| BackgroundGlioma, which is the most malignant tumor type, accounts for more than 70% of all brain tumors. The most common subtype (50%) of glioma is glioblastoma (GBM, WHO grade IV). The biological properties of glioblastoma primarily include high mortality and recurrence rates, uncontrollable invasiveness, strong angiogenesis and vasculogenic mimicry, immunosuppressive microenvironment and widespread hypoxia. Although the high mortality and recurrence rates of GBM primarily due to the uncontrollable invasiveness biological characteristics, but the microsurgery, radiation and chemotherapy, anti-invasion therapy, antiangiogeneic therapy, gene therapy and immune therapy of GBM wasn’t work well. The prognosis of GBM patients was still no obvious improvement, the median survival time was only about 12 months, and the 5-year survival rate was less than 5%. Hypoxia is a common feature in solid tumors due to their overwhelming progression and relatively inadequate blood supply. Tumor hypoxia is an independent prognostic factor associated with poor survival. Indeed, numerous studies have suggested that hypoxia activates many cellular processes in tumors, such as proliferation, survival, angiogenesis, migration, and invasion. Although several mechanisms have been proposed to elucidate the hypoxia-induced migration and invasion of tumor cells, an increasing need of studies for hypoxia-induced target molecules will provide new strategies to comprehensive therapy of GBM.The tumor microenvironment plays a critical role in tumor progression, and as the tumor rapidly outgrows its blood supply, malignant proliferative tumor cells are deprived of oxygen. Tumor hypoxia can powerfully induce cells to develop an aggressive and treatment-resistant phenotype that leads to rapid progression and poor prognosis. Brain gliomas, particularly the highly aggressive GBM subtype with its necrotic tissues, are affected similarly by hypoxia. Additionally, cell invasion, apoptosis, chemoresistance, and radiation resistance processes are affected by hypoxia. The extent of the influence of hypoxia on these processes makes it an attractive therapeutic strategy for glioma. While the low-grade glioma has a much lower hypoxia condition, which is insufficient to up regulate the miR-584-3p. While the WHO grade 2 tumors were characterized by modest cellular hypoxia (pO2s≈10%) and grade 3 tumors by modest-to-moderate hypoxia (p02s≈10%-2.5%). Severe hypoxia (≈0.1% oxygen) was present in 5 of 12 grade 4 tumors. Upon examining surgical specimens of human GBM, the cells surrounding the necrotic areas appeared to migrate away from these necrotic areas, possibly implicating hypoxia in the regulation of this process. Therefore, the severe hypoxic microenvironment, as an important incipient factor, significantly affects the biological properties of glioma cells, and leads to the tolerance of radiation and chemotherapy, and enhance of invasion and migration. At the same time, the severe hypoxic microenvironment also significantly changed the component of none-glioma cells and the characteristics of the cytokines in the microenvironment.miRNAs are a class of endogenous 20-to 22-nucleotide (nt)-long, single-stranded non-coding RNAs that have been identified as negative regulators of gene expression at the post-transcriptional level. These small molecules are incorporated into the RNA-induced silencing complex and bind to the seed sequence in the 3’-untranslated regions (3’ UTRs) of their target mRNAs to silence gene translation. Therefore, additional research is warranted to determine the important roles of numerous miRNAs in diverse tumor-related cellular processes, such as origin, proliferation, angiogenesis, survival, and metastasis. And an increasing number of studies have evaluated the roles of highly expressed microRNAs (miRNAs) in mediating the effects of hypoxia. In gliomas, various tumor-promoting and tumor-suppressing miRNAs have been identified, which may be involved in the regulation of proliferation, drug resistance and migration of hypoxic glioblastoma cells.The term