Migrating glioma cells express stem cell markers and give rise to new tumors upon xenografting

Glioblastoma (GBM) is the most frequent and malignant brain tumor with an overall survival of only 14.6 months. Although these tumors are treated with surgery, radiation and chemotherapy, recurrence is inevitable. A critical population of tumor cells in terms of therapy, the so-called cancer stem cells (CSCs), has been identified in gliomas and many other cancers. These tumor cells have a stem cell-like phenotype and are suggested to be responsible for tumor growth, chemo- and radio-resistance as well as recurrence. However, functional evidence for migrating glioma cells having a stem cell-like phenotype is currently lacking. In the present study, the aim was to characterize the phenotype of migrating tumor cells using a novel migration assay based on serum-free stem cell medium and patient-derived spheroid cultures. The results showed pronounced migration of five different GBM spheroid cultures, but not of the commercial cell line U87MG. An in vitro limiting dilution assay showed preserved but reduced spheroid formation capacity of migrating cells. Orthotopic xenografting in mice showed preserved but reduced tumorigenic capacity. Profiling of mRNAs revealed no significant deregulation of 16 predefined CSC-related genes and the HOX-gene list in migrating cells compared to spheroids. Determination of GBM molecular subtypes revealed that subtypes of spheroids and migrating cells were identical. In conclusion, migrating tumor cells preserve expression of stem cell markers and functional CSC characteristics. Since CSCs are reported to be highly resistant to therapy, these results emphasize that the CSC phenotype should be taken into consideration in future treatment of GBMs.     

Microarray-based analysis: identification of hypoxia-regulated microRNAs in retinoblastoma cells

Hypoxia is an essential feature of retinoblastoma and contributes to poor prognosis and resistance to conventional therapy. MicroRNAs (miRNAs) are small non-coding RNAs involved in a wide variety of biological processes, including cell differentiation, proliferation, death and metabolism. However, the relationship between hypoxia and the expression of miRNAs in retinoblastoma is not well understood. In this study, we aimed to analyze the pattern of miRNA expression in a retinoblastoma cell line under hypoxic conditions and to identify the miRNAs regulated by hypoxia, as well as their possible functions. miRNA expression profiling in retinoblastoma cells (HXO-RB44) under normal and hypoxic conditions was assessed by microarray techniques. The differentially expressed miRNAs were subjected to bioinformatic analyses to predict and categorise the key miRNAs and their target genes. A quantitative real-time RT-PCR approach was used to validate their expression. A Cell Counting kit was used to evaluate the functional significance of miR-181b in RB cell proliferation. There were 46 miRNAs that changed expression more than 2-fold in response to hypoxia (34 up-regulated and 12 down-regulated). We identified a cluster of miRNAs that includes miR-181b, miR-125a-3p, miR-30c-2, miR-497 and miR-491-3p as hypoxia-regulated miRNAs (HRMs) in retinoblastoma cells, of which miR-181b was the most typically differentially expressed miRNA under hypoxic conditions. Functionally, these HRMs are involved in apoptosis, cell adhesion, cell proliferation and mRNA processing, all processes that associate closely with the hypoxia response of cancer cells. Additionally, we found that administration of miR-181b inhibitor can suppress proliferation of retinoblastoma cells. These findings provide the first evidence that miRNAs play an important role in the hypoxia response of retinoblastoma cells. MiR-181b, the most typically up-regulated miRNA may aid in future clinical intervention of retinoblastoma.     

MicroRNAs 221 and 222 bypass quiescence and compromise cell survival

MicroRNAs (miRNA) have tumor suppressive and oncogenic potential in human cancer, but whether and how miRNAs control cell cycle progression is not understood. To address this question, we carried out a comprehensive analysis of miRNA expression during serum stimulation of quiescent human cells. Time course analyses revealed that four miRNAs are up-regulated and >100 miRNAs are down-regulated, as cells progress beyond the G(1)-S phase transition. We analyzed the function of two up-regulated miRNAs (miR-221 and miR-222) that are both predicted to target the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57. Our results show that miR-221 and miR-222 both directly target the 3' untranslated regions of p27 and p57 mRNAs to reduce reporter gene expression, as well as diminish p27 and p57 protein levels. Functional studies show that miR-221 and miR-222 prevent quiescence when elevated during growth factor deprivation and induce precocious S-phase entry, thereby triggering cell death. Thus, the physiologic up-regulation of miR-221 and miR-222 is tightly linked to a cell cycle checkpoint that ensures cell survival by coordinating competency for initiation of S phase with growth factor signaling pathways that stimulate cell proliferation.     

