Preliminary Study About The Impact Of PEBP4 Expression On Biological Behavior And Chemotherapy Sensitivity In Brain Glioma Cells

Being the most common tumor, gliomas may account for about 50% of the malignant tumors in the central nervous system. More than 14,000 new patients were diagnosed to glioma in United States each year, its annual national incidence rate of 0.005%. Glioblastoma patients accounted for 60-70% of the total, anaplastic astrocytoma accounted for 10-15%, anaplastic oligodendroglioma and glioblastoma, anaplastic astrocytoma oligodendrocytes approximately 10% the rest are rare tumors such as anaplastic ependymomas and anaplastic gangliogliomas. Because of diffuse and invasive growth, the surrounding normal brain tissue ill-defined and easy to relapse, the cure rate of glioma patients is low, resulting in poor prognosis and shorter overall survival. In spite of the optional treatment including surgical resection, radiotherapy and (or) chemotherapy and other comprehensive treatment, the median survival of glioblastoma patients is only 12 to 15 months, compared to 2-5 years with anaplastic glioma patients. Chemotherapy to gliomas in clinical, a considerable number of glioma resistance to chemotherapy is the main reason for the late ineffective treatment, and the first-line chemotherapeutic drugs just to maintain the actual efficiency of 20-30% recently. Therefore, an effective solution to drug resistance about chemotherapeutic drugs has become an important way to improve the survival rate of patients with glioma. Moreover the study of related regulation genes about glioma cells resistant behavior will help us further understand the molecular mechanisms of resistance, and could guide the clinical development of personalized chemotherapy and prognosis of glioma patients, which also provides a relatively ideal target gene for molecular biological therapy on gliomas in the future.As a new member of the phosphatidylethanolamine binding protein family, PEBP4 has been found in many human tissues, it is involved in physiological and pathophysiological processes membrane biosynthesis, neuronal development, sperm formation and apoptosis, it is a ubiquitous multifunctional complex protein in vivo. Recent studies have showed that PEBP4 plays a role in many physiological and pathological about anti-apoptotic in tumor cells, and it also play a role on tumor promotion in the development of tumor function. Highly expression of PEBP4 in tumor cells can inhibit apoptosis induced by TNF-α (tumor necrosis factor -α) or TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), such as inhibition expression of apoptosis-related proteins p53, p21CIP/WAF and (Bcl2- associated X protein) BAX, while increasing expression of apoptosis inhibitory protein Bcl2 and Bcl-XL. In addition, PEBP4 not only involve in suppressing MAPK signaling pathway, but also inhibit JNK signaling pathway and activate Akt pathway, which affects cell cycle and apoptosis of tumor cells. Thus PEBP4 is a critical factor which regulates multiple signaling pathways in tumor cells, and these upstream signaling pathways can regulate cell growth, division and apoptosis, the in-depth studying on PEBP4 will contribute to further understanding of complex biological mechanisms in tumor cells.In the present study, we will investigate the specific biological function of PEBP4 in glioma. In first, we will detect the gene and protein expression of PEBP4 at different WHO levels of glioma tissue and normal tissue, and analyze the relation between PEBP4 expressions with clinical data in particular prognostic. Secondly, the use of small interfering RNA interference and lentiviral vector, constructing different PEBP4 expression levels of cell lines, which provide a opportunity to observe its effect on glioma cell line proliferation, invasion and cell cycle of malignant biology. Subsequently, after detecting proliferation and invasion of PEBP4 interference glioma cell, and the relationship between the expression of cell cycle and apoptosis-related proteins, we will analyze PEBP4 molecules whether regulate ERK1/2 signaling pathway or not. Finally, PEBP4-disturbance