MiR-141-3p Promotes Glioma Cell Growth And Temozolomide Resistance By Directly Targeting P53

Research background: Glioblastoma multiforme(GBM)is the most common type of primary brain tumor and has shown the highest mortality rate among all brain malignancies.Temozolomide(TMZ),a DNA alkylating antineoplastic drug,is a promising chemotherapeutic agent that readily crosses the blood–brain barrier and is used as first-line therapy for the treatment of glioblastoma.Micro RNAs(mi RNAs),a series of small,highly conserved,non-coding RNA molecules 18–25 nucleotides in length,are known to activate or inhibit the progression of various cancers,including glioma,and have been proposed as novel targets for anticancer therapies in recent years.We found that the oncogenic mi R-141-3p was increased in clinical GBM samples and correlated with WHO grade.We also found that mi R-141-3p activated proliferation,and TMZ resistance.However,we don't know this mechanism.More than 30 years of research on tumor suppressor p53 have made p53 one of the most studied proteins in science and underlined its significance for understanding the mechanisms of GBM.Furthermore,in recent study,p53 has been found to sensitize temozolomide treatment in glioblastoma.So,we identified p53 as a direct target for mi R-141-3p,and showed that p53 was decreased in GBM with a negative correlation between mi R-141-3p and p53 level.Our findings suggest that mi R-141-3p could be a critical therapeutic target for GBM intervention.Objective: 1.To investigate the expression of mi R-141-3p in glioma and its effect on the proliferation and drug resistance of glioma cells;2.To explore the relationship between mi R-141-3p and p53,that is,whether mi R-141-3p affects the proliferation and drug resistance of glioma cells by directly targeting p53.Methods: 1.MRNA expression data for gliomas and normal controls and survival data of patients with glioma were downloaded from The Cancer Genome Atlas(TCGA)and Chinese Glioma Genome Atlas(CGGA)data portal.2.Fluorescence quantitative PCR reactions were performed using Taqman reverse transcription reagents and SYBR Green PCR Master Mix according to manufacturer's protocols.Various glioma cell lines were cultivated and analyzed for mi R-141-3p level through fluorescence quantitative PCR.3.Colony formation,CCK8 assay,5-ethynyl-2'-deoxyuridine(Ed U)assay were used to examined cell viability and the capacity of proliferation in U87,A172,LN229 and U251 glioma cells treated with anti-mi R-141-3p,U87/TMZ-resistant and A172/TMZ-resistant glioma cells treated with anti-mi R-141-3p and TMZ,U87 and A172 glioma cells treated with mi R-141-3p and p53 plasmids and U87 and A172 glioma cells treated with mi R-141-3p and p53 plasmids of TMZ cultivation.Then,cell flow cytometry was used to access cell cycles and apoptosis in glioma cells.Relative western expression for cyclin B1,cyclin E1,CDK2 and cleaved caspase 3 were analyzed by western blot analysis.4.We performed a Luciferase reporter assay to investigate p53 is a target of mi R-141-3p.The expression of mi R-141-3p and p53 levels were analyzed in 27 human glioma tissues including seven low-grade glioma tissues,20 high-grade glioma tissues and 5 adjacent normal brain tissues(NBT)removed from patients with fluorescence quantitative PCR and Western blot assays.The correlation of mi R-141-3p and p53 levels were also analyzed in GBM tissues.Relative western expression for p53 and its download protein p21 and bax were analyzed by western blot analysis.5.Nude mice were first intracranially xenotransplanted with luciferase-expressing U87 glioma cells transfected with anti-mi R-141-3p.After tumor formation,nude mice of TMZ treating group were treated with TMZ by oral gavage(100 ?M daily for 5 days per week for three cycles.After the start of treatment,tumor growth was monitored by a live animal bioluminescence imaging system every week.6.Immunohistochemical staining of nude mouse xenograft tumor tissues was performed with antibodies against p53,Ki67 and cleaved caspase 3 for apoptosis.Results: 1.Mi R-141-3p is increased in human glioma tissues.2.Mi R-141-3p activates glioma cell growth and TMZ resistance in vitro and vivo.3.p53 is a direct target of mi R-141-3p in glioma cells.4.Reintroduction of p53 could attenuate the oncogenic and TMZ resistant effect of mi R-141-3p.Conclusions: 1.Micro RNA-141-3p is increased in human glioma tissues.2.Micro RNA-141-3p negatively relates with expression of p53 and inhibits its downstreaming proteins bax and p21.3.The effects of micro RNA-141-3p promoting glioma cell proliferation and temozolomide resistance directly targets p53.