| Glioma is the most common malignancy of central nervous system. Approximately 40 percent are diagnosed as glioma in intracranial tumors. Difficulties on treatment are associated closely with the malignant biology phenotype of brain glioma, which including excessive proliferation, relentless invasion and antiapoptosis. Although the comprehensive treatment level of brain glioma is progressing continuously. However, the outcome of this malignancy is still not improved drastically. For example, patients with the most malignant histopathologic subtype, glioblastoma (GBM), carry the worst prognosis, with median survival period less than 12 months, despite surgical treatment combined with radiotherapy and chemotherapy. So, it has important science significance and clinical application value to identify new biomarkers of diagnosis and therapy of this malignancy for overcoming it.Wnt/β-catenin signaling is a conserved molecular mechanism in metazoan animals. This pathway broadly influence changes in gene expression that govern embryogenesis and postnatal responses, such as cell proliferation, cell-fate determination, cell survival, cell behavior and migration during morphogenesis. Abnormal Wnt/β-catenin signaling is associated with many human diseases, including cancer, osteoporosis, aging and degenerative disorders. As one essential regulator for the development of nervous system, Wnt/β-catenin signaling participates in the process of almost all aspects of neural development, including stem cell proliferation, maintenance and differentiation. Recently, Wnt/β-catenin signaling was reported to contribute to the formation of gliomas, and some proteins involved in the Wnt/β-catenin pathway were abnormally expressed in gliomas.FRAT1 has been identified as a positive regulator of the Wnt/β-catenin pathway, which can inhibit GSK3 activity and ultimately down-regulateβ-catenin. It has been proposed that activation of the Wnt signaling cascade can cause Dishevelled (Dvl) family protein to recruit FRAT/GBP into theβ-catenin degradation complex, leading to dissociation of GSK-3 from Axin and consequently stabilization ofβ-catenin. This may result in the cytoplasmic accumulation ofβ-catenin, which then translocates into the nucleus, where complexes with members of the TCF/LEF (T cell factor/lymphoid enhancer factor) family of transcription factors are formed and the transcription of Wnt responsive genes are then induced. Several transcriptional targets of Wnt/β-catenin signaling are known oncogenes in the pathogenesis of cancers. For example, two of the TCF/LEF target genes, c-myc and cyclin D1 are supposed to be involved in the oncogenic function of inappropriate Wnt signal activation. This mechanism is believed to contribute to tumor progression. Previous studies have shown that FRAT1 is strikingly overexpressed in several human cancers including esophageal cancer, cervical cancer, breast cancer, ovarian cancer, and so on. However, the relationship between FRAT1 and human gliomas is unclear.The possibility of FRAT1 as a new candidate biomarker for glioma remains to be substantially studied. For this, the current study was performed from four aspects as follows. 1. Significance of FRAT1 expression in human brain gliomasThree human brain glioma cell lines (U251, U87 and SHG-44), 84 cases of human brain glioma tissue and 6 cases of normal human brain tissue were investigated by immunohistochemistry for protein expression of FRAT1. The results showed that, 66.7% of tumor specimens expressed FRAT1 protein, whereas no FRAT1 protein expression was detected in normal human brain tissues. The immunoreactivity scoring (IRS) of FRAT1 protein in 84 cases of tumor specimens was 4.07±3.60, which is significantly higher than that of normal human brain tissues (P<0.01). There were especially significant difference (P<0.01 for all) respectively in the IRS of FRAT1 between benign and malignant brain glioma and among different pathological grades of glioma. FRAT1 protein was expressed in U251, U87 and SHG-44 cells, and expression level of FRAT1 protein in U251 cells is higher than others. The expression of Ki67 was investigated by immunohistochemistry, apoptotic cells were screened by TUNEL method, proliferative index (PI) and apoptotic index (AI) in brain glioma tissues were measured respectively. The results showed that, Ki67 expression could be detected in each tumor specimen, and the proliferative index (PI) of tumor specimens is (31.69±19.96) %. With the ascending of pathologic grade, the PI value of tumor specimens increased markedly (P<0.01); in addition, there was significant difference (P<0.01) between PI in FRAT1-positive group (42.43±17.51) % and that in FRAT1-negative group (14.27±7.43) %. PI of tumor specimens was positively correlated with FRAT1 IRS (P<0.01). Apoptotic tumor cells could be detected in all tumor specimens, and apoptotic index (AI) is (2.75±1.73) %. With the ascending of pathologic grade, AI increased markedly (P<0.01), and there was not significant difference (P>0.05) between AI in FRAT1-positive group (2.54±1.53) % and that in FRAT1-negative group (3.10±2.00) %. AI was significantly negatively correlated with FRAT1 IRS (P<0.05). Subsequently, 84 cases of human brain glioma tissue and 6 cases of normal human brain tissue were investigated by reverse transcription polymerase chain reaction (RT-PCR) for expression of mRNA of FRAT1 and by Western blot for expression of protein of