| BackgroundGlioma is the most common tumor of human brain accounting for 50% of primary brain tumor, with an incidence of approximate 5 per 100,000. Among the gliomas, more than half are Glioblastoma Multiforme (GBM). So far, the glioma therapy mainly includes microsurgery resection combined with post-operation radiotherapy and chemotherapy. However, the recurrent rate remains high and the prognosis usually remains poor, even if treated with combined therapy. In recent years, with the development of glioma research, based on anti-invasion, anti-angiogenesis, oncogene and immune-inhibitory microenvironments, several treatment strategies have been gained some achievements, but due to gliomas’characteristics of proliferation, drug resistance, invasion, immuno-inhibition, related clinical trials still failed to prolong patients’overall survival or improve their prognosis outstandingly.Glioma needs sufficient blood supply to grow, so the mechanism of angiogenesis remains one of hotspots in glioma research. However, those achievements proved effective in vitro and in vivo assays, such as anti-VEGF therapy, were found less useful. Recently, through extensive research, many teams have discovered a new mechanism for angiogenesis in malignant solid tumors, namely vasculogenic mimicry, which may cause the failure of Bevacizumab therapy. Therefore, further research in VM may provide new strategies for anti-angiogenesis.Numerous studies found that human gliomas, especially high-grade gliomas have a very active angiogenic activity, but the abundant blood supply for metabolically proliferation of glioma cells is still relatively insufficient, leaving glioma cells parenchyma generally in hypoxia state. Hypoxia is one of the important characteristics of solid malignancies. In the glioblastoma tissue, hypoxia area is widespread and is closely related to its biological characteristics. A large number of domestic and international studies have shown that hypoxia can significantly improve the proliferation of glioma cells, stimulate angiogenesis and promote invasion and migration. Meanwhile, hypoxia can also promote angiogenesis as a causative factor and induce glioma cells to upregulate VEGF expression and other angiogenic factors to form new tumor blood vessels, providing nutrients for tumor growth. Therefore, vascular mimicry, as a brand-new model of angiogenesis, is raising more and more attention.VM is an angiogenic pattern which is specifically for aggressive tumor. The formation of VM only depends on the migration and morphological transformation of tumor cells, without direct participation of vascular endothelial cells, to form a vessel-like networks which provide blood supplying for tumor cell growth. Without direct participation of vascular endothelial cells, the VM composed of tumor cells not only forms quickly but also has a strong ability to supply blood, which could meet the need of substantial and energy metabolism for tumor cells. This atypical vascular characteristic of VM could be one of the most important reasons for poor therapeutic effect of tumor angiogenesis. Although a variety of molecular signal pathways which participate in VM induction has been reported, the specific molecular signal pathways are still unclear and need more attention. In our research, we found that hypoxia is one of the vital factors for promoting the formation of VM in glioma, and the changes of microRNA induced by hypoxia could regulate this process.microRNA has been the research hotspot in recent years, due to it’s important regulatory role in all biological processes of cells. microRNA is a class of endogenous, single stranded, small, non-coding RNAs, which regulates gene expression at the post-transcriptional level. The main regulatory process of it is to bind with sequence of the seed-specific sequence in 3’untranslated region, and induces degradation or translational repression of the mRNA, thereby regulating the biological activity of tumor cells. Hence, the microRNA widely used in vitro experiment is designed by using this principle based on different experimental purposes. Owning to the proliferation characteristics of glioma, studies showed that severe hypoxia in glioma microenvironment can significantly affect the expression of microRNA in tumor cells. The key to the formation of VM is tumor cell migration and morphology transformation, thus, microRNA involved in glioma cell invasion, migration and it’s related target gene play an important role in the regulation of hypoxia on gliomas VM. In this research, we first found that microRNA-584-3p and microRNA-let-7f not only significantly inhibit the VM of glioma cells, but also regulate the cell invasion and migration of glioma.In addition to angiogenesis ability, the human brain glioblastoma has strong invasive characteristics, and it also