autophagy means "self-eating" and is a process that involves the formation of double-membrane vesicles containing damaged or old organelles (autophagosome). Following fusion to the lysosome (autolysosome), autophagy provides cells with reclaimed essential elements for survival through starvation, hypoxia, immune response and chemoradiotherapy. Because cancer cells are over-consumptive, these cells develop ubiquitous autophagy to overcome the relatively infertile tumor microenvironment. Although the autophagic process is composed of death mechanisms in normal and early tumor cells that prevents tumorigenesis through the selective cleanup of damaged organelles and specific oncogene proteins, this process is a double-edged sword in established, advanced tumor cells with increasing metabolic demands that need to be satisfied. In recent years, accumulating evidence has demonstrated that autophagy favors tumor development, and the inhibition of autophagy triggers apoptosis. Autophagy benefits glioma survival under hypoxic metabolic stress conditions and mediates resistance to anti-tumor therapies such as radiation and chemotherapy. Numerous studies have suggested that hypoxia activates multiple cellular processes in tumors, including autophagy. Nevertheless, the mechanisms by which hypoxia induces autophagy remain to be elucidated in detail. Interleukin-6 (IL-6), an overexpressed inflammatory autocrine and paracrine cytokine in glioblastoma, has been reported its promotive effect of proliferation, angiogenesis, apoptosis, invasion and migration. However, how IL-6 contributes to tumor progression by initiating autophagy has yet to be thoroughly investigated.In addition to the drug resistance, high invasion is responsible for the recurrence and mortality rate of glioblastoma. Invasion and migration, as the important malignant tumor phenotype, are regulated by the many factors in tumor microenvironment. Cell polarity determines the pattern of tumor invasion and metastasis. Hypoxia is the most important feature of glioma microenvironment, and in previous study, we found hypoxia could dramatically induce glioma anterior-posteroir polarity. The degree of cell polarity is in relation to hypoxia severity and the capacity of movment and invasion increases accordingly, but the detail remains to be elucidated.It has been reported that hypoxia can significantly affect the activities of RhoGTPase, the key regulators of the tumor cell cytoskeleton. The members of RhoGTPase family include the Rac, RhoA and Cdc42, and only the cooperation and orderly activation of them could initiate migration. Cdc42 activation determines the polarity and extends exploratory pseudopodia. Racl activation extends pseudopod forward. RhoA activation leads contraction at the tail and achieves movement. The inhibition of RhoGTPase will lead to migration and polarization failure of tumor cells. Although an increasing number of studies have evaluated the roles of highly expressed microRNAs (miRNAs) in mediating the hypoxia-induced migration and invasion of tumor cells, further investigations are needed.In the present study, we reported the microRNAs regulating mechanisms of hypoxia-induced autophagy, invasion and migration in glioblastoma cells. Firstly, we investigated the expression of hypoxia- induced miRNAs and mRNAs by high-throughput screening of all genomic miRNAs and mRNAs using a miRNA and mRNA microarray with normoxic and hypoxic glioblastoma cells, and then we identified several key targets of autophagy, invasion and migration with significantly increasing expression levels. Then, we provide evidence that the IL-6 and downstream MIR155-3p pathway has a central role within the hypoxia-induced autophagy, and we evaluated the tocilizumab therapeutic strategies using glioma cell xenograft mouse models. Finally, we reported that miR-584-3p is a novel tumor suppressor and a valuable prognostic marker for glioma. Our data highlight the importance of miRNAs in tumor development and provide new insights into understanding the molecular mechanism and adjuvant therapy