| Glioma,the most common primary central nervous system cancers,was progressive with poor survival.The incidence of glioma has increased dramatically in recent years,especially in younger people.GBM is one of the most aggressive tumors.Despite the advances in surgery,radiation therapy,and chemotherapy,the rate of tumor recurrence is high,which highlights the need for more effective therapies.Methylation of the MGMT promoter,co-deletion of complete chromosome arms 1p and 19 q,IDH1 mutation,and ATRX loss have become important molecular markers for diagnosis and prognosis.To develop more optimized and effective treatment strategies for glioblastoma,it is critical to gain deeper understanding of the molecular mechanism underlying glioma genesis and to identify targets for therapeutic intervention.The "Seed and Soil" theory,proposed by Stephen Paget over 120 years ago(1889),laid the foundations for the modern concept of the tumor microenvironment(TME).Exosomes are important components in TME.It is involved in several biological processes,such as growth,proliferation,invasion,and chemotherapy resistance through releasing many kinds of cell factors or miRNAs.Tumor cell derived exosome(T-exo),due to its structural feature and transport function,has been the focus of attention in tumor research area recently.The 2013 Nobel Prize in Physiology or Medicine went to three American and German scientists "for their discoveries of machinery regulating vesicle traffic,a major transport system in our cells".Exosomes are important nano-sized vesicles and attract more and more attentions.T-exo are produced to regulate all vital functions of tumor cells including growth,proliferation,migration,survival,malignancy,invasion,and resistance to host anti-tumor immunity and anti-cancer drugs.They are commonly used by tumor cells for signal communication at long distances to exchange by complex molecular messages and deliver a variety of essential bio-molecules.The use of exosomal nano-particle drug delivery system(DDS),which allow practitioners to use drugs to target specific areas of the body,is making measurable treatment impact.microRNA(miRNA)is a small non-coding RNA molecule,which is capable of binding to 3’ untranslated region(3’UTR)of target mRNA product to regulate gene expression mostly at post-transcriptional level.Because of its powerful role in all kinds of physiological pathological processes,miRNA attracts more and more attention.It is reported that miR-221 has oncogenic effect in rectal cancer,pancreatic cancer,breast cancer and prostate cancer.Next-generation sequencing was performed on RNA samples extracted from glioma tissues,and miR-221 was reported to be overexpressed in glioma patients,especially glioblastomas.Given that it has not been investigated in glioma,we recruit exosomal miR-221 as the research object to study the expression of miR-221 in human brain tissues or GBM-derived exosomes,and to explore the influence of T-exo on proliferation,invasion,apoptosis of glioma cells.DNM3(dynamin3)is a member of the motor protein family(including DNM1,DNM2 and DNM3)that participate in lots of membrane rearrangements such as cytolinesis,phagocytosis,budding of transport vesicles,and cell motility.Recently,DNM3 was implicated as having vital role in tumor cell proliferation and inducing apoptosis.Based on the full analysis of existing literature,we hypothesis DNM3 is linked to the development of glioma.As DNM3 is involved in the predicted results of Targectscan,we further explore the relationship between DNM3 and miR-221.Lucifearse assay was used to confirm the target site on the mRNA of DNM3.The expression level of DNM3,RELA and miR-221 was also determined.Finally transfection experiment was performed to demonstrate miR-221 influences glioma biological behavior by regulating DNM3.Part one The expression of miR-221 in human brain glioma tissues or GBM-derived exosome and the correlation between the expression level of miR-221 and glioma WHO grade.Objective: To explore the expression of miR-221 in human brain tissues or GBM-derived exosomes,and determine the correlation between the expression level of miR-221 and glioma WHO grade.Methods:1 Collection of samples: 48 