Study On The Biological Function And Mechanism Of MED27 In Glioma

Glioma is a highly malignant and invasive tumors,mostly originated in the brain.Because of its complex pathogenesis and the special location,early diagnosis is very difficult.Most patients have developed to late stage at the time of diagnosis.At present,the main clinical treatments of glioma are surgery,radiotherapy and chemotherapy.However,these therapies are difficult to achieve the desired results,resulting in a very poor prognosis.Thus,the treatment of glioma is still one of the great challenges in the field of neurosurgery.In recent years,with the concept of"precision medicine" being proposed,targeted therapy provides a potential treatment for patients with glioma.This provides a new possibility for improving the prognosis of patients with glioma.Therefore,it is a hot area to search for key molecules in the occurrence and development of glioma,which may provide a new target for early diagnosis and targeted therapy in patients with glioma.The mediator complex 27(MED27)is an important one of the MEDs.The current study found that it is widely present in the various organs and tissues of the human body with no tissue-specificity,playing a catalytic enzyme role in the beginning of the transcription.So far,however,there is still little study of MED27 in human diseases and its biological function is not clear.Previous studies have found that the replication capacity of Human Immunodeficiency Virus-1(HIV-1)was significantly reduced by knocking down MED family proteins(including MED27).However,the viability of HIV-1 infected cells was not affected significantly.It is hypothesized that the family proteins(including MED27)may be involved in the transcriptional process of HIV-1.It has also been found that MED27 interacts with thyroid hormone receptors and a variety of factors(including transcription factors and cofactors),playing a role as thyroid hormone receptors.In addition,the study about melanoma demonstrated that MED27 can activate NF-KB/iNOS signaling pathway to promote the biological progress of melanoma.Therefore,it is further speculated that MED27 may be used as a biology marker in early diagnosis and postoperative treatment of melanoma.However,the biological function and its related molecular mechanisms of MED27 in human glioma have been not reported until now.Our group has focused on the molecular study of glioma for many years,so we intend to further explore the biological functions and the potential molecular mechanism of MED27 in glioma based on the previous experimental basis and the reports of existing literatures.OBJECTIVE:1.To investigate the effect of MED27 on the proliferation,apoptosis and invasion of U87 glioblastoma cells.2.To investigate whether NF-?B/iNOS signaling axis is involved in the regulation of MED27 on the biological behavior of U87 glioblastoma cells.METHODS:PART 1:The expression of MED27 knockdown was evaluated by qRT-PCR to detect the construction of MED27 knockdown U87 cell model1.The cell model required for the experiment was established1.1 The second generation to eighth generation U87 cell lines were cultured in medium containing 10%fetal bovine serum(FBS)until the cell growth status was good,and the basal cell model required for this experiment was established.1.2 The U87 cell lines in the above growth state were randomly divided into three groups.The MED27 knockdown U87 cell model,the negative control U87 cell model and the blank control U87 cell model were established by transfection of MED27 siRNA,transfection of MED27 negative control siRNA and no treatment respectively.2.To evaluate whether the cell model was established successfully from RNA levelThe expression of MED27 mRNA in MED27 knockdown U87 cell model,negative control cell model and blank control cell model was detected by qRT-PCR to evaluate whether the cell model was established successfully.PART 2:The expression of MED27 protein was evaluated by Western Blotting to evaluate the construction of MED27 knockdown U87 cell model1.1 The second generation to eighth generation U87 cell lines were cultured in medium containing 10%fetal bovine serum(FBS)until the cell growth status was good,and the basal cell model required for this experiment was established.1.2 The U87 cell lines in the above growth state were randomly divided into three groups.The MED27 knockdown U87 cell model,the negative control U87 cell model and the blank control U87 cell model were established by transfection of MED27 siRNA,transfection of MED27 negative control siRNA and no treatment respectively.2?To evaluate whether the cell model was established successfully from protein levelThe expression of MED27 mRNA in MED27 knockdown U87 cell model,negative control cell model and blank control cell model was detected by Western Blotting to evaluate whether