underlying glioblastoma progression.Part I:mRNAs and microRNAs array analysis of differentially expressed microRNAs in hypoxic glioma cell lines and identification of microRNAs targets of autophagy, invasion and migration1.1 Objective(1) mRNAs and microRNAs array analysis of differentially expressed genes and microRNAs in hypoxic glioma cell lines. Conjoint analysis of two microarray data and high-throughput screening of autophagy, invasion and migration related microRNAs(2) Further laboratory test of microRNAs significantly affecting hypoxia-induced autophagy, invasion and migration in glioblastoma cells1.2 Methods(1) Hypoxia cell culture, RNA extraction, and Microarray analysis We determine the best hypoxia treatment time by the expression peak of hypoxia inducible factor-1α(HIF-1α) on 24h, extract total RNA, and microRNAs for Microarray testing.(2) Conjoint analysis of two microarray data and target microRNAs screenThe Box Plot and Scatter-Plot visualization were used for assessing the gene expression variation (or reproducibility) between arrays. Heat Map and Hierarchical Clustering arranged samples into groups based on their expression levels, which allows us to hypothesize the relationships between samples. According to the array results and the change ratio in the original data table (Ford-change), we used Volcano Plot method to screen out the hypoxic glioblastoma cells and selected the genes and microRNAs with significantly expression difference. Pathway analysis and GO Analysis were applied to determine the roles of these differentially expressed genes played in these biological pathways or GO terms. And with a large number of literature reading, we used correlation analysis of the differentially expressed genes and microRNAs to further narrowing the scope of research, and to find out the most likely regulators of hypoxia-induced autophagy, invasion and migration in glioblastoma cells.(3) Further laboratory testFurther verifying the levels of miRs in hypoxic U251, T98G, U87 and A172 cells by quantitative real-time PCR. Transfection of microRNA inhibitor and mimics to U251 cells and treatment under hypoxic condition. Detection of the conversion of LC3B-â… to LC3B-â…¡ and the degradation of SQSTM1/p62 (autophagy markers) by Western blot. Further verifying the levels of miRs in IL-6 treated hypoxic U251 cells by quantitative real-time PCR. Testing of U251 glioma cells migration by Transwell migration assays. 1.3 Results(1) mRNAs and microRNAs array analysis of whole genome mRNAs and microRNAs in hypoxic glioma cell linesAfter 24h hypoxic treatment mRNAs and microRNAs array analysis of whole genome mRNAs and microRNAs were tested. The total 27958 loci were detected with 25485 known sites, and 3104 increasing genes and 240 decreasing genes were found. The total 3555 miRs were detected with 2137 known sites, and 157 increasing genes and 85 decreasing genes were found.(2) Conjoint analysis of two microarray data and high-throughput screening of hypoxia-induced autophagy, invasion and migration related microRNAsThe microarray data quality of our hypoxic glioblastoma U251 cell samples was reliable. Volcano Plots shows that there were 304 significant differentially expressed genes, increasing 242, and decreasing 62. And about 84 significantly differentially expressed miRs, with increasing 67 and decreasing 17 were tested. And then we arranged the s significantly differentially expressed genes and miRs in big to small order.Pathway analysis of the differentially expressed genes found a total of 6 high concentration of pathways, with 5 up-regulated and one down-regulated. And GO analysis found out 218 up-regulated GO items, and 87 down-regulated GO items. Then according to a large number of literature review, and the migration or autophagy functions, we selected the most likely five miRs and 10 miRs regulating the autophagy and invasion respectively in glioblastoma cells.