The Mechanism Of DBC1 On The Proliferation And Migration In Glioblastoma Cells

Glioblastoma(GBM)is the most common and invasive type of primary brain tumor and the astrocytoma of the highest grade with an extremely poor prognosis.GBM is characterized by high proliferation,invasion into the surrounding normal tissue and vascularization.The average survival time of GBM is ranging from a few months to 3 years.Standard treatment for newly diagnosed GBM consists of surgical resection followed by radiation and chemotherapy.In addition,the FDA approved drug for GBM treatment is temozolomide with large setbacks.Although the surgical and pharmacological therapies have got some fruits,glioblastoma remains difficult to cure and has a low survival rate.Therefore,it's necessary to attach more importance to explort the molecular mechanisms underlying glioblastoma initiation and progression.Further identifying useful biomarkers,exploring novel targets is urgently needed for the long-term survival of patients with GBM.DBC1(Deleted in breast cancer 1)was named by its deletion in breast cancer.DBC1 promoted the survival of breast cancer cells by modulating estrogen receptor ? and ?.It has been suggested that DBC1 might be involved in the progression of hormone receptor-related human malignant tumors.Recently,a cooperative role of DBC1 with ndrogen receptor(AR)and concomitant expression of DBC1 and AR in advanced human malig-nant tumors have been reported.However,it was reported that DBC1 could be tumor suppressor.DBC1 inhibits the proliferation and invasive potential of gastric cancer cells and is correlated with poor prognosis.In addition,DBC1 could be tumor suppressor of SIRT1,which could inactivate various tumor suppressors.Moreover,DBC1 is a nuclear protein involved in various biological processes including DNA damage response,metabolism,epigenetics,nuclear receptor function.And depletion of DBC1 induced apoptosis of tumor cells and inhibited the proliferation of cancer cells.In this paper,the clinical tissue sample chip analysis showed that DBC1 was highly expressed and GBM patients had poor prognosis.At the same time,the expression levels of different glioblastoma cell lines were detected.The effects of DBC1 knockdown and recovery on proliferation and migration of glioblastoma cell lines LN-229 and U-87 MG were studied.The experimental results are as follows:1.The expression of DBC1 is closely related to the prognosis of GBM patientsWe detected by analyzing the clinical tissue microarray of glioblastoma,we found that the higher the malignant degree of glioblastoma,the higher the expression of DBC1.The expression of DBC1 in glioblastoma was generally detected by fluorescence quantitative PCR and Western blot,but it was low in normal glioblastoma cells,which was closely related to the degree of malignancy.2.DBC1 promotes cell proliferation and migration of glioblastomaIn order to explore the role of DBC1 in glioblastoma,we used sh-RNA technology to interfere with DBC1.After interfering with DBC1,MTT and BrdU experiments showed that the proliferation ability of glioblastoma was affected after interfering with DBC1.Flow cytometry analysis showed that LN-229 and U-87 MG interfering with DBC1 were blocked in G1 phase,and CDK2,CDK4 and CDK6 were significantly inhibited.And p21,known as the mammalian G1 checkpoint,was significantly up-regulated.After the restoration of DBC1,the proliferation ability of glioblastoma was restored and the cycle arrest was restored accordingly.In order to verify whether DBC1 is related to cell migration of GBM,migration assays were performed.The results suggested that DBC1 promotes the proliferation and migration of glioblastoma.3.DBC1 promotes tumorigenicity of glioblastomaTo further explore the effect of DBC1 on tumorigenicity of glioblastoma,soft agar and subcutaneous injection experiments were carried out on the LN-229 cell line of DBC1 by interfering and restoring.It was found that after interfering with DBC1,the tumorigenicity of cells was inhibited.When DBC1 was restored,the tumorigenicity of glioblastoma cells increased.4.DBC1 regulates cell proliferation of glioblastoma by regulating p21 throughubiquitination pathwayAfter interfering with DBC1,Western Blot experiments showed that there were significant changes in p21 and CDK2,CDK4,CDK6,etc.p21 was the first gene called CDK inhibitor,and it was also an important G1 checkpoint.We detected the level of p21 mRNA and found that there was no significant change in the level of p21 after interfering with DBC1.The change only occurred at the level of protein.Then we carried out MG132 and CHX experiments.We found that after interfering with DBC1,the level of p21 ubiquitination decreased,so DBC1 affected the expression of p21 through ubiquitination pathway.Then we interfered with p21 in the case of DBC1.We found that the related cyclin CDK2,CDK4 and CDK6 did recover,which further proved that DBC1 regulated cell cycle by regulating p21,and then affected the proliferation of glioblastoma cells.In conclusion,DBC1 regulates p21 through ubiquitination pathway,affects its protein level,and then affects the proliferation ability of glioblastoma.At the same time,interference with DBC1 can also effectively inhibit the migration ability of glioblastoma.In this study,the exploration of DBC1 enriches the theoretical basis for finding new therapeutic targets for glioblastoma.