The Mechanism Of Action Of SENP1 Expression Level For Glioma U251MG Cells And Its Relationship With Regulating Mechanism Of The AKT Signal Pathway

ObjectiveHuman brain glioblastoma accounted for 32% of all primary central nervous system tumors,81% of malignant tumors, originates from neuroectoderm, is the most common malignant brain tumor. According to the pathological grading standards proposed by WHO in 2007, can be divided into four grades: GradeⅠ for pilocytic astrocytoma, accounting for 5% of gliomas; gradeⅡ astrocytoma diffusion type, accounting for 30 to 40% of gliomas; Grade Ⅲ for anaplastic astrocytoma, about 15 to 25 percent, this grade tumors generally evolved from Grade Ⅱ; Grade Ⅳ for glioblastoma, intracranial gum account about 30% of stromal tumors. According to the relevant literature, the incidence of gliomas was 3.87 persons / 100,000, accounting for systemic cancer of 1% to 3%, after the comprehensive treatment with surgery, radiotherapy and chemotherapy the average survival is only 8 to 11 months, by this result, malignant glioma patients die as many as 60 million people each year, and that this number is still increasing year by year. Therefore, the human brain glioma study is an urgent issue. With the progress of the study, people will be more understanding of the occurrence and development of mechanisms of glioma, and provide a theoretical basis for the development of new anticancer drugs.By detecting expression level of SUMO-specific protease 1(SENP1)in normal brain tissue and different grades of astrocytoma tissues, to analyze the correlation with disease progression,and by using RNA interference to knock reduction the SENP1 expression in U251 MG glioma cells, we can study the impact of the proliferation and apoptosis in U251 cells for their expression levels, and molecular mechanisms which may exist.MethodsIn the m RNA and protein levels, we detect the differential expression of SENP1 in normal control group(three cases) and astrocytoma group(28 cases) specimens, by RT-q PCR and Western blottingThe si RNA specific for SENP1 was synthesized and transfected into U251 cells. U251 cells were inoculated in the 6-well plate respectively according to 2.5×105 cells/well in 2ml,after transfection with si-NC(normal control group), si-1 and si-2(SENP1 knock reduction group)for 12 hrs, when the cell fusion degree reached to over 90%, and the cells were incubated continually for 48 hrs for the detection at both m RNA and protein level.At the same time, silence SENP1 expression in U251 MG cell, the number of apoptotic cells was estimated by detecting the cell surface in FCM phosphatidylserine(PS).Finally, knockdown SENP1 in U251 cells, detect the protein expression and phosphorylation levels of intracellular AKT, as well as the protein expression level of p21, cyclin D1 which are this pathway downstream target factors by the Western blottingResultsAt the m RNA level,by q PCR detection From collected 31 cases of brain tissue samples we were found that: in the human brain astrocytoma, SENP1 m RNA expression levels were significantly higher than that in normal brain tissue(P<0.05), and with malignant tumors grade higher, the m RNA expression level higher than lower grades, in addition the phenomenon is much more obvious in the high-grade astrocytomas glioma(Ⅲ, Ⅳ level, WHO classification)(P<0.01); At the protein level, analysed the differences of protein levels by Western blotting between in normal SENP1 issues and different grade gliomas, the result is similar to the m RNA phenomenonAfter SENP1 si RNA transfected, SENP1 m RNA expression in U251 MG cells was inhibited(P<0.01), which means levels of protein expression were lower than normal.With analysis of apoptosis we found that compared with si-NC(normal control group), the si-1, si-2(SENP1 knockdown group) transfection U251 MG cells, which wre significantly increased the number of apoptotic, and statistical analysis of apoptosis rate was significantly increased(P<0.05) relative to the control group.After knockdown SENP1 in U251 MG cells, at the protein level by Western blot detecting the expression of Akt, cyclin D1, and p21,a cyclin-dependent kinase(CDK) negative regulator, the analysis results showed that compared with the normal control group the level of Akt phosphorylation was significantly reduced and the expression of its target factor cyclin D1 protein also decreased, and its downstream p21 protein expression which is associated with the cell cycle was increased.Conclusion1 The difference of SENP1 expression in normal human brain tissue and different grades glioma specimens are closely related with the tumor occurrence and development, which can be used as an important indicator of clinical monitoring and evaluation and prognosis.2 SENP1 can regulate anti-apoptotic pathway mediated by PI3K-Akt pathway, which means that it regulates Akt phosphorylation levels, to influence its downstream gene expression of p21 and cyclin D1, and then regulate U251 MG cell proliferation or apoptosis, further proved that it can promote the development and progression of cancer, SENP1 may be a new mechanism of molecular biology.