(3) Further laboratory test of microRNAs significantly affecting hypoxia-induced autophagy, invasion and migration in glioblastoma cellsFurther verifying the levels of miRs in hypoxic U251, T98G, U87 and A172 cells by quantitative real-time PCR, we finally focused on five most significantly differentially expressed miRs (autophagy related miR-155-3p and miR-224-3p, and the invasion related miR-210, miR-573-5p and miR-584-3p). After transfection of these miRs’ inhibitor and mimics to U251 cells and treatment under hypoxic condition, we found that hypoxia induced miR-155-3p and reduced miR-224 could regulate autophagy. While only miR-584-3p could affect the migration of glioblastoma cells. And the IL-6 could also up-regulate miR-155-3p.1.4 Conclusions(1) Hypoxic condition could significantly change the expression of various genes and miRs of glioblastoma cells. Hypoxia induced miR-155-3p and reduced miR-224 could regulate autophagy of glioblastoma cells. miR-584-3p is involved in the regulation of invasion and migration of U251 cells.(2) Microarray is an effective tool for the screening of molecular regulating targets.Part II:The Mechanism of Hypoxia-induced IL-6 is a Potent Autophagy Initiator in Glioblastoma by p-STAT3/MIR155-3p/CREBRF Pathway and the Therapeutic Efficacy of Tocilizumab2.1 Objective(1) Detection of the expression and the correlation tests of IL-6, STAT3, p-STAT3, LC3B and HIF-1α in human glioma specimens with different grades.(2) In vitro study of autophagy activation and IL-6 up-regulation in glioblastoma cells under hypoxic condition, the autophagy activation pathways in glioblastoma cells treated by exogenous IL-6 and endogenous IL-6, and the key regulatory proteins in the whole pathway of miR-155-3p induced autophagy of glioblastoma cells. In vitro study of IL-6 induced autophagy affects apoptosis in hypoxic glioblastoma cells.(3) Study the effects of IL-6 and MIR155-3p knockdown on in vivo tumor growth, the effects of Tocilizumab on in vivo tumor hypoxia-induced autophagy and growth and the combination therapy efficiency of temozolomide and tocilizumab in our xenograft tumor models.2.2 Methods(1) Hypoxia cell culture, treatments, and RT-PCR or Western blotCulture glioblastoma cells under 1% O2 hypoxic condition, and treat them with exogenous IL-6, IL-6 antibody, STAT3 inhibitor,3-MA, and BAF, then extract total RNA and protein for RT-PCR and Western blot.(2) Autophagy assays in vitroUsing glioblastoma cells (U251) that stably express a GFP-LC3B fusion protein, the localization of GFP-LC3B was examined by fluorescent microscopy. We detected the conversion of LC3B-I to LC3B-II and the degradation of SQSTM1/p62 (autophagy markers) by Western blot Western blot. Transmission electron microscopy for testing the autophagic level of IL-6 treated normoxia U251 and T98G cells. Treatment of normoxic and hypoxic cells with 3-methyladenine (3-MA) and BAF inhibited the initial stage of autophagy and autophagic flux, respectively.(3) Cell transfection and luciferase reporter assaysGFP-LC3B plasmid, IL-6 SiRNA, CREB3 SiRNA, miR-155-3p inhibitor, miR-155-3p mimics, STAT3 binding region mutation luciferase reporter plasmid, and miR-155-3p seed binding region mutation luciferase reporter plasmid transient transfection for testing the autophagic level, knockdown, overexpression, and luciferase reporter assays of treated U251 and T98G cells. IL-6 SiRNA Lentivirus, miR-155-3p inhibitor Lentivirus, and miR negative control Lentivirus stable transfection for in vivo tests.(4) ELISA assayExamination the secretion of IL-6 in glioblastoma cell culture supernatant (U251) under hypoxia and the peripheral blood serum of the mice by ELISA.(5) Immunochemical and immunofluorescence stainingOne hundred and one human glioma tissue samples and three normal brain tissues from decompression operation were obtained. And we detected the expression of IL-6, STAT3, p-STAT3, LC3B and HIF-1α in these human tissue specimens by immunochemical staining. Immunochemical staining of IL-6, p-STAT3, HIF-1α, and LC3B proteins in continuous paraffin sections of five high-grade glioma tissues and the co-localization of them. The expression of HIF-1α, IL-6, LC3B, STAT3, p-STAT3, Ki-67 and cleaved Caspase-3 in all xenograft specimens was examined by immunohistochemical staining. And we collected 5 WHO grade IV glioma fresh surgical removal tissue for fast frozen section, and staining most of the tumor associated cells’markers in the microenvironment. Co-localization of LC3B, ATG5 and CREB3 in 5 sample of WHO grade IV glioma tissues.