Study Of RTN4Expression In Regulation Of Malignant Behaviors Of Glioma And Its Possible Mechanism

Gliomas are common brain tumors with a high rate of recurrence and mortality.Though a lot of new technologies have been used for improving the therapeutic efficacy,the prognosis of glioma patients is not very well. Recent years, studies showed that the RB,PI3K and TP53pathway are critical to the oncogenesis and tumorgenesis of gliomas. A lotof studies have been focused on the molecular determinants of the malignant biologicalbehaviors of gliomas.RTN4is one of the members of the reticulon protein family which play an importantrole in mammalian central nervous system (CNS). Due to both alternative RNA splicingand different promoter usage, it has three splice isoforms, termed as RTN4A, RTN4B andRTN4C. RTN4was considered to be a tumor suppression gene in glioma and its splicingvariants exerted marked inhibition in cell growth. However, some studies showed thatRTN4A expression generally in gliomas. Contrary to the notion of RTN4as a tumorsuppressor gene, some studies have shown that RTN4A can be strongly expressed in mostof oligdendroglial tumors, even in glioblastoma. As a tumor suppression gene, whatfunctions RTN4exerted in glioma remain unknown. Review of RTN4functions inmalignant behaviors of gliomas is limited. In the current study, we will investigate the roleof RTN4in glioma via RT-PCR, MTT assay, colony formation assay, analysis of cell cycleand RNAi strategy.Part I RTN4expression pattern in gliomas and its relationship with pathologicaltypies and gradesPurpose: RTN4, one member of reticulon family, is involved in regulation of many cellbiological behaviors. But its expression pattern in gliomas remains unknown. In this part,we will use RT-PCR to examine RTN4expression in glioma tissues and cell lineages U251,U87, U373, A172. The relationship between its expression level and glioma pathologicaltypies and grades will be elucidated.Method:1) Collect10glioma tissue samples from patients via surgery. Each sample wasconfirmed by pathologists. By using real-time PCR, the expression level of RTN4wasdetected in glioma cells.2) Take advantage of RT-PCR, further we examined theexpression of RTN4mRNA in U251, U87, U373and A172cell lineages.Results: RTN4expression was general in gliomas. In some samples, RTN4expressed at ahigh level. RTN4expression in three low-grade gliomas (1.76±1.44) was found no statistical difference compare to its expression in7high-grade gliomas(2.34±2.53). Thereis statistical difference of RTN4expression between oligodendrogliomas（4.67±4.75）andastrocytomas（1.54±0.85）. In U251, U87, U373and A172cell lineages, RTN4expressionwas different in each lineage.Conclusion: RTN4expression can be found in glioma tissues and cell lineages and isextraordinary high in some samples.But there is no significant association between RTN4expression level and glioma pathological grades. However, we found significant differenceof RTN4expression between oligodendrogliomas and astrocytomas. Further studies wewill focus the question that why a tumor suppression gene overexpresses in gliomas. Part II Construction and identification of lentiviral-mediated RNA inte rferencetargeting RTN4mRNAPurpose: Previous studies we found RTN4expression pattern in glioma cell lines andtissues. To further clarify the function of RTN4in glioma, we first construct shRNAtargeting RTN4mRNA and block the endogenous RTN4expression in glioma cell lines.Method:1) Use the software to design appropriate siRNA targeting sequences. Thesesequences were inserted into plasmid via gene combination technology.2) Plasmid withshRNA targeting RTN4and control shRNA was sequencing. The sequeces were consistentwith those we designed. Then U251cell was transfected with these shRNAs. RT-PCR wasused to examine RTN4expression changes in U251cells after transfection.Results: ShRNA targeting RTN4and control shRNA were constructed successfully. Aftertransfection, RTN4expression in U251was inhibited (0.25±0.05) compared to the controlgroup (0.98±0.02)(P<0.05).Conclusion: We successfully constructed shRNA against RTN4mRNA. RTN4expressionin U251cells can be inhibited after transfection efficiently Part III Effects on malignant behavior of glioma cells induced by blocking RTN4mRNA expression with RNAi stragedy in glioma cell lineages. Purpose：In previous studies we found that RTN4expresses in gliomas generally. Then weconstructed shRNA aganist RTN4mRNA to knockdown endogenous RTN4expression inglioma cell lines. We will elucidate the functions of RTN4in glioma biological behaviors.Method: Transfected cell lines U251, U87and U373with shRNA against RTN4mRNA.MTT, colony formation were applied to assess the proliferation of glioma cells. Flowcytometry was applied to examine the cell cycle changes after transfection. Western blotwas used to measure the expression levels of downstream cell cycle related proteins.Results: After transfected with shRNA against RTN4mRNA, proliferation and the colonyformatting ability of U251, U87and U373glioma cells were inhibited compared to cellstransfected control shRNA(shCON). Cell cycle of U251cell was arrested at G0/G1phasewhen RTN4was knockdowned. By western blot we found CDK4and CDC25A wasdownregulated and p21was upregulated while RTN4expression was inhibited.Conclusion: Knockdown of RTN4can inhibit glioma cell proliferation via arrest cell cycleat G0/G1phase. Downregulation of CDK4and CDC25A and upregulation of p21wereinvolved in this procession.