human glioma samples and 11 non-neoplasm brain samples were collected from the neurosurgery department of the Second Affiliated Hospital of Hebei Medical University in 2015.23 females and 36 males were included,with ages ranging from 30 to 68.Diagnoses were established histologically by two pathologists according to the WHO classification guidelines.All samples were stored at-80℃ in liquid nitrogen immediately after resection.2 Venous blood samples were collected from 48 patients and 11 normal individuals as above.The cell-free serum was collected by holding at room temperature,while the plasma exosome was isolated by centrifugation,and then stored at-80℃ before use.3 SHG-44,U87 MG,HEB and U251 cells were cultured,and the exosome was isolated from the supernatant.4 Determine of miR-221 expression level: qRT-PCR was used to determine the expression level of miR-221 in tumor tissue or exsomes.3 Correlation analysis: t-test was used to analyze the correlation between the expression level of miR-221 and glioma WHO grade or different cell lines.Results:1 Relative miR-221 expression levels were analyzed by qRT-PCR in normal(n=11)and glioma tissues(n=48).The level of miR-221 was significantly upregulated in glioma samples than that in non-neoplasm samples.The differences between low grade glioma(gradeⅡ)and high grade glioma(grade Ⅲ and Ⅳ)were significant(P<0.01).2 The relative values of cell-free and exosomal miR-221 in the serum of cancer-free(normal,n=11)and glioma patients(n=48)were significantly different(P<0.01).3 Relative expression levels of exosomal miR-221 in plasma of normal(n=11)and glioma patients(n=48)were significant(P<0.01).More importantly,serum exosomal miR-221 level increased with the glioma grades.4 Relative expression levels of exosomal miR-221 in the supernatant of different glioma cell lines.miR-221 expression correlated with metastatic potential of the cancer cells(HEB<SHG-44<U251<U87MG).Conclusions:1 miR-221 is over-expressed in human glioma tissues and exosomes from plasma or different glioma cell lines.The expression of miR-221 in high grade glioma tissues is significantly higher than that in low grade glioma tissues.2 Expression level of exosomalmiR-221 is positively correlated with glioma WHO grade.Exosomal miR-221 from plasma might be used as potential diagnostic and prognostic biomarkers for glioma.Part two Influence of U87MG-derived exosomalmiR-221 on SHG-44 cells biological behaviorObjective: To explore the influence of U87MG-derived exosomal miR-221 on proliferation,invasion,chemoresistance of SHG-44 glioma cells.Methods:1 U87 MG and SHG-44 cells were cultured for in vitro functional experiment.Exosome extraction was performed from the supernatant of U87 MG.2 Anti-miR-221(miR-221 ASO)was transfected into the SHG-44 cells to downregulate the expression level of miR-221 in experimental group.Anti-miR-NC was transfected into control group.Meanwhile,SHG-44 cells in experiment groups were treated with U87 MG derived exosomes.3 CCK-8 experiment was performed to investigate the change of proliferation ability of glioma cells after downregulation of miR-221.4 Flow cytometry experiment was performed to investigate the change of apoptosis ability of glioma cells and temozolomide(TMZ)resistance after downregulation of miR-221.5 Both transwell invasion assay and scratch wound assay were used to evaluate the migratory ability of glioma cells after downregulation of miR-221.Results:1 qRT-PCR test showed that expression level of miR-221 in SHG-44 cells was significantly decreased after transfection of anti-miR-221 or incubation with U87MG-derived exosomes(P<0.01).2 CCK-8 assays showed that the proliferation ability of SHG-44 cells was significantly decreased after downregulating miR-221 expression(P<0.01).3 Flow cytometry tests showed that the apoptosis ability of SHG-44 cells stayed unchanged,while downregulatedmiR-221 can sensitize glioma cells to TMZ.4 Transwell invasion assay and scratch woundassay showed that the invasion ability of SHG-44 cells was significantly decreased after downregulating miR-221 expression(P<0.01).Conclusion: Downregulation of miR-221 could significantly decrease proliferation and invasion.Apoptosis of SHG-44 