(6) Cell viability and apoptosis assaysA CCK-8 assay was utilized to determine cell viability of IL-6 antibody blocked normoxia and hypoxia U251 and T98G cells. A Annexin V-FITC/PI flow cytometry and TUNEL staining assay was utilized to determine apoptosis of IL-6 antibody blocked normoxia and hypoxia U251 and T98G cells.Western blot of apoptosis related caspase3 and PARP was utilized to determine apoptosis of IL-6 antibody blocked normoxia and hypoxia U251 and T98G cells. The expression of Ki-67 and cleaved Caspase-3 in all xenograft specimens was examined by immunohistochemical staining.(7) Glioblastoma cell subcutaneous xenografts in vivo mice modelThe effects of IL-6 and MIR155-3p knockdown, Tocilizumab, and combination therapy efficiency of temozolomide and tocilizumab in our in vivo xenograft tumor models were tested by immunohistochemical staining and tumor volume examining.2.3 Results2.3.1 Detection of the expression and the correlation tests of IL-6, STAT3, p-STAT3, LC3B and HIF-1α in human glioma specimens with different gradesThe expression of IL-6, p-STAT3, LC3B and HIF-1α in 101 human glioma specimens with different grades and 3 normal brain tissues were positively correlated with the WHO grade of glioma. Immunohistochemical staining of continuous paraffin sections revealed the co-localization of the IL-6, p-STAT3, HIF-1α, and LC3B proteins in high-grade glioma tissues, particularly in the hypoxic area around tumor vessels. As IL-6 can be derived from many sources, including glioma cells and macrophages, we double stained these markers and found that the IL-6 was primarily secreted from tumor cells, with only 25% of the total amount of IL-6 being secreted by non-tumor immune cells. Pearson’s or Spearman’s rho rank correlation tests showed that IL-6, p-STAT3, HIF-1α, LC3B and grade were positively correlated. Most correlation indexes were approximately 0.7 to 0.8, indicating a strong positive relationship.2.3.2 Autophagy activation and IL-6 up-regulation in glioblastoma cells under hypoxic conditionConsistent with the GFP-LC3B puncta-formation assay, hypoxia led to a significant time-dependent up-regulation of LC3B-II and down-regulation of SQSTM1. Treatment of normoxic and hypoxic cells with 3-methyladenine (3-MA) inhibited the initial and late stages of autophagy, respectively, whereas treatment of normoxic and hypoxic cells with bafilomycin (BAF) blocked autophagic flux and resulted in an increase in hypoxia-induced autophagy. ELISA showed the secretion level of IL-6 was low under normal culture conditions, and hypoxia treatment induced a significant increase in IL-6 secretion.2.3.3 Activation of the IL-6/p-STAT3 pathway in hypoxia-induced autophagy in glioblastoma cellsIL-6 could independently induce autophagy in GBM cells by utilizing GFP-LC3B transient transfection, the LC3B conversion assay and transmission electron microscopy to visualize the aggregation of expressed LC3B in U251 cells. Western blot analysis showed the blockade of both endogenous and exogenous IL-6 repressed autophagy in GBM cells. Bafilomycin Al (BAF) was used to inhibit autophagic flux. The results of Western blot analysis suggested that the IL-6 antibody inhibited hypoxia-induced autophagy by blocking the IL-6/p-STAT3 pathway. Additionally, IL-6 siRNA against endogenous IL-6 also blocked activation of the IL-6/p-STAT3 pathway and hypoxia-induced autophagy in glioblastoma cells.2.3.4 MIR155-3p-induced autophagy and the function of IL-6/p-STAT3 pathway in hypoxic glioblastoma cellsMIR155-3p expression was time dependent in hypoxia-treated U251 cells. MIR155-3p expression was dose dependent in IL-6-treated U251 cells, and suppression of IL-6 significantly reduced MIR155-3p expression. Stattic blocked the IL-6-and hypoxia-induced