Down-regulation And Inhibited Affection Of RRP22 In Astrocytomas

Background:Astrocytomas are the most common neoplasm of the central nervous system. Although progress has been made, the survival rate of astrocytoma is still poor. Malignant astrocytomas, such as glioblastoma multiforme(GBM, WHO grade IV), are one of the most aggressive brain tumors with the poorest prognosis in spite of treatment with surgery, radiotherapy, conventional chemotherapy. Therefore, improving the prognosis of patients with astrocytomas relies on effective therapies that are directed against unique molecular aberrations. With developments in molecular neurosurgery and recent advances in the understanding of genetic aberrations within gliomas, patient-targeted therapies have emerged to increase the therapeutic efficacy. However, most of the genetic aberrations found in gliomas were not specific, which is necessary for an accurate diagnosis. Ras was the first identified oncogene in human tumors, the aberrations have been identified in about 15% malignant tumors. In recent years, scholars have put forward the concept of Ras superfamily. Ras superfamily is an important kind of functional proteins. They play a important role in the regulation of cell growth, differentiation, survival, proliferation and other functions. Some members, such as NRAS or BRaf, are oncogenic; other members, such as RIG and ARHI, have been identified as tumor suppressors. RRP22, which is located on chromosome 22 on the 12q site, is a novel member of the Ras superfamily and exclusively expressed in the central nervous system. Current researches on the RRP22 were few, and often focused on the gene sequence and expression in several tumor cell lines. Howerver, there is nothing correlative report on its expression and mechanisms in human glioma. We have speculated that RRP22 play a important role in inhibited growth, decreased invasiveness and induced cell death.Objective:In our study, the RRP22 mRNA levels in astrocytomas of different clinicopathologic grades were determined for the first time. While RRP22 was transfected into a malignant cell line of astrocytomas, U251, the biological effects on tumor cells were analyzed.Methods:To examine whether RRP22 was down-regulated in astrocytomas, a quantitative RT-PCR assay was performed to analyze the mRNA level of RRP22. RRP22 mRNA levels were examined in 5 normal brain tissue samples and 61 astrocytoma tissue samples. Triplicate PCRs were performed on each sample, and the CT for RRP22 and?-actin were obtained. We calculated the mean CT for each sample and analyzed the mRNA level using the equation -??CT RRP22 (-??CT RRP22=-(?CT RRP22-?CT max),?CT RRP22=CT RRP22- CT?-actin). RRP22 was subcloned into pZsGreen1-C1, and the result were confirmed using restriction endonuclease digestion and gene sequencing. U251 cells were transfected with the recombinant plasmid pZsGreen1-C1-RRP22 by using Lipofectamin method, and quantitative PCR was used to confirm the results. Then, biological effects on tumor cells were analyzed using the method of MTT, Transwell, electron microscopy and flow cytometry respectively.Result:The RRP22 level in tumor samples was significantly down-regulated than that in normal brain tissues (P<0.01). A significant association was found between RRP22 mRNA levels and the pathological grades (grades?,?and?(or?), P<0.01). However, there was no significant difference between malignant grades?and?(p>0.05). When the RRP22 was over-expressed, RRP22 exhibited the properties of growth inhibition, decreased invasiveness, and cell death induction.Conclusion:1. The expression of RRP22 were found down-regulated in astrocytomas;2. The expression of RRP22 in astrocytomas is not only significantly decreased but is also correlated with the pathologic stage. Howerver, there was no significant difference between malignant grades?and?. The results showed that the aberrations of RRP22 may be used for accurate diagnosis in future;3. When RRP22 in astrocytoma cells was over-expressed,we foud inhibited growth, decreased invasiveness and induced cell death. Due to the unique expression of RRP22 in the brain, our results strongly suggest that RRP22 might be a specific biomarker for the diagnosis of astrocytomas and a therapy target in future studies.