cell lines and glioma cells will beco-cultured in temozolomide, and observe the effect of PEBP4 expression on chemosensitivity. The experiment consists of three parts:The first part, PEBP4 expression levels at different levels of brain glioma and their correlation with prognosis will be observed; the second part, the carrier glioma cell lines infected with lentivirus by PEBP4 interference, observation PEBP4 expression on glioma cell proliferation, and invasion of the cell cycle, while the use of shRNA interference cell PEBP4 expression studies ERK1/2 pathway glioma proliferation, invasion and apoptosis; the third part, to observe the expression levels PEBP4 glioma cell line for TMZ susceptibility and possible mechanisms discussed shRNA-PEBP4 joint temozolomide effect on the ability to clone cell lines.Part I Expression of PEBP4 in glioma clinical tissue specimensObjective:Study PEBP4 mRNA and protein expression levels in different grade glioma clinical tissue samples to explore the relationship between PEBP4 expression and clinicopathological factors and the impact on prognosis.Methods:Immunohistochemistry and Western blot PEBP4 in protein expression levels 58 cases of glioma at different levels, as well as mRNA expression level of Real-time PCR detection of PEBP4. Verify the relationship between clinical and pathological factors and their influence on the prognosis of glioma patients by statistical methods.Results:The mRNA expression in tumor tissues may PEBP4 level was positively correlated with glioma (p<0.05), while the expression PEBP4 relative to normal brain tissue was significantly higher, which is consistent with the expression of mRNA, and the PEBP4 with the expression of glioma becomes high level and significantly increased (p <0.05). Immunohistochemical detection found PEBP4 mainly expressed in the cytoplasm and cell membrane, diffuse distribution, brown or brown particles are mainly concentrated in the cell membrane in the cytoplasm. Clinical study found, PEBP4 expression and patient age, gender, KPS scores were not statistically significant, but is closely related with glioma WHO grade (p= 0.023). Univariate and multivariate Cox regression analysis showed survival time of patients with glioma WHO grade (p= 0.013) and PEBP4 expression (p= 0.006) significantly correlated. Further multivariate Cox regression analysis showed that, in all clinical pathological factors and biochemical parameters, PEBP4 expression was the only independent prognostic factor for judging glioma. Moreover, high expression PEBP4 is the poor prognosis of glioma patients independently.Results:With the pathological grade of glioma increased, while the expression PEBP4 consequent increase, it is an independent prognostic factor intervention impact, and high expression of poor prognosis PEBP4 prompt expression of glioma patients.Conclusions:With the increasing of glioma pathological grade, the expression PEBP4 is greatly enhanced correspondingly. The high expression of PEBP4 is identified as an independent predictor for poor prognosis of patients with gliomas. Meanwhile, high PEBP4 is associated with shorter survival time in patients with gliomas.Part II Researches on function and molecular mechanisms of PEBP4 in glioma cellsObjective:Using a lentivirus vector to inhibit the expression of U251 and U373 glioma cells in PEBP4 explore, invasion of PEBP4 change on line U251 and U373 glioma cell proliferation and apoptosis. Meanwhile shRNA interference PEBP4 expression of glioma cell lines U251 and U373 cell cycle, proliferation, invasion and apoptosis related important factor expression. And further study of the cell line U251 PEBP4 down expression, ERK1/2 signaling pathway related proteins change.Methods:PEBP4 interference using lentiviral vector-infected U251, U373 cell line was constructed PEBP4 low expression in glioma cell lines. MTT cell proliferation assay, Transwell invasive test to detect levels of cell cycle was measured by flow cytometry. Western blot evaluate the tumor cell proliferation, invasion and apoptosis-related protein p-Rb, Cyclin D1, E-cadherin, Bcl-2 caspase-3 and change, as well as measuring the