up-regulation of MIR155-3p in U251 and T98G glioma cells. The p-STAT3-binding element mutation and luciferase reporter assays indicated the direct promoting effect of p-STAT3 on the MIR155-3p promoter. The GFP-LC3B puncta-formation assay showed that the MIR155-3p inhibitor significantly suppressed U251 cell autophagy under hypoxic conditions, an effect that was partially rescued by exogenous IL-6. The GFP-LC3B puncta-formation assay showed that the MIR155-3p mimics further enhanced glioma cell autophagy under hypoxic conditions, partially rescuing the anti-autophagic effects of IL-6 RNAi and the recombinant human IL-6 antibody.2.3.5 The key regulatory proteins in the whole pathway of miR-155-3p induced autophagy of glioblastoma cellsWe searched for potential MIR155-3p target genes in miRDB and identified the best potential target gene, CREBRF, with a full target score and three perfectly strong binding seed sequences. The luciferase activity in MIR155-3p-transfected cells decreased to approximately half of the activity observed using the control miRNA. The level of CREBRF protein was decreased significantly in MIR155-3p mimic-transfected glioma cells compared to those transfected with the negative control miRNA but increased significantly in MIR155-3p inhibitor-transfected glioma cells. As CREBRF is a negative regulator of CREB3, the CREB3 level showed an opposite trend. The cells transfected with CREB3 siRNA exhibited a reduction in CREB3 protein and significantly reduced autophagy under conditions of both normoxia and hypoxia. Quantitative real-time PCR in T98G and U251 cells, revealed a significant decrease in ATG5 after CREB3 knockdown that was confirmed by Western blot in both cell lines. Co-localization of LC3B, ATG5 and CREB3 in a sample of WHO grade IV glioma tissues.In conclusion, it is tempting to postulate that the hypoxia/IL-6/p-STAT3/MIR155-3p/CREBRF/CREB3/ATG5 pathway plays a pivotal role in hypoxia-induced autophagy in glioma cells (All confirmed in primary glioma cells).2.3.6 IL-6 induced autophagy affects apoptosis in hypoxic glioblastoma cellsA CCK-8 assay was utilized to determine cell viability, and the IL-6 antibody significantly suppressed GBM cells only under hypoxic conditions. Hypoxia-induced apoptotic cell death was augmented in the presence of the IL-6 antibody, as demonstrated by Annexin V-FITC/PI and TUNEL assays. Application of the IL-6 antibody resulted in the activation of apoptotic signaling pathways, including activated caspase 3 and PARP.2.3.7 The effects of IL-6 and MIR155-3p knockdown on in vivo tumor growthKnockdown of IL-6 and MIR155-3p could inhibit tumor growth in vivo by blocking autophagy and inducing apoptosis in glioma cells. IL-6 knockdown reduced the expression of IL-6, p-STAT3 and LC3B and induced apoptosis in xenograft specimens, as examined by immunohistochemical staining. MIR155-3p knockdown reduced the expression of LC3B and induced apoptosis in xenograft specimens, as examined by immunohistochemical staining.2.3.8 The effects of Tocilizumab on in vivo tumor hypoxia-induced autophagy and growthTocilizumab markedly inhibited tumor growth in U251 cell xenografts, as measured by tumor volume. IL-6 in the peripheral blood serum of the tocilizumab group was maintained at a lower level, as examined by ELISA, relative to the control group. Tocilizumab reduced the expression of IL-6, p-STAT3 and LC3B in all xenograft specimens, as evaluated by immunohistochemical staining. Tocilizumab inhibited the proliferation and induced apoptosis in xenografts. The expression of Ki67 and cleaved caspase 3 in all xenograft specimens was examined by immunohistochemical staining.2.3.9 Investigation of the combination therapy efficiency of temozolomide and tocilizumab in our xenograft tumor modelsTocilizumab and temozolomide combination therapy markedly inhibited tumor growth in U251 cell xenografts, as measured by tumor volume. Tocilizumab reduced the expression of IL-6, p-STAT3 and LC3B and induced the apoptosis in temozolomide-treated xenograft specimens, as examined by immunohistochemical staining.2.4 Conclusions(1) The expression of IL-6, p-STAT3, HIF-1 a, LC3B and the WHO grade of glioma are positively correlated. The IL-6 is primarily secreted from tumor cells with only 25% of the total amount of IL-6 being secreted by non-tumor immune cells. The co-localization of the IL-6, p-STAT3, HIF-1a, and LC3B proteins in high-grade glioma tissues is in the hypoxic area around tumor vessels.