Expression Of CuG-binding Protein 1 In Human Astrocytoma And The Mechanism Of Effect Of Its Knockdown On U251 Cells.

Recently, the mechanisms of tumorigenesis have been the focus of scholars at home and abroad, and it has been given increasing attention to elucidate tumor genesis and metastasis from the perspective of tumor molecular biology. In China, the neurogliomas is the first neoplasm of nervous system with high incidence, rapid process, and poor prognosis, and the morbidity tends to rise at present. As a member of RNA binding protein CELF family, CUGBP1 can regulate the alternative splicing by stimulating inclusion or exclusion of the non-consecutive exons in the nucleus pre- mRNA. In the cytoplasm, it can adjust their translation or degradation by binding to the specific sequence in certain target mRNAs. It can also influence the mRNA editing through RNA editing enzyme complex. CUGBP1 is involved in embryonic and cardiac development, skeletal muscle and adipose tissue differentiation and germ cell formation, and also play a great role in the liver cells and mammary epithelial cell proliferation, tumor genesis, as well as the deterioration of certain tumors. Whether CUGBP1 is abnormally expressed in human glioma cells or tisses and involved in the glioma genesis and metastasis has not been documented at home and abroad.SignificanceWe have found CUGBP1 is highly expressed in glioma cells and its expression level is related with the pathological classes of human glioma. At the same time, our research has explained the correlation between CUGBP1 and genesis and metastasis of neoroglioma from the point of cell proliferation, apoptosis and the related genes alteration at their mRNA expression.ObjectiveCUGBP1 is expressed in different glioma cell lines, glioma brain tissue and normal brain tissue.Furthermore, cell proliferation, apoptosis and related gene expression are observed to have changed after CUGBP1 gene knockdown. We aim to analyze the correlation between CUGBJP1 and glioma genesis so as to elucidate the mechanism and effect of CUGBP1 in glioma genesis and metastasis. MethodsDetection of CUGBP1mRNART-PCR and real-time quantitative RT-PCR were used to observed and compare the expression of CUGBP1 at the mRNA level in different glioma cell lines, neuroglioma and normal brain tissue.By contrast of the housekeeping gene GAPDH, we detected the expression of CUGBP1 in different glioma cell lines through semi-quantitative RT-PCR.We detected the expression of CUGBP1 in different pathological class of neuroglioma by real-time quantitative RT-PCR, by contrast of normal brain tissue and GAPDH.Construction of RNA interfering lentivirus & Knockdown efficiencyTo further explorate the correlation between CUGBJP1 and glioma genesis and its mechanism, we congstructed CUGBP1-shRNA lentivirus and transfected them into human glioma cell line of U251, by contrast of Scr-shRNA lentivirus vector transfected U251 cells. We also identified their efficiency of transfection and knockdown.Interferring target sequences were designed, then connected to lentiviral vector and packaged into virus particles.The results of transfection were assessed through observation and cell counting by common microscopy and fluorescence microscopy.The effects of knockdown on CUGBP1 gene expression were detected in transfected human glioma cells by real-time quantitative RT-PCR.Cell proliferationCUGBP1 was closely related with proliferation of in liver cells and mammary epithelial cell in certain pathological contexts. We wondered whether there existed similar phenomenon in human neuroglioma. We knocked down CUGBP1 gene through transfecting CUGBP1-shRNA lentivirus vector into glioma cells to reveal its relationship with cell proliferation.Cell counting was performed and taken down for CUGBP1-shRNA lentivirus transfected glioma cells after transtection at different time point (1d, 2d, 3d, 4d and 5d) through CELLOMICS, by contrast of scr-shRNA lentivirus transfected cell. Their growth curves were drawed and the effects of knockdown on glioma cell growth were analyzed.BrdU were added into the culture medium of CUGBP1-shRNA lentivirus vector transfected glioma cells 2-24 hours before detecting, and