Study On The Influence Of Silencing Of Wip1 Gene Coordinates With Ionizing Radiation On Glioma Cells Cultured In Vitro

Glioma is the most common malignant tumor in the central nervous system with strong invasiveness and easy to relapse. With the development of the techniques of neurosurgery and oncology, surgery combined with radiotherapy as well as chemotherapy has become a routine treatment for glioma. However, the prognosis is still poor, which affects people’s health seriously.Wipl (Wild-type p53-induced phosphatase 1) belongs to PP2C (protein phosphatase 2C) family and is coded by PPM1D gene.It is single structure and it is not sensitive to Okadaic acid. The Wipl protein of Wipl is consist of 605 amino acid residues. It could be divided into 2 domains.It could be induced by p53 and overexpressed in many kinds of tumors with poor prognosis.Chkl (checkpoint kinase 1) and Chk2 (checkpoint kinase 2) are both the targets of Wipl and were significant to the normal life of the cells.They determine the cell cycle, proliferation and apoptosis of the cells. Chk1 and Chk2 could influence the proliferation ability of tumor cells after ionizing radiation.The purpose of this study is to find the function of Wip1 gene in the radioresistance of glioma cells and the possible mechanism so that we could provide the theroretical basis in the treatment of glioma.The thesis is included in three parts:Chapterl Establishment of Wipl gene silencing model of glioma cell line and the study on the influence of silencing of Wip1 in combination with ionizing radiation to the proliferation of U-251 glioma cells.Chapter2 Study on expression of Chkl in U-251 glioma cells intervened by Wipl gene silencing in combination with ionizing radiation.Chapter3 Study on expression of Chkl in U-251 glioma cells intervened by Wipl gene silencing in combination with ionizing radiationAll data were handled by SPSS19.0 ststistical software. Normal distribution measurement data was expressed with mean±standard deviation (χ±S). Statistical comparisons were conducted using 1-way ANOVA. The intergroup comparisons were tested by LSD tests. The difference was statistically with P<0.05.Chapterl Establishment of the Wipl gene silencing model of glioma cell line and the study on the influence of silencing of Wipl in combination with ionizing radiation to the proliferation of U-251 glioma cells.ObjectivesEstablish the Wipl gene silencing model of the U-251 glioma cell line.Study the influence of silencing of Wipl in combination with ionizing radiation to the proliferation of U-251 glioma cells.Methods:We cultureed the U-251 cells and used lentivirus to infect the cell to establish the model of Wipl silencing U-251 glioma cells. The efficiency of the cell infection rate was observed under fluorescence microscope. The cells were divided into four groups:Wipl silencing in combination with ionizing radiation group (IR+Wipl-), Wip1 silencing group (Wip1-), ionizing radiation group (IR) and negative control group (NC). CC8 method was used to detect the ability of cell proliferation of each group.Results:1, Three or four days after the U-251 cells was infected by lentivirus, Cells were observed under the fluorescence microscope to determine the efficiency of infection by the GFP positive expression. The results show that the efficiency of infection is over 90%2, The cells’proliferation activity decreased after the silencing of Wip1 gene in combinatioin with ionizing radiation:cck8 method was used to detect the proliferation activity of the U-251 glioma cells. The rank of the proliferation rate of the cells (from low to high) were Wip1 silencing in combination with ionizing radiation group (IR+Wip1-), Wip1 silencing group (Wipl-), ionizing radiation group (IR) and negative control group (NC), respectively. Over time, the difference between groups increased gradually. The difference after 24 hours (P<0.05) 48 hours (P<0.05),72 hours (P<0.05),96 hours (P<0.05),120 hours (P<0.05) were statistically significant. After 24 hours,48 hours,72 hours,96 hours and 120 hours of ionizing radiation, the proliferation rate of IR group and Wip1-group was lower than NC group (P<0.05). A after 24 hours,72hours,96 hours and 120 hours of ionizing radiation, the proliferation rate of IR+Wipl-group was lower than IR group (P<0.05), Wipl-group (P<0.05) and NC group (P<0.05). After 48 hours of ionizing radiation, the proliferation rate of IR+Wip1-group was lower than IR group (P<0.05) and NC group (P<0.05), and the difference was statistically significant.Conclution:The Wipl gene silencing model of the U-251 glioma cells was construsted successfully throuth the infection of lentivirus. Both silencing of the Wipl gene and ionizing radiation could inhibit the proliferation ability of the U251 cells. Besides, the proliferation activity of U-251 glioma cells decreased significantly after the intervene of the silencing of Wip1 gene incombination with ionizing radiation. The expression of Wip1 gene affects the radioresistance