(2) The hypoxia/IL-6/p-STAT3/MIR155-3p/CREBRF/CREB3/ATG5 pathway plays a pivotal role in hypoxia-induced autophagy in glioma cells in vitro. IL-6 plays an important initiation role in this process. Inhibition of IL-6 induces autophagy-enhanced apoptosis in GBM cells.(3) IL-6 and MIR155-3p knockdown in glioma cells can block autophagy, induce apoptosis and inhibit tumor growth in vivo. Tocilizumab inhibits tumor growth by antagonizing hypoxia-induced autophagy and promoting apoptosis in a xenograft tumor model. The anti-IL-6 therapy Tocilizumab reduces temozolomide-induced autophagy and improve its efficiency in vivo.Part III:MicroRNA-584-3p reduces the migration and invasion of human glioma cells by targeting hypoxia-induced ROCK13.1 Objective(1) Analyzing miR-584-3p and ROCK1 expression in clinical samples of surgically removed glioma tissues with different WHO grades. Analysis the miR-584-3p associated clinical survival information of the patients by Kaplan-Meier estimates.(2) The mechanism of miR-584-3p regulated migratory and invasive capacities and cytoskeletal reorganization of hypoxic glioblastoma cells in vitro.(3) Inhibiting effects of miR-584-3p on hypoxia-induced invasion in glioma cells in vivo.3.2 Methods(1) Hypoxia cell culture, treatments, and RT-PCR or Western blotCulture glioblastoma cells under 1% O2 hypoxic condition, and treat them with transfection vectors and ROCK1 inhibitor Y27632, then extract total RNA and protein for RT-PCR and Western blot.(2) Migration and invasion assays and cytoskeletal stainingEffects of miR-584-3p on migratory capacities of hypoxic glioblastoma cells tested by wound healing assay and Transwell migration assays. Effects of miR-584-3p on invasive capacities of hypoxic glioblastoma cells tested by Matrigel invasion assays. We stained the fixed cells cytoskeletal to visualize stress fibers by phalloidin.(3) Cell transfection and luciferase reporter assaysmiR-584-3p inhibitor, miR-584-3p mimics, miR-584-5p inhibitor, miR-584-5p mimics, ROCK1 SiRNA, and miR-584-3p seed binding region mutation luciferase reporter plasmid transient transfection for testing the knockdown, overexpression, and luciferase reporter assays of treated glioblastoma cells. miR-584-3p inhibitor Lentivirus, miR-584-3p mimics Lentivirus, and miR negative control Lentivirus stable transfection for in vivo tests.(4) Immunochemical staining and quantitative real-time PCR of glioma tissue samplesTwenty-six human glioma tissue samples from decompression operation were obtained. And we detected the expression of ROCK1 in these human tissue specimens by immunochemical staining. The levels of miR-584-3p was tested by quantitative real-time PCR.(5) Survival analysisFollow-up result of 26 patients with glioma included survival and the time of death. Significant differences in miR-584-3p expression were observed between the low-grade (â… -â…¡) and high-grade (â…¢-â…£) glioma patients and associated clinical survival information of the patients was analyzed using Kaplan-Meier estimates.(6) Glioblastoma intracranial brain tumor xenografts in vivo mice modelThe mice were randomly divided into three groups (control, miR- 584-3p mimics and miR-584-3p inhibitor) with five mice in each group. For investigation in vivo, intracranial brain tumor xenografts were obtained by transfected U87 cells stereotactically implanting into five-week-old male BALB/c nude mice brains. We adopted a noninvasive and harmless method to continually observe the intracranial tumors size of model mice by T2 weighted magnetic resonance imaging (T2WI-MRI). 