Single Minded 2-s(SIM2-s) Gene Is Expressed In Human GBM Cells And Involved In GBM Invasion

BackgroundGlioblastoma Multiforme (GBM) is the common malignant epithelial tumor in adult central nervous system, and accounts for nearly 10% in cranial tumors. GBM is developed by malignant change of astrocytoma and owns the most malignancy. The age of onset in GBM patients is usually between 45 to 70 years old. The medical history of GBM patients is very short correlated with a high mortality rate. GBM patients' average lifetime is about 6 months and half of them is less than 3 months. The current therapy of GBM generally is surgical resection, followed by adjuvant radiotherapy and chemotherapy. Despite recent advances in both diagnostic modalities and therapeutic strategies, it can not be radically cured. Novel therapies are needed to improve the prognosis and life quality of patients with GBM. Recently, it is well known that the basic reason of poor prognosis on GBM is such characteristics as low differentiation, anti-apoptosis and high invasion. In the process of searching newly emerging gene therapy, many genes associated with the malignant characteristics of GBM such as VEGF are chosen as the target for gene therapy. However, because the normal cells in many organs expressed VEGF, so it is difficult to utilize for the clinical diagnosis and therapy. If we can find a GBM specific target gene, it will be a breakthrough on the GBM gene therapy.Human Single-minded 2 (SIM2-s) gene is a member of SIM gene family, which located in the Down's Syndrome Critical Region (DSCR) of chromosome 21. Due to the selective shearing, the SIM2 gene product exists at least 2 different subtype, SIM2-s and SIM2-1. Both SIM2-s and SIM2-1 express in lung, kidney, skeletal muscle, testicle and tonsil, and there is low-level expression of SIM2-1 in bone marrow. In the development of embryonic, both SIM2-s and SIM2-1 express in fetal heart and fetal kidney. And research showed that SIM2 gene plays an important role in brain development and neuron differentiation. In the research of cancer incidence rate of Down's syndrome patients, there is a 20-fold elevated risk of leukemia in people with Down's syndrome, whereas solid tumor formation is relatively rare. The reasons include tumor suppressor genes on chromosome 21, a slower rate of replication and higher likelihood of apoptosis. In the further research of chromosome 21, the SIM2 gene may be involved in the proceed of solid tumor. There is a serial of studies showed that SIM2 gene highly expressed in colon carcinoma, prostatic carcinoma and pancreatic carcinoma, metastasis and cell line, while do not express in the corresponding normal tissues. And the expression of SIM2-s showed a stage-specific in colon carcinoma and prostatic carcinoma. In a univariate life span analysis of 103 prostatic carcinoma patients, the positive SIM2-s expression of tumor tissue is associated with the decreased life span. In the univariate analysis of preoperative serum PSA, histology classify, patho-stage and SIM2-s expression, only the histology classify and SIM2-s expression associated with life span. Research confirmed that human GBM cell line T98G expressed SIM2-s intensively. Thus, SIM2-s may be a potential target for GBM therapy.Until now, there are several strategies for blocking genes, including dominant negative, antisense nucleotide, biochemical agent, double-stranded or single-stranded "decoy" oligonucleotides and RNA interference (RNAi) technology. RNAi is a manner of post-transcriptional gene silencing and is one of the anciently and evolutionarily highly conserved phenomenon in living nature. RNAi inhibits gene expression pathway with specificity and high performance which is mediated by small interfering RNAs (siRNAs). siRNAs identificate and cut homologous target mRNA. On account of rejecting or closing specific gene expression by using RNAi, it has been widely applied to explore gene function, infectious disease and gene therapy of malignant tumor. In the present study, the RNAi technology targeted inhibiting SIM2-s was employed to investigate the influence on human GBM cell invasion. The study was divided into three parts.Part I Expression of SIM2-s in Human GBM Samples and T98G and U251 Cell LinesObjectiveTo detect the expression level of SIM2-s in human GBM cell lines and clinical specimens. This study tried to provide a basis for further evaluation of SIM2-s as a therapeutic agent against GBM.Methods1. SIM2-s mRNA and protein expression of 115 cases clinical specimens were detected by RT-PCR, Western blot analysis and Immunohistochemistry analysis and expression positive rate was evaluated.115 cases clinical specimens including 63 cases gliomas,21 cases meningiomas,23 cases pituitary tumors and 8 cases normal brain tissues. There are 43 cases GBM of 63 cases gliomas.2. SIM2-s mRNA and protein expression in Human GBM cell lines T98G and U251 were detected by RT-PCR and Western blot analysis.Results1. No SIM2-s expression was detected in normal cortex, meningioma and pituitary tumor at mRNA level or protein level. However, the expression of SIM2-s in glioma samples and GBM cell lines were