of the glioma cells.Chapter2 Study on expression of Chkl in U-251 glioma cells intervened by Wipl gene silencing in combination with ionizing radiation.ObjectivesAfter U-251 glioma cells were intervened by Wipl gene silencing in combination with ionizing radiation.We Study the possible mechanism of Wipl gene in radioresistance of U-251 glioma cells through detecting the expression of mRNA and protein of Chkl.MethodsThe RNA and protein of U-251 glioma cells were extracted 24 hours after silencing of Wipl gene in combination with ionizing radiation. Real time Q-PCR was used to analyse the RNA expression of Chkl and Western Blot was used to analyse the protein expression of Chkl.ResultsThe results of the Chkl expression of mRNA analysed by Realtime PCR and the expression of protein analysed by Western blot:The chkl mRNA expression of the NC group was asserted as 1. The relative expression of chkl mRNA of IR+Wipl group,Wipl-group, IR group was 1.712±0.057、2.914±0.087、0.815±0.018, respectively. The difference between groups was statistically significant (P<0.05). Wipl group is higher than NC group and IR group (P<0.05) is lower than NC group (P<0.05). IR+Wipl-group is lower than Wip1-group but higher than IR group and NC group. In the experiment, western blotting identified an approximately 54 kDa molecular mass Chkl fragment. The relative expression levels of Chkl of IR+Wipl-group, Wipl-group, IR group and NC group were were. 0.571±0.072,0.631±0.052,1.056±0.121 and 0.,950±0.105, respectively. The difference between groups was statistically significant (P<0.05). IR group is higher than NC group (P<0.05) but Wipl-group is lower than NC group (P<0.05). IR+Wipl-group is lower than IR group (P<0.05) and Wipl-group (P<0.05). The difference was statistically significant.ConclutionsAfter the intervene of silencing of Wip1 in combination with ionizing radiation, the mRNA expression of Chkl increased but the protein expression of Chkl decreased. Silencing of Wipl in combination with ionizing radiation could inhibit the proliferation rate of glioma cells through the inhibition of the protein expression of chkl.Chapter3 Study on expression of Chk2 in U-251 glioma cells intervened by Wipl gene silencing in combination with ionizing radiation.ObjectivesAfter U-251 glioma cells were intervened by Wip1 gene silencing in combination with ionizing radiation.We Study the possible mechanism of Wipl gene in radioresistance of U-251 glioma cells through detecting the expression of mRNA and protein of Chk2.MethodsThe RNA and protein of U-251 glioma cells were extracted 24 hours after silencing of Wip1 gene in combination with ionizing radiation. Real time Q-PCR was used to analyse the RNA expression of Chk2 and Western Blot was used to analyse the protein expression of Chk2.ResultsThe results of the Chk2 expression of mRNA analysed by Realtime PCR and the expression of protein analysed by Western blot:The chk2 mRNA expression of the NC group was asserted as 1. The relative expression of chk2 mRNA of IR+Wipl group,Wipl-group, IR group was 0.541±0.057,0.739±0.036,0.777±0.016, respectively. The difference between groups was statistically significant (P<0.05). IR group, Wip1-group and IR+Wip1-group were lower than NC group (P<0.05).IR+Wipl-group was lower than IR group and Wipl group. In the experiment, western blotting identified an approximately 63 kDa molecular mass Chk2 fragment. The relative expression levels of Chk2 of IR+Wipl-group, Wip1-group, IR group and NC group were were 0.450±0.062,0.756±0.063,0.591±0.075 and 0.,837±0.084, respectively. The difference between groups was statistically significant (P<0.05). IR group is lower than NC group (P<0.05) and Wipl-group is lower than the other 3 groups (P<0.05). The difference was statistically significant.ConclutionsAfter the intervene of silencing of Wipl in combination with ionizing radiation, the mRNA expression of Chk2 increased but the protein expression of Chk2 decreased. Silencing of Wipl in combination with ionizing radiation could inhibit the proliferation rate of glioma cells through the inhibition of the protein expression of chk2.Full Text Conclusions1. The Wipl gene silencing model of U-251 glioma cell was established by the infection of lentivirus.2. The U-251 glioma cells’proliferation activity decreased after the silencing of Wipl gene in combinatioin with ionizing radiation.Wip1 gene plays an important role in the radioresistance of glioma cells.3. After the intervene of silencing of Wipl in combination with ionizing radiation, the expression of Chkl mRNA increased but the expression of chkl protein decreased. The proliferation ability of glioma cells could be inhibited through the in hibition of the protein expression of chkl.4. After the intervene of silencing of Wipl in combination with ionizing radiation, the expression of mRNA and protein of chkl both decreased.The proliferation ability of glioma cells could be inhibited through the in hibition of the protein expression of chk2.