A Study Of The Therapeutic Potential Of NcRNA Interventions In Glioblastoma

Objectives:1.To verify the inhibitory effects of miR-23 b sponge-expressing lentiviruses on the expression levels of miR-23 b,VHL and the downstream targets of VHL,and the angiogenic,invasive and migratory capabilities of glioblastoma cells.2.To determine the association between SNORD76 and HOTAIR alterations,the functional implication and underlying mechanism of SNORD76 in gliomagenesis and the in vivo function of SNORD76-expressing lentivirus on the U87-MG orthotopic tumors,and to investigate the clinical relevance of SNORD76 in gliomas samples.Methods:1.After infection with the scrambled control-expressing or miR-23 b sponge-expressing lentivirus in U87-MG,LN229 and U251,q RT-PCR was utilized to quantify the miR-23 b expression and Western blot was adopted to determine the change in the proteins associated with angiogenesis,invasion and migration.Tubule formation,transwell and wound healing assay were then performed to further examine the effects of miR-23 b sponge on glioma cell angiogenic,invasive and migratory phenotypes in vitro.2.U87-MG derived orthotopic tumor mice models were constructed to evaluate the effect of miR-23 b sponge in vivo.In vivo imaging analysis of the mice was performed every 15 days for 45 days.Immunohistochemistry was utilized to confirm the change in the expression of proteins that had been observed in the Western blot analysis.The vascular formation was determined by immunohistochemical staining with CD31 antibody while the invasive and migratory abilities were defined by H&E staining.3.q RT-PCR analysis was performed to quantify the expression of HOTAIR,GAS5 and SNORD76 in 5 glioma cell lines(U87-MG,U87-EGFRv III,LN229,U251 and SNB19),and in glioma cells treated with HOTAIR-expressing(low HOTAIR expression),HOTAIR si RNA-expressing(high HOTAIR expression)or nonsense control-expressing(both)lentivirus.4.MTT assay and soft-agar colony formation assay were performed to measure the proliferative ability of glioblastoma cells.The glioblastoma cells with low SNORD76 expression were treated with SNORD76-expressing or nonsense control-expressing lentivirus,while those with high SNORD76 expression were treated with SNORD76 si RNAs or nonsense control.5.After infection with SNORD76-expressing or nonsense control-expressing lentivirus in U87-MG and U251,PI-staining flow cytometry analysis was performed to examine the change in cell cycle,and then western blot anaylsis were utilized the evaluate the expression of cell cycle associated proteins.6.In vivo imaging analysis of the mice bearing orthotopic tumors derived from U87-MG cells co-infected with lentiviruses expressing luciferase and a nonsense or SNORD76 was performed every 10 days for 30 days.Histochemical staining was utilized to visualize the orthotopic tumors and the cell cycle-associated proteins altered after the introduction of SNORD76.7.Quantification of the expression of SNORD76 and GAS5 was performed on surgically resected glioma tissues of WHO grade II(n = 20),grade III(n = 22),and grade IV(n = 21).Results:1.The introduction of miR-23 b sponge reduced miR-23 b expression by ~50% in all three glioblastoma cell lines,and thus elevated the expression of tumor-suppressive proteins(VHL and E-cadherin)and abrogated the expression of oncogenic proteins(HIF-1α,VEGF,MMP2,MMP9,β-catenin and ZEB1).As a result,the angiogenic,invasive and migratory capabilities were impaired by downregulation of miR-23 b,which was confirmed by tubule formation,transwell and wound healing assay.2.In vivo imaging analysis revealed the suppressed growth rate of the miR-23 b sponge-treated U87 cells,and treatment with the miR-23 b sponge was associated with a significantly longer survival.Immunohistochemical staining of paraffin-embedded tumor sectioned demonstrated a reduction in CD31-positive vessels after miR-23 b sponge treatment,and H&E staining of the miR-23b-downregulated tumors revealed less peripheral invasion.3.After infection with a lentivirus expressing HOTAIR si RNA,the HOTAIR expression reduced by approximately 50% in U87-MG and LN229.Consequently,SNORD76 in both cell lines increased by approximately 2-fold;however,in U87-MG,GAS5 showed an unexpected decrease,while in LN229 no significant change was detected.On the contrary,forced HOTAIR expression by lentiviruses expressing full transcript of HOTAIR led to a significant decrease in SNORD76 expression while no significant change in GAS5 expression was observed.4.Forced SNORD76 expression in U87-MG and U251 reduced the proliferation rate of both cell lines.In addition,anchorage-independent growth was also impaired in SNORD76 overexpressing groups,which were represented by a lower volume and number of spheroids compared with cancer cells infected with a nonsense control.In LN229 and SNB19 that express higher endogenous SNORD76,knockdown of SNORD76 using the selected si RNAs increased the proliferation rate and anchorage-independent growth,which led to a more malignant phenotype.Flow cytometry analysis showed that overexpressing SNORD76 resulted in the accumulation of cells in S phase at 48 h following lentivirus infection.Western blot analysis indicated that enforced SNORD76 expression elevated the expression of Rb and p Rb and downregulated the expression of cyclin A1,cyclin B1 and p107.5.In vivo imaging analysis of the mice 1,10,20 and 30 days after implantation revealed that the growth of orthotopic tumors was significantly inhibited by forced expression of SNORD76.Likewise,treating with SNORD76 was associated with a significantly longer survival.H&E staining showed that the SNORD76-treated tumor volume was reduced.Immunohistochemistry showed decreased expression of cyclin A1,cyclin B1 and p107 and the increased expression of Rb and p Rb in response to SNORD76 treatment.6.Compared with gliomas of WHO grade II and III,glioblastoma(WHO grade IV)displayed a significantly lower expression of SNORD76,while there was no significant difference between tumors with lower grades.In addition,the expression of GAS5,the host gene of SNORD76,showed no significant difference in the clinical samples.Conclusions:1.miR-23 b sponge elevates the expression of miR-23b-targeted VHL and diminishes the angiogenic,invasive and migratory phenotypes of glioblastoma cells in vitro.2.Ectopic expression of miR-23 b sponge in vivo impairs the growth of U87-MG cell derived orthotopic tumor and prolongs the overall survival of the mice.3.SNORD76 expression inversely associates with HOTAIR alteration in glioma cells.4.SNORD76 overexpression arrests glioma cells in S phase of the cell cycle by affecting the Rb-associated cell cycle regulation.5.SNORD76 suppresses the in vivo tumorigenicity of U87-MG cells and associates with a significantly longer survival of the mice.6.SNORD76 downregulation is associated with an aggressive phenotype in glioma.