MicroRNA-134Regulates Biological Activities Of Glioma Cells Via Reducing Nanog Expression

Objective To detect the expression and significance of miR-134in differentpathological glioma tissues in vivo and glioma cell line U87and U251in vitro.MiR-134expressing U87stable cell line was generated for the effect of microRNA-134on NANOG mRNA and protein level with lentivirus transfection method. And toinvestigate the effect of miR-134induced Nanog knockdown on proliferation andapoptosis in U87glioma cell in vivo and vitro, which lay the foundation for thesubsequent further researches about Nanog related pathway in glioma gene targetedtherapy.Methods (1) The expression of miR-134was detected by SYBR green real-timequantitative reverse transcription-PCR (Real-time PCR)analysis in the pathologicalspecimens from42cases of glioma tissues and11cases of normal brain tissuesrespectively.(2) pLenti-miR-134-GFP or pLenti-GFP were transfected into U87gliomacells respectively. MiR-134expressing U87stable cell line was generated withlentivirus transfection method. Then Nanog mRNA and protein expression weredetected by RT-PCR and Western blotting, respectively.(3) MTT assay was used todetect the modification of glioma cell proliferation; Effects of migration and invasion bymiR-134on U87cells was evaluated by Transwell assay; Cell apoptosis wasinvestigated by Flow cytometry (FCM) and transmission electron microscopy; Theinhibiting effect of miR-134ectopic expression on the growth of glioma xenograft tumors in nude mice were also observed.Results (1) Real-time PCR analysis revealed that the expression of miR-134wassignificantly lower in glioma samples compared with normal brain tissues(P<0.05), andwas lower in grade Ⅲ and grade Ⅳ gliomas compared to gradeⅠand gradeⅡtumors(P<0.05). Meanwhile, remarkable downregulation of miR-134could also be observed inglioma cell lines U87and U251(P<0.05).(2) After U87-miR-134stable cell line wasgenerated by Blasticidin S, green fluorescent signal was detected in more than98%GFP-labeled miR-134-transfected U87cells. MiR-134expression was significantlyincreased in U87-miR-134cells, while in Vector-control cells it did not changeobviously. Furtuer results revealed that miR-134can both restrain the mRNA andprotein expression of Nanog in glioma cells by RT-PCR and Western blotting.(3) Wefurther detected the effect of miR-134mediated NANOG decrease on the proliferativeability of glioma cells by MTT assay. The result revealed that U87-miR-134cellsshowed a significant reduction in cell viability compared to Vector-control or the Blankgroup (P<0.05). To confirm the effect of miR-134in vivo, tumor xenograft animalmodel was performed, which showed that high miR-134level significantly sloweddown tumor growth in vivo (P<0.05), while there was no statistical difference betweenVector-control and Blank groups (P﹥0.05). The average tumor weight in theVector-control group was significantly higher than U87-miR-134group (P<0.05).Transwell assay (without Matrigel) showed that the migratory speed of U87-miR-134was markedly slower than that of control cells (P<0.05). Furthermore, Transwell matrixpenetration (coated with Matrigel) assay showed that the upregulation of miR-134dramatically reduced the invasiveness of U87cells (P<0.05). To confirm the effect ofmiR-134mediated apoptosis of U87glioma cells, flow cytometry, DAPI staining andtransmission electron microscopy methods were used. The percentage of the apoptoticcells were significantly higher in U87-miR-134compared to the Vector-control and the Blank group by flow cytometry using Annexin V-PE/7-amino-actinomycin Ddouble-stained U87cells.(P<0.05). Typical apoptotic morphological changes werefound in U87-miR-134cells which included shrinkage, deformation, and detachmentfrom culture dishes. Nuclear condensation and chromatin margination were evidentlyobserved using an inverted fluorescence microscope, and a transmission electronmicroscopy.Conclusion (1) These results suggested that miR-134could be closely related to humanglioma and the low level of miR-134might be bound up with glioma oncogenesis andits invasive propensity.(2) Successful establishment of miR-134expressing U87stablecell line, which lay a foundation to the subsequent further explore on the role ofmiR-134target genes in biological behaviour of gliomas.(3) These findings indicatedthat exogenous miR-134was able to inhibit glioma growth in vitro and vivo. MiR-134could substantially inhibit migration and invasion of U87glioma cells in vitro. Thesestatistical results and morphological changes mentioned above elucidated the apoptoticinducing ability of miR-134to repress tumor proliferation.