5μm thick serial sections were cut and stained with hematoxylin and eosin (H&E) and then evaluated using a microscope. Further immunochemical staining also showed the relationship between miR-584-3p and ROCK-1 levels.3.3 Results3.3.1 Verifying the levels of miR-584-3p in hypoxic glioblastoma cellsWe verified the levels of miR-584-3p in hypoxic U251 and U87 cells by quantitative real-time PCR, and the validated expression results were consistent with the microarray results. miR-584-3p expression peaked at approximately 24 h of hypoxia treatment in glioma cells. The up-regulated miR-210 indicated the qualified hypoxia condition, and the miR-584-5p expression also increased similarly which suggested that the up-regulation of miR-584-3p was at the level of transcription. miR-210 expression also peaked at approximately 24 h of hypoxia treatment in glioma cells.3.3.2 Analyzing miR-584-3p expression in clinical samples of surgically removed glioma tissues with different WHO gradesTo analyze miR-584-3p expression in clinical samples of surgically removed glioma tissues from 26 patients, RT-PCR was performed. Interestingly, significant differences in miR-584-3p expression were observed between the low-grade (â… -â…¡) and high-grade (â…¢-â…£) glioma patients. miR-584-3p expression was significantly lower in low-grade glioma tissues than in high-grade glioma tissues. miR-584-3p expression levels displayed a dispersed distribution among high-grade glioma patients.3.3.3 miR-584-3p associated clinical survival information of the patients was analyzed using Kaplan-Meier estimatesFollow-up result of 26 patients with glioma included survival and the time of death. Seven patients of high-grade (â…¢-â…£) glioma group were dead. Associated clinical survival information of the patients was analyzed using Kaplan-Meier estimates. Unexpectedly, the subgroup of high-grade (â…¢-â…£) glioma patients with high miR-584-3p expression presented a significantly prolonged postoperative survival time.3.3.4 Effects of miR-584-3p knockdown on migratory and invasive capacities of hypoxic glioblastoma cellsThe wound healing assay and Transwell migration assays performed in U251 and U87 glioma cells revealed that the miR-584-3p inhibitor significantly promoted their migration, even under normoxic conditions and evidently potentiated hypoxia-induced pro-migratory effects. The Matrigel invasion assays performed in U251 and U87 glioma cells revealed that the miR-584-3p inhibitor significantly promoted their invasion, even under normoxic conditions and evidently potentiated hypoxia-induced pro- invasive effects.3.3.5 Effects of miR-584-3p over-expression on migratory and invasive capacities of hypoxic glioblastoma cellsThe wound healing assay and Transwell migration assays revealed that miR-584-3p overexpression significantly inhibited U251 and U87 glioma cell migration under normoxic conditions and strongly antagonized the hypoxia-induced pro-migratory effects. The Matrigel invasion assays revealed that miR-584-3p overexpression significantly inhibited U251 and U87 glioma cell invasion under normoxic conditions and strongly antagonized the hypoxia-induced pro-invasive effects.3.3.6 Effects of miR-584-3p overexpression on cytoskeletal re-organization of hypoxic glioblastoma cellsTo determine the role of miR-584-3p in motility-related morphological changes, we stained the cytoskeletal using phalloidin. Our results showed that miR-584-3p knockdown facilitated stress fiber formation. miR-584-3p overexpression with miR-584-3p mimics impaired hypoxia-induced stress fiber formation.3.3.7 The mechanism of miR-584-3p regulated migratory and invasive capacities of hypoxic glioblastoma cellThe miR-584-3p hinder the migratory and invasive capacities of glioma cells and affect the formation of actin stress fibers by modulating the RhoA/ROCK pathway. And miR-584-3p knockdown could induce ROCK-1 expression. After treated with Y-27632 for 12h, the wound healing assay and Transwell migration assays of U251 cells revealed that the pro-migratory effect of the miR-584-3p inhibitor was significantly inhibited by ROCK1 block. The same as Y-27632, ROCK1 k... |
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