change ERK1/2 signaling pathway. Further experiments using the U0126 treated cell lines U251, Western blot to detect changes in ERK1/2 and related proteins, again using the MTT assay the proliferation of tumor cells to change.Results:shRNA-PEBP4 interference vector with satisfactory results, the success of lentivirus infection after U373 and U251 cells significantly reduced PEBP4 expression. MTT assay results suggest that the expression of interference PEBP4 U251 and U373 glioma cell proliferation was significantly decreased (p<0.05). Invasive tests showed the downregulating after PEBP4 invasion ability of tumor cells was significantly reduced (p <0.01). Cell cycle detected after PEBP4 interfere with viral vector transfection U251 and U373, cells showed a significant G0/G1 phase arrest, S phase and G2/M phase shortening (p<0.05). Western blot further confirmed, PEBP4 Disturbance U251 and U373 cells, p-Rb cell cycle related factors and downregulation of Cyclin D1, and the invasion-associated protein E-cadherin increases, increasing pro-apoptotic factor caspase-3 expression, anti-withered death factor Bcl-2 expression decreased, while increased ERK1/2 phosphorylation level, while the total ERKl/2 is not affected. After using the U0126 to inhibit ERK1/2 pathway upregulation of caspase-3, Bcl-2 expression increased, and the test results indicate a trend similar PEBP4 after disturbance, while PEBP4 expression did not change significantly. Last added U251 cell line after shRNA-PEBP4 treated ERK1/2 inhibitor U0126, shR-PEBP4+NC group relative shR-ctrl+U0126 group, the proliferation of tumor cells decreased significantly, suggesting that the ERK1/2 is PEBP4 of downstream effector molecules.Conclusions:PEBP4 possibly indirectly affect cycle progression of tumor cells, which change the ability of glioma cell about proliferation, invasion and apoptosis by regulating ERK1/2 signaling pathway.Part Ⅲ The effect of PEBP4 expression on glioma cells sensitivity to temozolomideObjective:Discussion PEBP4 disturbance on glioma cell lines U251MG and U373MG to chemotherapy temozolomide sensitivity.Methods:Use PEBP4 interference lentivirus vector-infected U251, U373 cell lines established PEBP4 different expression levels of U251, U373 cell lines. This cell line with various concentrations of temozolomide total days of incubation, MTT assay on the third day of the inhibition rate and calculate the respective IC50 values. Colony formation test detect the clone formation ability of U251, U251-shRNA, U251-GFP, and U373, U373-shRNA, U373-GFP in temozolomide. Western blot detection U251, U373 cells after PEBP4 interference multidrug resistance protein (P-gp, MRP1) and repair enzyme (MGMT, MMR) changes in DNA damage.Results:Using three days MTT method to measure the inhibition of tumor cell growth rate and calculate the IC50, using result in U251 and U373 cell lines alone TMZ resistance index was 23.26±2.44ug/mL and 21.65±2.01ug/mL, while U251 U373 and empty vector group and TMZ combined effect of resistance index were 21.33±1.27ug/mL and 22.78±1.88ug/mL, however, interfere with the expression of the group and TMZ joint PEBP4 after U251 and U373 cells to TMZ resistance index were was 9.78±1.03ug/mL and 8.06±1.82ugmL, equivalent to the first two group index decreased significantly (p <0.05). In temozolomide concentrations 0,0.05,0.10,0.15 umol/L when cultured 72h, the survival rate of cloned cells U251-shRNA group were 0.745±0.09,0.34±0.08,0.09± 0.01,0.015±0.001, U373-shRNA cells group clone survival rates were 0.713±0.16,0.29± 0.07,0.06±0.01,0.01±0.001, two groups of cell lines were significantly lower than the same concentration compared to the control cell clone survival rate was significantly reduced (p<0.05). After the suppression PEBP4 expression, U251 and U373 cell lines P-gp and MRP1 protein expression had no change, and protein expression of MGMT and MMR were significantly decreased.Conclusions:PEBP4 downregulation can reduce glioma cells temozolomide resistance. shRNA-PEBP4 with temozolomide can significantly reduce the cloning ability of tumor cells, which may impact the DNA repair enzymes and apoptosis of glioma cell.