significant. At mRNA level, we found 7.7% of Grade I-II,14.3% of Grade II-III and 41.9% of Grade IV samples were SIM2-s positive, and 15.4% of Grade I-II,14.3% of Grade II-III and 60.5% of Grade IV samples were positive at protein level.2. SIM2-s expression was detected in human GBM cell lines T98G and U251 at both mRNA and protein levels.Conclusion1. SIM2-s selectively expressed in gliomas compared with other common brain tumors and normal cortex. And the rate of positive expression increased significantly in correlation with the malignancy of glioma. And it provide a basis for the investigation of GBM gene therapy by inhibiting SIM2-s expression.2. SIM2-s expressed in human GBM cell lines T98G and U251, and they can be used for our further research.Part II Effects of Silencing SIM2-s Expression by siRNA Technology on GBM Cell Proliferation and InvasionObjectiveTo study the transfection efficiency of SIM2-s siRNA in T98G and U251 cell lines. To detect the silencing potency of SIM2-s siRNA in T98G and U251 cell lines. To detect the effect of silencing SIM2-s expression in GBM cell proliferation and invasion.Methods1. Fluorescence siRNA was transfected into T98G and U251 cell lines mediated by LipofectamineTM2000. The efficiency of transfection was observed under the fluorescent microscope.2. After transfected with siRNA, SIM2-s expression in T98G and U251 was detected by RT-PCR and Western blot analysis.3. After transfected with siRNA, the proliferation of T98G and U251 was detected by cell counting assay and MTT assay.4. After transfected with siRNA, invasion of T98G and U251 was detected by Transwell assay.Results1. SIM2-s siRNA could be highly efficiently transfected into GBM cell lines by LipofectamineTM2000.2. SIM2-s siRNA could efficiently silence the expression of SIM2-s in human GBM cell lines T98G and U251. SIM2-s siRNA caused a 60% to 80% decrease of target gene in mRNA level and a 40% to 50% decrease in protein level.3. After transfected with SIM2-s siRNA, there were no proliferation differences in human GBM cell lines T98G and U251.4. After transfected with SIM2-s siRNA, the invasion of human GBM cell lines T98G and U251 decreased significantly.Conclusion1. SIM2-s siRNA can be highly efficiently transfected into GBM cells mediated by LipofectamineTM2000.2. SIM2-s siRNA can efficiently silence the expression of SIM2-s in human GBM cells.3. There is no difference in human GBM cells proliferation after transfected with SIM2-s siRNA.4. The invasion of human GBM cells decreased significantly after transfected with SIM2-s siRNA.Part III Investigate of the Mechanisms on GBM Cells Invasion Decreasing after Silencing SIM2-s Expression by siRNAObjectiveTo detect the invasion associated genes such as matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 after silencing SIM2-s by RNAi technology for elucidating the mechanisms of GBM cells invasion decreasing.Methods1. After transfected with SIM2-s siRNA, RT-PCR and Western blot analysis were applied to detect the expression of invasion associated genes such as MMP-2, MMP-9.TIMP-1,TIMP-2.2. In order to detect the change of MMP activity of cell-culture supernatants, unreduced samples were applied by SDS-PAGE gelatin zymography using a 10% polyacrylamide gels containing lmg/ml gelatin following the previously published procedure.Results1. After silencing SIM2-s by RNAi, the decreased invasion of human GBM cells was indeed associated with MMPs. MMP-2 mRNA levels of GBM cells transfected with SIM2-s siRNA were apparently downregulated compared with that of mock and control RNA. On the contrary, the mRNA levels of TIMP-2 were increased. The mRNA expressions of MMP-9 and TIMP-1 remained unchanged in GBM cells treated with SIM2-s siRNA. The Western blot analysis showed that the active form of MMP-2 was downregulated in GBM cells transfected with SIM2-s siRNA compared with that of control RNA, while TIMP-2 was increased, which was consistent with the mRNA level.2. The enzymatic activity of MMP-2 in the conditioned medium from each cell line detected by the gelatin zymography was clearly decreased under SIM2-s siRNA treated.Conclusion1. After silencing SIM2-s in human GBM cells by siRNA, the decreased potential of invasion maybe associated with downregulation of MMP-2 and upregulation of TIMP-2.2. After silencing SIM2-s in human GBM cells by siRNA, the enzymatic activity of MMP-2 in the conditioned medium from each cell line decreased significantly, which confirmed the decreased potential of invasion was associated with MMPs in GBM cells.In summary, our data showed that SIM2-s expressed selectively in gliomas especially GBM cells and the positive rate of SIM2-s was associated with the gliomas WHO grade. Meanwhile, we provided the evidence of a functional role of SIM2-s on invasive potential in GBM cells. The present study revealed that knock down of SIM2-s decreased invasion of GBM cells, which may associated with downregulation of MMP-2 and upregulation of TIMP-2. These results is helpful to throw lights on future studies that should extend these findings to invivo tumor models, and may provide a theoretical guidance for clinical application on GBM gene therapy.