Expression Of HMGN5 And Preliminary Study On Its Function Molecular Mechanism In Glioma

Objective Glioma is the most common and aggressive primaiy intracranial tumor,accounting for 50% of adult malignant brain tumors.Due to the invasiveness of glioma,therapeutic efficacy and prognosis of patients with glioma are still unsatisfactory,especially for patients with glioblastoma multiform,the survival being less than 2 years.Fortunately,deep research in molecular biology have provided broad prospects in gene therapy.HMGN5 is a member of high-mobility group protein(HMGN),which can change the structure of chromatin and regulate the biological function and molecular transcription via combining with nucleosome.And research has confirmed that HMGN5 plays an important role in the development of many tumors(lung cancer,prostatic cancer,bladder cancer,breast cancer and osteosarcoma).However,It is not clear how HMGN5 contributes to glioma.HMGN5 gene expression level in glima tissue and the connection with the pathological features were investigated;the influence on RNA interference silence HMGN5 gene in glioma cells proliferation,cell cycle and forability were also studied as theoretical basis for glioma targeted therapy.Methods1.Identification of expression of HMGN5 in Glioma samples Western-Blot and Real-time PCR were performed to measure the expression of HMGN5 protein and m RNA in glioma tissues and normal brain tissues and the relativity between the expression of HMGN5 and pathological features of glioma was analysed.2.To investigate the expression and biological function of HMGN5 in human glioma cell lines(U251,U87)Western-Blot and Real-time PCR were performed to assess the expression of HMGN5 protein and m RNA in human glioma cells;interfering sequences was designed and Western-Blot and Real-time PCR were performed to check the efficacy of HMGN5 silencing;CCK-8 assay were carried out to study the proliferation of U251 and U87 cells after HMGN5 silencing;the cell cycle changes were analysed by PI staining;FCM was used to assay the change of apoptotic rate;scratch migration assay and transwell migration assay were performed to study the proliferation and invasion of U251 and U87 cells after HMGN5 silencing.3.Priliminary investigation of molecular basis mediated by HMGN5 in glioma The protein related to cell proliferation,invasion and migration,as well as PI3K/Akt and MAPK pathway,were tested by Western blot after down-regulating the expression level of HMGN5.Results1.The expression levels of HMGN5 protein and m RNA using Western-Blot and Real-time PCR was higher in glioma samples than normal brain tissue,with a significantly frequency(P<0.001).HMGN5 protein was highly expressed in 73.3%(88/120)of glioma samples,while in 20.0%(2/10)of normal brain samples,a significantly frequency(P<0.001).The expression level of HMGN5 was positively correlated with the status of pathology classification(WHO I-II vs.WHO III-IV)(P=0.0004)in glioma patients.2.HMGN5-si RNA significantly reduced the expression levels of HMGN5 protein and m RNA in U87,U251 cells(P<0.01).CCK-8 assay showed that OD490 value for HMGN5-si RNA group significantly reduced(P<0.01).PI staining showed significant G0 / G1 phase retardation in the HMGN5-si RNA group,compared to control group.FCM assay showed that the apoptotic rate of HMGN5-si RNA group higher than the control group.(P<0.01).3.HMGN5 inhibition significantly increased TMZ-induced cytotoxicity in a time-dependent manner inboth cell lines.The combination of HMGN5 si RNA with TMZ significantly enhanced apoptosis of glioma cells compared to those treated with TMZ alone.These results suggest that HMGN5 inhibition was able to sensitize TMZ-induced apoptosis.(P<0.01).4.The number of cells crossed the scratch deceased drastically,with HMGN5-si RNA group compared to control group(P<0.05).5.Knocking down HMNG5 expression decreased the expression of BCL2,Cyclin D1,MMP-2,MMP-9 and increased the expression of Bax,p21.6.HMGN5 significantly decreased the expression of phosphorylated PI3 Kand AKT,p-MEK1 and p-ERK1/2.(P<0.05).Conclusions1.The expression levels of HMGN5 protein and m RNA was higher in glioma samples than normal brain tissue,and The expression level of HMGN5 was positively correlated with the status of pathology classification in glioma patients.2.2.HMGN5-si RNA can significantly reduce the expression levels of HMGN5 protein and m RNA in U87,U251 cells,and restrain cell proliferation,promote cell apoptosis,decreae cell invasion and migration,and sensitize TMZ-induced apoptosis.3.Further mechanism research indicated that HMGN5 may effect the expression of BCL2,Cyclin D1,MMP-2,MMP-9,Bax,p21 protein via PI3K/AKT or MAPK pathway,and in the end to effect the development of glioma.