Study On Brain Tumor Stem Cell Related Gene, MicroRNA Expression And RNAi In Glioma

Backgroud:Glioma is the most common primary malignant tumor of the nervous system. Traditional treatment includes surgical resection, radiotherapy and chemotherapy. However, because of the high invasiveness of the tumor cells, the postoperative recurrence rate is very high. According to the Brain Tumor Stem Cell (BTSC) theory, we have a new perspective about gliomas'occurrence and development mechanisms. Also it offers the hope for promoting a new type of molecular targeted therapy.In the development of nervous system, Hedgehog (HH) signaling pathway plays a key role on neural stem cells. Meanwhile, microRNA is a hot research area in the recent days. It is considered that microRNA plays important role in BTSC self-renewal and differentiation. Some microRNA can influence the expression of important genes of human glioma stem cells through negative regulation. Therefore. understanding the BTSCs in glioma-related genes, microRNA expression and the role and mechanisms of intervention in the treatment of gliomas is of research significance.Objectives:This study aims to separate and identify the BTSCs in the human glioma cells (primary cells or U87-MG cell line). Glil and MiR-210 expression are evaluated in BTSCs and normal brain tissues. By using Glil-siRNA to block the pathway in differentiation of BTSCs, we identify its influence on the pathway's activity, proliferation, apoptosis and migration.Methods:The BTSCs were isolated from human glioma cells by magnetic activated cell sorting (MACS). We identified the characteristics of BTSCs by testing the expression of CD 133+through flow cytometer and xenograft tumor formation in nude mice. The expression level of Glil and MiR-210 in BTSC and gliomas of different histological grade was evaluated by real-time PCR. We also applied Glil-siRNA to block the HH pathway, and identified its influence on the expression level of Glil and MiR-210, proliferation, apoptosis and migration of glioma cells.Results:(1) MACS method can isolate CD133+brain tumor stem cells successfully from glioma cells.(2) Brain tumor stem cells have high expression of Glil. But the expression of Glil has not related to the histological grade of the tumor.(3) Brain tumor stem cells have high expression of MiR-210. But the MiR-210 expression is not correlated with the histological grade of the tumor.(4) Glil-siRNA can suppress the HH signaling pathway successfully. But the MiR-210 expression is not influenced.(5) Glil-siRNA inhibition of HH signaling pathway can inhibit the growth of glioma cells(6) Glil-siRNA inhibition of HH signaling pathway can promote apoptosis of glioma cellsConclusions:MACS method can isolate CD133+brain tumor stem cells successfully from glioma cells. This sub-group of cells exhibited characteristics of brain tumor stem cells. Brain tumor stem cells have high expression of Glil mRNA in primary glioma cells and U87-MG cell line. However, the expression of Glil has not related to the histological grade of the tumor. Brain tumor stem cells have high expression of MiR-210 in primary glioma cells. The MiR-210 expression is not correlated with the histological grade of the tumor. Glil-siRNA transfected glioma cells can inhibit the HH signaling pathway. The proliferation of glioma cells was inhibited. Apoptosis was increased, invasive ability was reduced, but MiR-210 expression was not significantly changed.