Study Of Hedysarum Polybotrys Polysacchcrides On Glioma Cells By Regulating MiR-34a-5p/UHRF2 And Related Signal Pathway

BackgroudHuman malignant glioma is the most fatal primary central nervous system tumor in adults.The postoperative,chemotherapy and radiotherapy outcomes for glioma were extremely poor,with a median survival of only 12months to 15months.It has become a hot topic to explore the pathogenesis of glioma and find new effective therapeutic targets in current gliomas research.Gliomas have significant heterogeneity characteristics,which shows that the gene expression and phenotype of the daughter glioma cells are completely distinct from that of the parent glioma cells,leading to significant deterioration in the proliferation,apoptosis,cell cycle,invasion and drug resistance of glioblastoma cells.Heterogeneity is the reason why malignant glioma cells evolve continuously and adapt to external microenvironment,and finally escape clinical treatment.It may provide a better way to treat gliomas by elucidating the molecular mechanism of the pathological development of gliomas from a genetic perspective.Hedysarum polybotyls polysacchides(HPS)are bioactive macromolecules derived from the roots of Hedysarum polybotrys.Numerous studies have shown that HPS can both kill tumor cells directly and inhibit tumor development indirectly through immunomodulation.However,the effect of HPS on glioma has not been reported.UHRF2(ubiquitin like with PHD and RING finger domains 2)is a ubiquitin-like protein,which has many complex functions such as cell cycle regulation,genomic stability regulation and epigenetic regulation.UHRF2 plays a central role in the regulation of cell cycle and is considered as a potential tumor suppressor gene.MicroRNAs(miRNAs)are short,single-stranded,non-coding RNAs containing 18?27 nucleotides in length.MiRNAs target mRNAs to induce post-transcriptional silencing and regulate the protein expression.In recent years,miRNAs have been recognized as a new target for cancer treatment.Part ? The Expression Profiling of UHRF2 and Its Related miRNAs in Human Glioma TissuesObjectiveThis stutdy aims to observe the expression profiling of UHRF2 and its related miRNAs in human glioma tissues and non-neoplastic brain tissues.MethodsWe collected 30 surgically resected gliomas and 10 non-neoplastic brain tissues(NBT),includingl4 cases of low-grade glioma tissues(LGG,including WHO grade ?glioma and WHO grade ? glioma)and 16 cases of high-grade glioma tissues(HGG,including WHO grade ? glioma and WHO grade IV glioma).Quantitative real-time PCR(qRT-PCR)was used to analyze the levels of UHRF2 mRNA and UHRF2-related miRNA(hsa-miR-196b-5p,hsa-let-7d-5p,hsa-miR-98-5p,hsa-miR-34a-5p).The level of UHRF2 protein and its downstream cyclin E1 and cyclin D1 protein were detected by western blot.Meanwhile,Pearson's analysis was used to the correlation between UHRF2 and cyclin E1 or UHRF2 and cyclin D1.Results1.The expression levels of UHRF2 mRNA and protein in NBT,LGG and HGG decreased gradually,indicating that the UHRF2 level was negatively correlated with the malignant progression of human glioma.2.The protein levels of cyclins E1 and cyclins D1 increased gradually in NBT,LGG and HGG,indicating that cyclins E1 and cyclins D1 levels were positively correlated with malignant progression of human glioma.3.In human glioma tissues,UHRF2 is negatively related to cyclin E1 or cyclins D1,suggesting that the changes of UHRF2 in human glioma may cause cyclin E1 and cyclin D1 changes.4.Compared with NBT,four miRNAs related to UHRF2 were up-regulated in glioma,and hsa-miR-34a-5p was the most obvious.Conclusion1.UHRF2 level was negatively correlated,and cyclins E1 and cyclins D1 levels were positively correlated with the malignant progression of human glioma.The changes of UHRF2 in human glioma may cause cyclin E1 and cyclin D1 changes.2.Hsa-miR-34a-5p was the most up-regulated miRNAs related to UHRF2.Part II The Effect of HPS on Human Glioma Cells And Its Effect on UHRF2ObjectiveTo explore the anti-proliferation effects and cell cycle regulation of HPS on human glioma in vitro levels.MethodsHuman glioma U251 cells were treated with 0?g/ml,50?g/ml,250?g/ml,500?g/ml HPS.And then the effect of HPS on the proliferation of U251 cells was detected by MTT assay.U251 cells were treated with 500?g/ml HPS for 48 hours.The effect of HPS on the cell cycle of U251 was detected by PI staining and flow cytometry.The protein levels of UHRF2,cyclin El,cyclin Dl,p53,Bcl-2 and Bax were detected by western blot.The mRNA levels of cyclin El,cyclin Dl,p53,Bcl-2 and Bax were detected by qRT-PCR.U251 cells were transfected with pcDNA-3.1 plasmid containing UHRF2 cDNA,and the effects of UHRF2 overexpression on the protein levels of cyclin El and cyclin D1 were detected by western blot.Results1.The growth inhibition rate of U251 cells treated with 0?g/ml,50?g/ml,250?g/ml,500 ?g/ml HPS increased gradually.U251 cells treated with 500?g/ml HPS wereblocked in G0/G1 phase of cell cycle.These