contributes to the progress of glioma malignancy. The cell morphology transformation and biological characteristics are closely related to the invasion and migration, while strong invasion is one of the most important malignant phenotype of glioma. Recent studies have shown that tumor cells with EMT (Epithelial-Mesenchymal transition) have higher degree of malignancy. Moreover, the entire process, tumor cells simulating vascular endothelial cells to form vascular mimicry phenotypes, is the mutual transformation between epithelial and mesenchymal essentially. Thus, the regulation of glioma cell invasion, migration and EMT molecular and signal transduction pathway may play an important role in the formation of VM. Consequently, the microRNAs related to regulating glioma cells invasion and migration should have an important regulatory role in the formation of VM.The research revealed that hypoxia induces vascular mimicry of glioblastoma and further explored the key regulatory microRNAs associated with VM and the specifically molecular mechanism of them. The study was mainly divided into the following sections. Firstly, we studied the promoting effect of hypoxia on forming VM of glioblastoma cells and the close relations between vascular mimicry and the ability of glioma cell invasion and migration. Secondly, through related experiments of VM based on the microRNAs (microRNA-584-3p and microRNA-Let-7f), we had found their invasion and migration regulatory ability in glioblastoma, which confirmed both could inhibited the hypoxia-induced formation of VM in glioblastoma. Lastly, we conducted further research on the specifically molecular mechanism of microRNA-584-3p and microRNA-Let-7f regulating glioblastoma. This research was intended to provide some theoretical evidences for the anti-angiogenic therapy of human brain glioma.Part I microRNA-584-3p reduces the vasculogenic mimicry of human glioma cells by regulating hypoxia-induced ROCK1 dependent stress fiber formation1. Analyzing the connection between glioma WHO classification and VM expression in glioma specimens.We detected the VM in 81 human glioma specimens with different grades and 3 normal brain tissues. We collected glioma and normal brain specimens from different glioma patients and did IHC staining of CD34 and PAS in them. We found that the VM expression is positively correlated with glioma grades.2. Detecting microRNA-584-3p gene expression in VM positive and negative specimens.We detected the microRNA-584-3p expression in VM positive and negative glioma specimens. The result showed that microRNA-584-3p gene expression is lower in VM positive patients than it in VM negative patients (p<0.05)3. Analyzing the Correlation between VM expression and the prognosis of glioma patients.Kaplan-Meier analysis of VM expression and the survival time of glioma patients demonstrated that VM+patients had a shorter postoperative survival time (p<0.01), in which the median survival time is only 328 days. (The VM+patients with III and IV grade glioma had even shorter survival time of 281 days).4. The study of relationship between microRNA-584-3p expression and glioma invasion, migration,Cytoskeleton and VM.We found that neither overexpression nor inhibiting the expression of microRNA-584-3p would affect the cell viability of A172 and U87 cells (Results obtained from CCK8). we transiently transfected A172 glioma cells with the miR-584-3p inhibitor or mimics and then stained them to visualize stress fibers using phalloidin, our results showed that miR-584-3p knockdown facilitated stress fiber formation and the maintenance of movement morphology of glioma cells and that miR-584-3p overexpression impaired hypoxia-induced stress fiber formation.We then seeded A172 cells which were transfected with microRNA-584-3p mimics or inhibitor to Matrigel coated 96-well plate and cultured the cells for 6 hours. We found that the microRNA-584-3p mimics group significantly inhibited the VM in GBM cells and the microRNA-584-3p inhibitor promoted the VM in GBM cells.5. Exploring the effect of hypoxia on GBM VMWe seeded A172 and U87 cells to Matrigel coated 96-well plate and cultured the cells under normoxia or hypoxia for 6 hours. We found that hypoxia promoted the VM in GBM cells.6. The study of how microRNA-584-3p affects the VM of GBM under hypoxiaWe seeded A172 and U87 cells which were transfected with microRNA-584-3p mimics or inhibitor to Matrigel coated 96-well plate and cultured the cells under normoxia or hypoxia. (A172 cells:4h、6h、10h、12h; U87 cells:6h、8h、10h、12h). Our results clearly demonstrate that miR-584-3p knockdown could markedly promote VM formation of human glioma cells and aggravate the hypoxia-induced VM promoting effects. While miR-584-3p over-expression could antagonize VM formation, especially hypoxia-induced VM formation on human glioma