The Effect And Mechanism Of BTBD10 On Chemosensitivity Of Human Glioma To TMZ

Glioma accounts for approximately 40~50% of primary brain tumor and is the most common malignant tumor of the central nervous system type.Although in the past few decades,medical equipment and surgical technology has been rapidily development,but the mortality and disability rate caused by malignant glioma is still high,the 5 year survival rate is less than 10%.Especially glioblastoma(GBM)has the worst prognosis while it is the highest degree of pathological malignant glioma.There are many reasons for which,including tumor diffuse infiltrative growth,blood brain barrier effects of chemotherapy drugs,inadequate dose and so on.Compared with surgical resection and radiotherapy,contribution of chemotherapy in improving the survival time of patients with glioma is limited.The resistance to chemotherapy is an important factor to restrict its further use in improving curative effect.At present,glioma stem cells,apoptosis,autophagy,multidrug resistance,DNA damage repair system are researched more on the mechanisms of chemotherapy resistance.They,involving related genes and signal transduction pathway,is not completely clear.As a new generation of alkylating agent,temozolomide is currently standard chemotherapy drugs for glioma in clinical,but the differences of the sensitivity and resistance distinguish the prognosis of the patients.Therefore,further study on the molecular mechanism of glioma chemotherapy resistance can open up a new road in improving the efficacy of chemotherapy in patients with.In a previous study,we used c DNA microarray analysis in glioma samples,and found a new BTB/POZ domain-containing protein named BTBD10,which expression was significantly down regulated than the normal tissue.Literature review informed,the BTB / POZ protein family members participate in a variety of cellular processes and its possible mechanisms include mediating protein-protein reaction,interaction with DNA through the formation of dimers or multimer,genes on and off,the regulation of the genes expression,and cell proliferation,differentiation and apoptosis.So,we choose the BTBD10 as the research object,using RT-PCR,Northern blotting,Western blot and the construction of BTBD10 lentiviral expression vector techniques,to identificate the BTBD10 expression amount and its impact in cell proliferation and apoptosis on the brain glioma tissues and U251 Cell Lines.The experimental results show that BTBD10 was down regulated compared to the normal brain tissue in all gliomas,and its level of expression was negatively correlated with the malignant level of tumor,namely the higher the level of BTBD10 expression the lower the malignant level of tumor.At thesame time,confirmed from the U251 cells with overexpression of BTBD10,BTBD10 can inhibit the activity of the PI3K/Akt signaling pathway,thus inhibit the proliferation and promote the apoptosis of glioma cells.Therefore,we can conclude that BTBD10 as a tumor suppressor gene may become a potential target for glioma therapy.Of course,to really understand the related function and the mechanism of BTBD10 we also need to continue in-depth research.For example,what kind of impact on the chemosensitivity of glioma will produce in different BTBD10 expression levels,and what mechanism will realize this impact.All these are unclear.This study intends to screen two cell lines with lower expression of BTBD10 from four GBMs cell lines,and construct the GBMs cell lines with over expression of BTBD10 through transfecting lentiviral vector.Next,we will determine the changes of sensitivity to chemotherapeutic drugs TMZ and the differences of proliferation and apoptosis level.Finally,through the detection of protein related to chemoresistance mechanisms including apoptosis,autophagy,multidrug resistance and DNA repair system to explore the possible mechanism of overexpress BTBD10 induced changes in chemotherapeutic sensitivity of GBMs cell lines.The experiment is divided into two parts: the first part is to investigate the chemotherapeutic sensitivity of GBMs cell lines with overexpression of BTBD10;the second part is to explore the mechanism of chemosensitivity to TMZ in U251 and U87 cell after overexpression of BTBD10.Part ? Effect on Chemosensitivity of GBMs cells after overexpression of BTBD10Objective: In this part of experiment,lentiviral vector containing BTBD10 was constructed after screening GBMs cell line to verify the TMZ chemotherapy sensitivity changes on GBMs cells.Methods: Real-time PCR methods was used to examine the m RNA of BTBD10 in U87?U251?A172?U373 GBMs cells.To construct lentiviral vector plasmid containing BTBD10,selectively transfect two cell lines which express low level of BTBD10.Real-time PCR and Western-blot were used to test transfection efficiency.The U251 and U87 cells were divided into three groups: the blank group(CON)?the empty vector group(NC)and overexpression group(OE).The identification of TMZ chemotherapy sensitivity was based on IC50 value determined by CCK-8.Results: In the four GBMs cell lines,BTBD10 m RNA expression levels of U251 and U87 were lower than that in A172,U373,and there is a significant difference(P?0.05).The BTBD10 overexpression vectot was constructed and transfected into U251,U87 cells successfully.The BTBD10 m RNA expression levels were 11.43 and43.92 times in U251,U87 cells after transfection.At the same time,the BTBD10 protein expression levels were significantly increased,consistenting with the trend of BTBD10 m RNA expression levels.In U251 group,the IC50 values to TMZ of OE group,CON group and NC group were 10.31mg/L,19.78mg/L,19.84 mg/L.In U87 group,the IC50 values to TMZ of OE group,CON group and NC group were 8.628 mg/L,16.36 mg/L,16.71 mg/L.In these two cells,the IC50 values of OE group was significantly lower than that of CON group and NC group.Conclusion:.The expression level of BTBD10 was positively correlated with the sensitivity of GBMs cells to TMZ,and over expression of BTBD10 could increase the sensitivity of U251 and U87 cells to TMZ.Part ? To explore the mechanism of chemosensitivity changes in U251and U87 after overexpression of BTBD10Objective: To investigate proliferation?invasion and apoptosis of U251?U87 cells with overexpression of BTBD10 and the possible mechanism of increased sensitivity to TMZ chemotherapy.Methods: The MTT method?Transwell method and flow cytometry were used to detect proliferation?invasion and apoptosis of U251?U87 cells after application of TMZ.At the same time,Westtern blot was used to detect apoptosis related proteins BCL-2?Caspase3,autophagy related proteins LC3 ? Beclin1,multidrug resistance proteins ABCC1?ABCB1,DNA damage repair proteins MGMT?ERCC2.Results: MTT results showed that in U251,U87 cells,according to the change times of OD490,the proliferation rate of cells in OE group was obviously lower than that in CON group and NC group,reaching statistical difference(P?0.05).Transwell results suggest that in U251,U87 cells,comparing with the transfer cell numbers,there was no significant differences between the OE group and CON group,as well as NC group(P?0.05).In U87 cells,the comparison of transfer cell number between the three groups hasno significant difference(P ? 0.05).The results of flow cytometry suggest that in U251,U87 cells the apoptosis rate of OE group increased significantly compared with CON group and NC group,reaching statistically significant difference(P ? 0.05).Resistance related protein detection results indicate,when U251,U87 cells over express BTBD10,Bcl-2?ABCC1?ABCB1 expression was reduced,Caspase3 expression was elevated,also OE group compared with CON group and NC group reaching statistically difference(P ? 0.05).While the expression of LC3,Beclin1,MGMT,ERCC2 have no significant difference(P?0.05)..Conclusion:.After overexpression of BTBD10 in U251,U87 cells,the proliferation was inhibited,apoptosis level was increased and the invasion was not changed significantly.The sensitivity changes to TMZ may be related with the means of apoptosis and multidrug resistance.