the OD values of BrdU incorporation were detected by pairs antibody sandwich enzyme immunoassay, by contrast of Scr-shRNA lentiviral vector transfected cells. Further more, the effects of knockdown on DNA synthesis and cell proliferation of neuroglioma were analyzed.Cell cycle and cell apoptosis CUGBP1 was related with the expression or activity of certain proteins such as P21, which were associated with cell cycle withdrawal and resulted in that the risk of mutation or dysplasia was increased in myoblast of myotonic dystrophy. We wondered whether there existed similar phenomenon in human neuroglioma. We knocked down CUGBP1 gene through transfecting CUGBP1-shRNA lentivirus vector into glioma cells to discover its relationship with cell cycle and cell apoptosis.PI were added into the CUGBP1-shRNA lentivirus vector transfected glioma cells, subsequently, the proportion of cell cycle distribution was detected in these cells through FACS assay, by contrast of Scr-shRNA transfected cells. The effects of knockdown on cell cycle distribution were analyzed.Annexin V-APC was added into the CUGBP1-shRNA lentivirus vector transfected glioma cells. The ratio of cell apoptosis was detected in these cells through FACS assay, by contrast of Scr-shRNA transfected cells. Further more, the impacts of knockdown on cell apoptosis were analyzed.The expression of Cell cycle and apoptosis related gene To further explore the correlations and its mechanisms between CUGBP1 and glioma cell proliferation and apoptosis-related downstream genes, we examined the expression of these genes after knockdown of CUGBP1 gene.The expression of apoptosis repression related genes BCL2 was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The impact of the knockdown on expression of BCL2 in neuroglioma cells was analyzed.The expression of cell cycle suppressor genes CDKN1A was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of CDKN1A in neuroglioma cells was analyzed.The expression of DNA synthesis related gene PCNA was examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of PCNA in neuroglioma cells was analyzed. The expression of DNA synthesis related genes MCM2-7 were examined in CUGBP1-shRNA lentivirus vector transfected glioma cells through real-time quantitative PCR, by contrast of Scr-shRNA transfected cells. The effect of the knockdown on expression of MCM2-7 in neuroglioma cells was analyzed.ResultsDetection of CUGBP1mRNABy contrast of the housekeeping gene GAPDH, the CUGBP1 were high expressed at mRNA level in human glioma cell lines U87, U251, U373 and human glioblastoma cell line A172 through RT-PCR assay.The expression of CUGBP1 mRNA in human neuroglioma increased as 2.96 times much as that in normal brain tissue via real-time quantitative RT-PCR assay, by contrast of housekeeping gene GAPDH. Further more, while the tumor grade increases, CUGBP1 mRNA in neurogliomas tends to increase, with about 2.26 fold increase in glioma grade one, 2.36 fold in grade three and 4.27 fold in grade four.Knockdown efficiencyFluorescence-positive cells accounted for more than 95% among CUGBP1-shRNA lentivirus or Scr-shRNA lentivirus transfected glioma cells 72 hours after transfection by common microscopy and fluorescence microscopy, indicating successful transfection. By contrast of GAPDH, the expression of CUGBP1mRNA in CUGBP1-shRNA lentivirus transfected glioma cells, compared with that in control cells, decreased by about 92% through real-time quantitative RT-PCR assay, suggesting that CUGBP1-shRNA lentivirus effectively transfected glioma cell lines and inhibited expression of CUGBP1mRNA significantly as a reliable tool for gene silencing.Cell proliferationWe examined CUGBP1-shRNA and Scr-siRNA lentiviral vector transfected cells once a day for 5 days and took pictures by CELLOMICS. While Scr-shRNA lentiviral vector transfected cells tended to gradually increase and up to 3 times by the fifth day, CUGBP1-shRNA lentiviral vector transfected cells remained no change and even decrease on fifth day, suggesting that the knockdown of CUGBP1 gene inhibited proliferation of glioma cells. By contrast of Scr-shRNA lentivirus transfected cells, we detected OD values ofBrdU incorporation in CUGBP1-shRNA