cells stayed unchanged,while downregulatedmiR-221 can sensitize glioma cells to TMZ.miR-221 influences glioma on the level of proliferation,invasion and TMZ resistance.Part three DNM3 is a downstream target of miR-221 and the effects of RELA on miR-221 transcription.Objective: To determine whether miR-221 influences glioma cells biological behavior by regulating DNM3 and whether RELA modulate miR-221 transcription.Methods:1 Search the online tool Targetscan to predict the downstream target site,and screen the results according to relative papers.2 miR-221 overexpression lentiviral vectors were constructed.miR-221 overexpressing cell line were established via stable infection.3 Dual-luciferase tests were used to explore the relationship between DNM3 and miR-221.4 qRT-PCR and Western blot test was performed to determine the expression level of miR-221 and DNM3 in tumor tissues and SHG-44 cells,and analyze the correlation between miR-221 and DNM3.5 The role of DNM3 in glioma cell proliferation,apoptosis and migratory was studied using the CCK-8 assay,flow cytometry,transwell assay and scratch wound assay.6 Bioinformatic analyses were performed to predict the upstream transcription factor(p65,RELA)binding site in the miR-221 promoter region.Using luciferase test,the binding of candidate transcription factors to the miR-221 promoter region was further confirmed.Real-time PCR was used to detect the change in expression of the miR-221 under modulation of RELA.Results:1 DNM3 is presumed to contain target site of miR-221 accord to the Targetscan result and relative document data.2 miR-221 overexpression cell line were established via stable infection.qRT-PCR test showed that expression level of miR-221 in stableSHG-44 cells was 45.5 folds than that of the control(Vector)cells(P<0.01).3 miR-221 could significantly decrease luciferase activity in SHG-44 cells transfected with wild type DNM3 3’UTR,comparing to miR-NC(P<0.01).Mutation on binding sites could significantly abrogate the effect of miR-221 on DNM3 3’UTR(P<0.01).4 DNM3 expression profile was determined by qRT-PCR in human tissues(n=59).DNM3 level decreased with the glioma grades.Spearman’ s analysis indicated a negative correlation between miR-221 and DNM3 expression in glioma cells(Spearman r=-0.908,P<0.01).5 DNM3 was highly expressed after transfection of DNM3.The ability of cell proliferation was significantly downregulated after increasing DNM3 expression.Cell apoptosis detection by flow cytometry also revealed that DNM3 decrease TMZ resistance in glioma cells(P<0.01).Transwell migration assay and scratch wound assay indicated that overexpressing DNM3 inhibits the migration and invasion of glioma cells.6 Through bioinformatic analyses,RELA binding site were identified in the upstream promoter region of human miR-221.Dual-luciferase reporter assays also indicated that RELA could bind to the promoter region of both miR-221 gene.Real-time PCR results indicated that RELA overexpression in SHG-44 cells induce miR-221 expression.Conclusions: DNM3 is a downstream target of miR-221 and RELA modulate miR-221 transcription.Part four miR-221 upregulation promotes tumor growth in vivoObjective: To determine whether miR-221 influences glioma cells biological behavior in animals.Methods:1 miR-221 overexpression lentiviral Vectors were constructed.miR-221 overexpression cell line was established via stable infection.2 The miR-221 expression profile was determined by qRT-PCR in miR-221 overexpressing SHG-44 cell.3 Glioma subcutaneous model in nude mice was established with miR-221 overexpressing SHG-44 cells.4 The growth rate and volume of tumors in vivo are compared with the controls.Results:1 qRT-PCR test showed that expression level of miR-221 in stable SHG-44 cells was 12.5 folds than that of the control(Vector)cells(P<0.01).2 miR-221 overexpressing tumors demonstrated a much faster increase in tumor size.Accordingly,the miR-221 overexpressing tumors collected at 20 days after inoculation weighted over two-times higher than the control tumors comparing to control(P<0.01).Conclusions: MiR-221 upregulation promotes tumor growth in vivo,and miR-221 is also a pro-tumorigenic factor in vivo. |
PDF Full Text Request