cells.7. The study of the mechanism of how microRNA-584-3p modulate the VM of GBM under hypoxia.Our previous study have shown that ROCK1 is the target of microRNA-584-3 and Y27632 can inhibit ROCK1 signaling. In this study, we found that Y27632 can inhibit the VM of GBM and had even better therapeutic efficiency than microRNA-584-3 mimics. And the pro-VM effect of microRNA-584-3 inhibitor can also be blocked by Y27632.In summary, our study demonstrates that VM expression is positively related to glioma grades and the prognosis of glioma patients is negatively correlated with VM expression. The VM+ patients had lower microRNA-584-3 expression than VM-patients and the overexpression of microRNA-584-3 can suppress the VM in GBM and vice versa. Inhibiting ROCK1 can block the anti-VM effect of microRNA-584-3p which implied a potential role of ROCK1 in how microRNA-584-3p modulating VM. Collectively, we found that microRNA-584-3p may inhibit the VM in GBM through blocking ROCK1 and microRNA-584-3p is a cancer suppressor gene.Part Ⅱ microRNA-Let-7f reduces the vasculogenic mimicry of human glioma cells by regulating periostin (POSTN) dependent migration1. Analyzing the connection between glioma WHO classification and VM expression in glioma specimens.We detected the VM in 45 human glioma specimens with different grades and 2 normal brain tissues by immunochemical staining. We collected glioma and normal brain specimens from different patients and did IHC staining of CD34 and PAS in them. We found that the VM expression is positively correlated with glioma grades.2. Detecting microRNA-let-7f gene expression in VM positive and negative specimens.We detected the microRNA-let-7f in VM positive and negative glioma specimens (45 patients). The result showed that microRNA-let-7f gene expression is lower in VM positive patients and higher in VM negative patients, and the difference was statistically significant (p<0.05)3. Correlation between VM expression and the prognosis of glioma patients.To analyze whether VM abundance in clinical samples associated with the clinical survival information of the 81 patients, Kaplan-Meier test were used here. Kaplan-Meier analysis of VM expression and the survival time of glioma patients demonstrated that VM+ patients had shorter a lower postoperative 1-year survival rate. (p<0.05)4. The study of relationship of microRNA-let-7f and glioma invasion, migration and VM.We used the microRNA-let-7f inhibitor and mimics. As a proof of principle, the transient transfection of 80 nM inhibitor or mimicsdid not affect glioma cell viability (Results obtained from CCK8). And we observed a significant pro-migratory effects of the microRNA-let-7f inhibitor and anti-migratory effects of its mimics in cell detachment test and transwell assays of A172 cells, we transiently transfected A172 glioma cells with the microRNA-let-7f inhibitor or mimics and then seed them to Matrigel coated 96-well plate and cultured the cells for 6 hours. We found that over-expression of microRNA-let-7f group significantly blocked the VM. While the microRNA-let-7f knockdown group significantly promoted the VM.5. The study of how microRNA-let-7f affects the VM of GBM under hypoxiaWe seeded A172 and U87 cells which were transfected with microRNA-let-7f mimics or inhibitor to Matrigel coated 96-well plate under normoxia or hypoxia. we investigated the time-dependent effects of miR-Let-7f knockdown and overexpression in A172 glioam cells. (A172 cells:4h、6h、10h、2h; U87 cells:6h、8h、10h、 12h).Interestingly, the VM had a dynamic process with increased observation time, our results clearly demonstrate that miR-Let-7f knockdown could markedly promote the VM forming capacity of human glioma cells and aggravate the hypoxia-induced VM promoting effects. While miR-Let-7f over-expression could antagonize the VM formation, especially the hypoxia-induced VM on human glioma cells. Our previous study has shown that POSTN is a downstream molecule of microRNA-let-7f. In this experiment, we found that inhibiting POSTN expression can also impair the VM in GBM under hypoxia which implied that microRNA-let-7f modulate hypoxia-inducible VM though POSTN.Above all, Our study demonstrates that VM expression is positively related to glioma grades and the prognosis of glioma patients is negatively correlated with VM expression. The VM+ patients had lower microRNA-let-7f expression than VM-patients and the overexpression of microRNA-let-7f can suppress the VM in GBM and vice versa. Inhibiting POSTN can also block the VM formation in GBM under hypoxia. Collectively, we found that microRNA-let-7f may promote the VM in GBM through modulating POSTN, and our findings suggest that miR-Let-7f may serve as a potential complementary therapeutic target in the anti-angiogenesis treatment of gliomas via suppressing VM. |
PDF Full Text Request