lentivirus transfected glioma cells through double-antibody sandwich enzyme immunoassay. OD value in CUGBP1-shRNA lentivirus transfected cells decreased on first and third day and even much lessen on third day, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene inhibited DNA synthesis, thereby inhibiting proliferation of glioma cells.Cell cycle and apoptosisBy contrast of Scr-siRNA lentivirus transfected cells, we examined the cell cycle of CUGBP1-shRNA lentivirus transfected glioma cells through FACS assay. The cell ratio in cell cycle S phase among CUGBP1-shRNA lentivirus transfected glioma cells increased significantly, reaching 67.71%, while the cell ratio in the G0 / G1 and G2 / M phase was significantly reduced, suggesting that the knockdown of CUGBP1 gene resulted in abnormal cell cycle distribution of glioma cells. The cells in S phase increased, implying that the S phase arrest occurred.By contrast of Scr-shRNA lentivirus transfected cells, we examined the cell apoptosis of CUGBP1-shRNA lentivirus transfected glioma cells through FACS assay. The apoptosis ratio among CUGBP1-shRNA lentivirus transfected glioma cells increased significantly, reaching 73.8%, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly increased apoptosis, thereby indicating that the gene can inhibit glioma cell apoptosis.The expression of Cell cycle and apoptosis related gene.By contrast of housekeeping gene GAPDH, we detected the expression of BCL2 mRNA in CUGBP1-shRNA lentivirus and Scr-siRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of BCL2 mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of BCL2 gene, thereby indicating that CUGBP1 gene can promote the expression of BCL2, thus inhibit apoptosis and promote tumor cell growth.By contrast of housekeeping gene GAPDH, we detected the expression of CDKN1A mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of CDKN1A mRNA significantly increased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly promoted the expression of CDKN1A gene, thereby indicating that CUGBP1 gene can inhibit the expression of CDKN1A, thus deinhibit cell cycle and promote tumor cell growth.By contrast of housekeeping gene GAPDH, we detected the expression of PCNA mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of PCNA mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of PCNA gene, thereby indicating that CUGBP1 gene can promote the expression of PCNA , thus promote DNA synthesis and tumor cell growth.By contrast of housekeeping gene GAPDH, we detected the expression of MCM2-7 mRNA in CUGBP1-shRNA lentivirus and Scr-shRNA lentivirus infected glioma cells through real-time quantitative RT-PCR. The expression of MCM2-7 mRNA significantly decreased in CUGBP1-shRNA lentivirus infected glioma cells, compared with that in control cells, suggesting that the knockdown of CUGBP1 gene in the glioma cell line significantly inhibited the expression of MCM2-7 gene, thereby indicating that CUGBP1 gene can promote the expression of MCM2-7, thus promote cell DNA synthesis and tumor cell growth.Conclusion:Firstly, our study innovatively discovers that CUGBP1 is highly expressed in different glioma cell lines and human neuroglioma. Furthermore, its expression tends to increase with the glioma pathological grade.Secondly, CUGBP1-shRNA lentiviral vector can efficiently transfect human glioma cell lines and stably knock down CUGBP gene.Thirdly, the knockdown of CUGBP1 gene can inhibit DNA synthesis for glioma cell, thus inhibiting the growth of glioma cells.Forthly, the knockdown of CUGBP1 gene may cause S phase arrest and inhibit cell cycle process. Moreover, its knockdown also significantly increases the apoptosisl ratio of neuroglioma cells.Last but not least, the knockdown of CUGBP1 gene can downregulate the expression of apoptosis inhibitor gene BCL2 and DNA synthesis related genes PCNA and MCM2-7, also upregulate the expression of cell cycle inhibitative gene CDKN1A. CUGBP1 may promote cell DNA synthesis, promote cell cycle process and inhibit glioma cell apoptosis, thus promote development of glioma cells. Additionly, CUGBP1 may serve as one of predicting indicators or a potential gene therapeutic target for treatment of malignant glioma.