by using luciferase reporter system. Immunofluorescence and immunoprecipitation were introduced to study the regulatory mechanisms between β-catenin and PPARα.3. Establishing intracranial model for glioma and investigate the inhibitory effects of combination therapy of mi R-21 inhibitor and nimotuzumab.4. Confirming putative downstream pathways of HOTAIR by bioinformatics. Treating U87 and LN229 cells by using lentivirus containing HOTAIR si RNA segments, DZNep(small molecule inhibitor for EZH2) and 2-PCPA(small molecule inhibitor for LSD1), to examine its effects on cell cycle progression.5. Restore the expression of HOTAIR 3’ and 5’ functional domains to detect whether si-HOTAIR induced cell cycle progression inhibition could be partly reversed.6. Investigate the anti-tumor effects of si RNA HOTAIR in established intracranial glioma model.Results: 1. The expression of p-EGFR and p-AKT was reduced significantly in mi R-21 inhibitor transfected glioma cells; cell proliferation was slowed down, cell cycle was arrested at G1, more apoptotic cells were detected(p<0.01). 2. Western blot and luciferase reporter analysis showed that VHL and PPARA were direct targets for mi R-21. 3. Infecting lenti-AS-mi R-21 or transfecting VHL expression plasmid could all inhibit the activity of canonical Wnt/β-catenin signaling. Western blot and immunofluorescence showed that mi R-21 could regulate EGFR/AKT signaling pathway through PPARα/AP-1. 4. Immunoprecipitation results showed that β-catenin formed a complex with AP-1, and mi R-21 could regulate the formation of this complex. 5. Combination of mi R-21 inhibitor with nimotuzumab worked better than single drug therapy. 6. Bioinformatics showed that HOTAIR regulated genes were cell cycle related. 7. Small molecule inhibitor of EZH2, DZNep could mimic the effects of si RNA HOTAIR to arrest cell cycle at a G1 phase. 8. Restoration of 5’ domain of HOTAIR could partly reverse the cell cycle blockage effects of si RNA HOTAIR. 9. Intracranial model results confirmed that si-HOTAIR could inhibit tumor growth in vivo.Conclusions: 1. mi R-21 regulates EGFR/AKT signaling through VHL/β-catenin and PPARα/AP-1 complexes. 2. There is a feedback loop between mi R-21 and EGFR 3. Combination therapy of mi R-21 inhibitor and nimotuzumab works better than single drug therapy. 4. Lnc RNA HOTAIR regulated genes are cell cycle related; HOTAIR modulate glioma cell cycle progression through the binding of its 5’ domain with PRC2complex. 5. Knocking down the expression of HOTAIR inhibits the proliferation of glioma in vivo.