Study On The Expression Of HMena In Glioma And Invasive Pattern Of Malignant Glioma

Malignant glioma is the most common rumors of the central nervous system. It accounts for 12% to 15% of the intracranial tumor. Its diffusely infiltrative growth characteristics results in the difficulty to resect totally during the operation. The prognosis is dismal and the median survival of patients ranges from 12 to 15 months despite of aggressive surgery combined with radiation and chemotherapy. Invasive nature of malignant glioma is a major of factors causing therapeutic failure. The invasive of tumor cells to adjacent parenchyma is a complex process. Of many factors involved, cell moving plays an important role in this process. hMena is a regulator of cell moving, which regulates the remodeling of actin skeleton and forming lamellipodia at the leading edge of the motile cell. It is reported that hMena is over expressed in many tumors including mammary tumor and pancreatic cancer. But in glioma, it has not been reported yet. In addition, the brain tumor stem cells are also indicate in some studies that they are involved in the invasive growth pattern.The present study included three parts:1. To observe the expression and distribution characteristics of human ortholog of mammalian enabled (hMena) in human glioma and analyze the correlation of hMena expression with the pathological grade of glioma,65 specimens of glioma with different pathological grades and 5 control brain tissues were collected. In 6 of the 21 glioblastoma patients, multi-specimens were obtained under assistance of neuronavigation system during the operation. We found that hMena expression was negative in control brain tissue but positive in different grades of glioma. The expression rate of hMena was positively correlated with the increasing grade of WHO classification (rs=0.682, P=0.000). Positive cells only distributed around the vessels within the tumor mass in low grade glioma, while in high grade glioma, these cells were able to be detected not only in the tumor but also in the boundary zone and adjacent brain parenchyma. In the tumor mass, hMena are expressed highly and diffusely. In the junction zone, hMena positive cells formed radiolitic pattern around the vessels. In adjacent brain parenchyma, single positive cell was scattered. hMena expression was markedly elevated in WHO Grade III and IV glioma compared with WHO Grade II and I glioma.2. In the first part of the research, we found that the distribution characteristics of hMena positive cells are extremely similar to that of BTSCs in malignant glioma. To explore the relationship between BTSCs and hMena expression, MACS was used to isolated CD 133+cells from U251 cells and LN229 cells. Western blot was used to detected the hMena expression both in CD133+ cells and CD133-cells, cell motility assay was used to assess their migrating ability. Result:36-48h after MACS, BTSCs formed cell sphere, which floated in the medium. The spheres of cells were closed to each other tightly. Immunofluorescence indicated that the cell sphere expressed CD 133 and nestin. Western blot indicated that hMena was over expressed in CD 133+ cells comparing to CD 133- cells both in U251 cells and LN229 cells (t=26.7, P<0.0001; t=13.08,P=0.0002). And CD133+ cells are more motional than CD133-cells (t=16.09, P<0.0001).3. To explore the role of hMena in the cell moving, siRNA was used to knock down the expression of hMena. Immunofluorescence was used to detect the distribution characteristics of hMena and lamellipodia formation in U251 cells. Cell motility assay was used to assess the migrating ability of U251 cells. Result:U251 cells of hMena siRNA group expressed hMena in low level compared to nonsense siRNA group. They changed to round morphology and were not able to form obvious lamellipodia, and their moving ability was weak comparing to the cells of nonsense siRNA group (t=7.506, P<0.0001). hMena are concentrated around the invading front of the lamellipodia in U251 cells of nonsense siRNA group indicated by immunofiuorescence, while this phenomenon was not able to be seen in hMena knocked down cells by siRNA.According to the research, we draw the conclusions:Firstly, the expression rate of hMena in glioma was positively correlated with the WHO grade of central nervous system tumor. Secondly, hMena positive cells are cluster around the vessels in malignant glioma and the distribution pattern is similar to BTSCs. This suggests these cells might come from the tumor mass. Thirdly, expression of hMena in high level in tunor cells benefits for cell moving and hMena could use for tracing the moving cells in the tumor.