results suggested that HPS inhibited the growth of U251 cells.2.After 500 ?g/ml of HPS treatment,the expression level of UHRF2 in U251 cells increased significantly,cyclin E1 and cyclin D1 levels decreased significantly,the expression levels of pro-apoptosis-related protein Bax anti-tumor related protein p53,increased,while the expression of anti-apoptosis-related protein Bcl-2 decreased.3.UHRF2 overexpression induced the decrease of cyclin expression in U251 cells,which is similar to the increase of UHRF2 level and the decrease of cyclin expression after HPS treatment.These results suggest that HPS can reduce cyclin expression and inhibit cell growth by up-regulating UHRF2.Conclusion1.HPS blocked U251 cells in G0/G1 phase of cell cycle and inhibited the cells growth.2.HPS reduced cyclin El and cyclin D1 levels by up-regulating UHRF2,which was similiar to that by UHRF2 overexpression.Part ? Study on the Mechanism of HPS on Human GliomaCells by Inhibiting MiRNA-34a-5p and Increasing UHRF2ObjectiveTo explore the potential molecular mechanism of HPS on Human Glioma Cells from the perspective of UHRF2 and its related miRNAs.MethodsThe wild type(wt UHRF2 3'-UTR)and mutant(mut UHRF2 3'-UTR)luciferase reporter gene vectors were constructed,and co-transfected with miRNA-34a-5p mimic or NC mimic.The luciferase activity of co-transfected cells was detected after 48 h.After transfection with miRNA-34a-5p mimic,U251 cells were treated with HPS 500?g/ml for 48h.The expression level of miRNA-34a-5p was detected by qRT-PCR.The protein levels of UHRF2,cyclin El and cyclin D1 were detected by western blot.The proliferation level of U251 cells was detected by MTT assay.Results1.After co-transfection of wt UHRF2 3'-UTR with miRNA-34a-5p mimic,the luciferase activity was significantly reduced,which confirmed that miRNA-34a-5p directly targeted UHRF2 in glioma U251 cells.2.After treatment of U251 cells with 500?g/ml HPS,the miRNA-34a-5p level was significantly reduced.MiRNA-34a-5p expression was significantly up-regulated after HPS treatment combined with miRNA-34a-5p mimic transfection.3.MiRNA-34a-5p overexpression down-regulated UHRF2 expression enhanced by HPS,and up-regulated cyclin El and cyclin D1 reduced by HPS.4.HPS-inhibited U251 cell proliferation can be reversed by miRNA-34a-5p overexpression.Conclusion1.HPS inhibited miRNA-34a-5p expression.2.MiRNA-34a-5p reversed the HPS-induced UHRF2 and HPS-reduced cyclin E1/D1 by targeting UHRF2.Part ? Tumor Biological Characteristics Effects of HPS onNude Mice Bearing Glioma TumorObjectiveTo investigate of the anti-proliferation effects and cell cycle regulation of HPS on human glioma in vivo levels.MethodsU251 cells were inoculated under the skin of nude mice to establish a xenograft model of human glioma.Then,150mg/kg and 400mg/kg HPS were given intragastorally.Meanwhile,400mg/kg HPS ad ministration and miRNA-34a-5p mimic intratumor injection were also designed.Tumor volume was detected every 4 days and tumor weight was weighed on day 24.The expression levels of cyclin E1,cyclin D1,p53,Bcl-2 and Bax were detected by western blot.Results1.150 mg/kg,400 mg/kg HPS inhibited the growth and tumor weight of xenograft tumors in vivo,and this effect was correlated with the dose of HPS.However,miRNA-34a-5p overexpression reversed the antitumor effect of HPS and induced tumor growth.2.In the 150mg/kg and 400mg/kg HPS-treated groups,the levels of cyclin E1 and cyclin D1 in the tumor decreased significantly,the expression levels of pro-apoptosis-related protein Bax and anti-tumor related protein p53 were increased,and the levels of anti-apoptosis-related protein Bcl-2 were decreased.3.However,miRNA-34a-5p overexpression reversed the role of HPS,up-regulating cyclin E1,cyclin D1,Bcl-2,and down-regulating Bax and p53 levels.Conclusion1.HPS inhibited the in vivo tumor growth by inhibiting cyclin E1/D1.2.MiRNA-34a-5p reversed the HPS-induced tumor inhibition in vivo.Part ? Effects of HPS on Immune Regulation in Nude MiceBearing Glioma TumorObjectiveTo explore the effects of HPS on immune system regulation in human glioma model animals.MethodsThe phagocytic activity and phagocytic index of peritoneal macrophages in nude mice were detected by chicken erythrocyte stimulation.The serum was collected from each group of nude mice.The concentrations of TNF-a and caspase-3 in serum were detected by ELISA.Results1.150mg/kg,400mg/kg HPS can improve the phagocytic activity and phagocytic index of peritoneal macrophages and enhance the immune function of nude mice.However,miRNA-34a-5p overexpression decreased phagocytic activity and phagocytic index of peritoneal macrophages and decreased immune function in nude mice.2.150mg/kg,400mg/kg HPS can increase the concentration of TNF-a and caspase-3 in the serum of nude mice bearing tumor.However,miRNA-34a-5p overexpression inhibited the HPS-induced up-regulation.ConclusionHPS increased the immune function in nude mice bearing glioma tumor,whereas miRNA-34a-5p decreased the immune function.