MiR-218-5p Inhibits The Stem Cell Properties And Invasive Ability Of A2B5~+/CD133~- Subgroup Human Glioma Stem Cells

Background and objective: Malignant gliomas, which are the most common primary tumors of the central nervous system, are characterized by high capacity for invasion, proliferation and recurrence. Presence of glioma stem cells(GSCs) are the root of the occurrence and recurrence of glioma, the key to treat gliomas is completely remove the tumor, and the complete elimination of glioma stem cells. Depending on glioma stem cell surface marker expression, may be divided into different subsets GSCs, relatively few studies on the biological characteristics of present different subsets of GSCs. Micro RNAs(mi RNAs) act as oncogenes or tumor suppressor genes, and regulate proliferation, apoptosis, invasion, differentiation, angiogenesis,chemotherapy and radiotherapy resistance and behavior of glioma stem cells, which are important in glioma development and recurrence. In recent years, we obtained a new glioma cell line, SHG-139, from clinical specimens. Subsequently, SHG-139 glioma stem cell spheres(SHG-139s) were successfully collected after culture in neural stem cell medium(NSCM) and propagated for 10 passages. The molecular biological characteristics of SHG-139 s, in addition to the pathological and histological characteristics of intracranial xenografted tumor were confirmed in our laboratory. Surprisingly, we identified SHG-139 s as an A2B5+/CD133-glioma stem cell line, which is in contrast to the majority of GSCs, which express the CD133 marker. Using Gene Chip analysis, we subsequently showed that mi R-218-5p was expressed at significantly lower expression in SHG-139 s compared with SHG-139.Therefore, this study was conducted to investigate the hypothesis that mi R-218-5p plays an important role in maintaining the stemness and invasive ability of SHG-139 s.Methods: q RT-PCR was used to detect mi R-218-5p expression in noncancerous brain tissue, human glioma tissue, human glioma cell lines and human glioma stem cell lines. Lentivirus vectors encoding mi R-218-5p and anti-mi R-218-5p were constructed and stably transfected into A2B5+CD133- SHG-139 s cells. Neurosphere formation and transwell assays, and immunofluorescence and q RT-PCR analyses were used to explore the role of mi R-218-5p in SHG-139 s.Results: q RT-PCR analysis showed that mi R-218-5p expression was lower in human glioma tissues and cells than in noncancerous brain tissue and normal human astrocyte cells, and lower in A2B5+/CD133-(SHG-139s) cells than in CD133+ cells(SU2, U87s) cells. Immunofluorescence staining and q RT-PCR showed that mi R-218-5p reduced stem cell marker(A2B5, Nestin, PLAGL2, ALDH1, SOX2) expression compared with controls; however, immunofluorescence staining analysis showed that upregulated mi R-218-5p expression led to no difference in CD133 expression. Mi R-218-5p reduced SHG-139 s cell invasiveness in transwell assays and reduced MMP9 expression detected in q RT-PCR and immunofluorescence analyses. All differences were statistically significant.Conclusion:(1)The expression of mi R-218-5p was low in glioma tissue and glioma cells.(2) The expression of mi R-218-5p was low in SHG-139 s human glioma stem cells.(3)Mi R-218-5p is implicated as a tumor suppressor gene in glioma that functions by upregulating mi R-218-5p expression, which inhibits the stem cell and invasive properties of SHG-139 s.