FRAT1. The results showed that, both mRNA and protein of FRAT1 were expressed in human brain glioma tissue, while none was expressed in normal human brain tissues. With the ascending of pathologic grade of tumor specimen, the expression level of FRAT1 mRNA and protein increased (P<0.05). Furthermore, three human brain glioma cell lines (U251, U87 and SHG-44) were investigated by RT-PCR for expression of mRNA of FRAT1 and by Western blot for expression of protein of FRAT1. The results showed that, both mRNA and protein of FRAT1 were expressed in U251, U87 and SHG-44 cells, and expression level of FRAT1 mRNA and protein in U251 cells is higher than others. The results suggested that FRAT1 mRNA and protein were highly expressed in human brain glioma tissues and cell lines; in addition, the expression level of FRAT1 mRNA and protein increased markedly with the ascending of pathologic grade of tumor specimens. The results also suggested that FRAT1 protein expression was positively correlated with tumor proliferation, as well as negatively correlated with apoptosis of tumor cells, which indicated FRAT1 may be a valuable biomarker of brain gliomas.2. Establishment of human brain glioma cells in vitro by RNAi targeting FRAT1 geneAccording to FRAT1 cDNA sequence, the specific RNAi fragments targeting FRAT1 were designed and synthesized, which were cloned into pRNAT-U6.1/Neo vector, and the eukaryotic expression vector pRNAT-FRAT1 of FRAT1 shRNA was constructed. Sequencing proved the correct sequence of this vector. The pRNAT-FRAT1 vector, blank pRNAT-U6.1/Neo vector and pRNAT-NC vector were transfected respectively into human brain glioblastoma U251 cells by LipofectamineTM 2000 medium, and the permanent transfectants U251-S, U251-neo and U251-NC cells were established through selecting with G418. RT-PCR results showed that mRNA expression of FRAT1 was inhibited markedly in U251-S cells, whereas no obvious changes of FRAT1 mRNA expression in U251-neo and U251-NC cells were detected. The results of Western blot indicated that protein expression of FRAT1 was also suppressed significantly in U251-S cells, whereas no obvious changes of FRAT1 protein expression in U251-neo and U251-NC cells were detected. Based on the above results, FRAT1 RNAi vector was successfully transfected into glioma cells. Subsequently, the expression of FRAT1 mRNA and protein was inhibited, which may be an excellent cell model that could be used to exploit the effects of FRAT1 expression on the biologic features of brain gliomas.3. RNAi-mediated inhibition of FRAT1 leads to decreased proliferation, migration and invasion in glioma cellsThe cell growth curve drawn by MTT test showed that U251-S cells grew significantly slow than U251, U251-neo and U251-NC cells (P<0.01). The results of plate colony formation and Soft-Agar Assay showed that the colony number of U251-S cells markedly decreased than those of U251, U251-neo and U251-NC cells (P<0.01). The cell cycle analysis by flow cytometry (FCM) showed that the G0/G1 phase cells significantly increased, whereas G2/M phase cells markedly decreased in U251-S cells, as compared with those in U251, U251-neo and U251-NC cells. The invasiveness of cells was measured by using the monolayer wound healing assay and transwell invasion assay. The migration of U251-S cells declined notably (P<0.01). There were no obvious differences in migration among U251, U251-neo and U251-NC cells. Matrigel invasion assay showed that FRAT1 targeting shRNA significantly suppressed the invasiveness of U251 cells. The number of the invaded cells declined notably in U251-S cells (P<0.01) with contrast of the control groups. No obvious differences in invasion were observed among U251, U251-neo and U251-NC cells. These data indicated that FRAT1 inhibition suppresses migration and invasion of human brain glioma cells in vitro.4. Impact on tumorigenesis and proliferation of human brain glioma cells in nude mice by RNAi targeting FRAT1 geneU251, U251-neo, U251-NC and U251-S cells were inoculated respectively in flank subcutaneous tissue of nude mice to establish xenograft models of human brain glioma. The tumor growth status was observed and tumor volume was measured termly, and the tumor growth curve was drawn. The animals were killed and the tumor weight was investigated 40 days after inoculation. Subsequently, the transplanted tumors were obtained to perform FRAT1 immunohistochemical staining. The results showed that the tumorigenesis time delayed, tumor grew slow, both tumor volume and tumor weight decreased significantly (P<0.01) in U251-S group as compared with those in U251, U251-neo and U251-NC cells groups. FRAT1 protein expression was downregulated markedly in U251-S group in comparison with that in U251, U251-neo and U251-NC cells groups. The above results suggested that RNAi targeting FRAT1 can suppress significantly tumorigenesis and growth of human brain glioma in nude mice, which may be associated with marked inhibition of proliferation and invasion of human brain glioma cells.In summary, FRAT1 overexpression may be closely related with aggressive proliferation, invasion and anti-apoptosis of human brain gliomas. The above results provide important supplements for the research of Wnt/β-cantenin pathway. Meanwhile, FRAT1 may act as a valuable biomarker for molecular diagnosis of glioma and a potential target for gene therapy of glioma.
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