the cell model was established successfully.PART 3:To investigate the effect of MED27 on the proliferation,apoptosis and invasion of U87 cells1?To construct the required cell modelsConstruct the MED27 knockdown U87 cell model,the negative control U87 cell model and the blank control U87 cell model through the siRNA transfection techniques.2?To measure the proliferation of U87 cells after knocking downMED27Measure the proliferation of MED27 knockdown U87 cell model and the negative control U87 cell model by MTT Assay after transfecting siRNA.3?To detect the apoptosis of U87 cells after knocking down MED27After transfecting siRNA,Caspase 3 Activity Assay was used to measure apoptosis changes of MED27 knockdown U87 cell model and negative control U87 cell model.4?To detect the invasion ability of U87 glioma cells after knocking down MED27Transwell Chamber Assay was used to measure apoptosis changes of MED27 knockdown U87 cell model and negative control U87 cell model after transfecting siRNA.PART 4:To explore the potential mechanism of MED27 on proliferation,apoptosis and invasion of U87 cells1?The expression of NF-?B was detected after knocking down MED27 in U87 cellsAfter transfection of siRNA,Western Blotting was used to detect and compare the expression changes of transcription factor NF-?B in MED27 knockdown U87 cell model and negative control U87 cell model.2.To investigate the effect of MED27 expression on iNOS in U87 cellsAfter transfection of siRNA,the expression changes of transcription factor iNOS in MED27 knockdown U87 glioma cell model and negative control U87 glioma cell model were compared by Western Blotting.RESULTS:PART 1:MED27 mRNA expression level was significantly reduced after MED27 siRNA transfectionAfter successful construction of the three U87 glioma cell models above,we found that MED27 siRNA transfection in U87 cells significantly reduced the expression of MED27 mRNA compared with the negative control group by qRT-PCR(P<0.05).Meanwhile,compared with the blank control group,the negative control group had no significant effect on the expression of MED27 mRNA in U87 cells(P>0.05).PART 2:MED27 protein expression level was significantly reduced after MED27 siRNA transfectionAfter successful construction,we found that expression level of MED27 protein was significantly reduced in MED27 knockdown U87 cells group compared with the negative control group(P<0.05).Meanwhile,compared with the blank control group,the negative control group had no significant effect on the expression of MED27 protein in U87 cells(P>0.05).PART 3:MED27 siRNA transfection can reduce the proliferation and invasion ability of U87 cells,promote its apoptosis.1?The proliferation ability of U87 glioma cells was significantly decreased after knocking down MED27After constructing the above three U87 cell models successfully,we found that the proliferation ability of MED27 knockdown U87 cells group was significantly decreased compared with the negative control group by MTT Assay(P<0.05).2?The biological activity of Caspase 3 in U87 cells was significantly enhanced after knockdown of MED27We found that compared with the negative control group,the expression of Caspase 3 was significantly increased in U87 cells transfected with MED27 siRNA by using Caspase 3 Activity Assay(P<0.05).3?The invasion ability of U87 cells was significantly enhanced after knocking down MED27After constructing the above three cell models successfully,Transwell Assay showed that the invasive ability of MED27 knockdown U87 cells group was significantly decreased compared with the negative control group(P<0.05).PART 4:The expression of NF-?B and iNOS was significantly decreased after siRNA transfection of U87 cells1?The expression of NF-?B in U87 cells was decreased after knocking down MED27We found that the expression of NF-?B was significantly decreased in MED27 knockdown U87 cells group compared with the negative control group by Western Blotting after successful construction of the three U87 cell models above.2?The expression of iNOS in U87 cells was decreased after knocking down MED27We found that the expression of iNOS was significantly decreased in U87 glioma cells transfected with MED27 siRNA compared with the negative control group by Western BlottingCONCLUSION:1.Through functional experiments,we found that MED27 can inhibit U87 cells apoptosis,promote its proliferation and invasion ability.2.Through the mechanism experiments,we further speculated that MED27 may play its roles of inhibiting apoptosis and promoting proliferation and invasive ability of U87 cells through the activation of NF-?B/iNOS signaling pathway.3.In this study,we concluded that MED27 may be a critical biological marker in the occurrence and development of gliomas.It plays a role as oncogene and may become a new target for glioma targeted therapy.