Comprehensive analysis of microRNA expression profile in malignant glioma tissues

Malignant gliomas represent the most devastating group of brain tumors in adults, among which glioblastoma multiforme (GBM) exhibits the highest malignancy rate. Despite combined modality treatment, GBM recurs and is invariably fatal. A further insight into the molecular background of gliomagenesis is required to improve patient outcomes. The primary aim of this study was to gain broad information on the miRNA expression pattern in malignant gliomas, mainly GBM. We investigated the global miRNA profile of malignant glioma tissues with miRNA microarrays, deep sequencing and meta‐analysis. We selected miRNAs that were most frequently deregulated in glioblastoma tissues, as well as in peritumoral areas, in comparison with normal human brain. We identified candidate miRNAs associated with the progression from glioma grade III to glioma grade IV. The meta‐analysis of miRNA profiling studies in GBM tissues summarizes the past and recent advances in the investigation of the miRNA signature in GBM versus noncancerous human brain and provides a comprehensive overview. We propose a list of 35 miRNAs whose expression is most frequently deregulated in GBM patients and of 30 miRNA candidates recognized as novel GBM biomarkers.     

miR-27a在猿猴病毒小T抗原诱导支气管上皮细胞恶性转化中的作用

目的:检测猿猴病毒40(simian virus 40,SV40)小T抗原(small T antigen,ST)诱导人支气管上皮细胞(human bronchialepithelial cell,HBE)恶性转化中miRNAs的表达谱,寻找与细胞转化相关的miRNAs。方法:选择HBE、HBER和HBERST细胞株,提取总RNA,利用miRNA芯片和实时荧光定量PCR技术检测和验证永生化HBE、HBER和HBERST细胞中差异表达的miRNAs。通过细胞生长曲线检测、细胞周期分析、细胞克隆形成试验等确证与SV40 ST诱导HBE细胞恶性转化相关的miRNAs。结果:在HBE、HBER和HBERST细胞856个miRNA的表达谱中筛选出6个与SV40 ST诱导细胞转化相关的miRNA,2个表达上调(miR-20a和miR-27a).4个表达下调(let-7d,let-7f,miR-1246和miR-3746)。抑制miR-27a能减缓HBERST细胞的增殖速度(P0.01),延长细胞在G0～G1期的停留时间(P0.01)和降低HBERST细胞在软琼脂上形成克隆的数目(P0.01)。结论:miR-27a的异常表达参与了SV40ST诱导的HBE细胞恶性转化。     

miR-155, identified as anti-metastatic by global miRNA profiling of a metastasis model,inhibits cancer cell extravasation and colonization in vivo and causes significant signaling alterations

To gain insight into miRNA regulation in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using global miRNA profiling, 28 miRNAs were found to exhibit significantly altered expression between isogenic metastasizing and non-metastasizing cancer cells, with miR-155 being the most differentially expressed. Highly metastatic mesenchymal-like CL16 cancer cells showed very low miR-155 expression, and miR-155 overexpression in these cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. Our experiments addressing the underlying mechanism of the altered tumor burden revealed that miR-155-overexpressing CL16 cells were less invasive than CL16 control cells in vitro, while miR-155 overexpression had no effect on cancer cell proliferation or apoptosis in established lung tumors. To identify proteins regulated by miR-155 and thus delineate its function in our cell model, we compared the proteome of xenograft tumors derived from miR-155-overexpressing CL16 cells and CL16 control cells using mass spectrometry-based proteomics. >4,000 proteins were identified, of which 92 were consistently differentially expressed. Network analysis revealed that the altered proteins were associated with cellular functions such as movement, growth and survival as well as cell-to-cell signaling and interaction. Downregulation of the three metastasis-associated proteins ALDH1A1, PIR and PDCD4 in miR-155-overexpressing tumors was validated by immunohistochemistry. Our results demonstrate that miR-155 inhibits the ability of cancer cells to extravasate and/or colonize at distant organs and brings additional insight into the complexity of miR-155 regulation in metastatic seeding.     

miR-153靶向ZEB2抑制胶质母细胞瘤侵袭与转移

目的:探究miR-153对人胶质母细胞瘤（glioblastoma,GBM）侵袭转移的影响及相关作用机制。方法:利用qRT-PCR检测miR-153在GBM中的表达;将miR-153转染至胶质瘤U251细胞后,应用qRT-PCR 验证转染效率;应用Western blot、Transwell及划痕实验检测U251细胞的上皮间质转化、细胞侵袭及转移能力的变化;应用qRT-PCR及Western blot检测ZEB2的mRNA和蛋白表达;利用质粒转染技术过表达miR-153后,同时过表达ZEB2,再应用Transwell及划痕实验检测U251细胞的侵袭及转移能力的变化。结果:与对照相比,miR-153在GBM中低表达;过表达miR-153显著抑制胶质瘤U251细胞的上皮间质转化、细胞侵袭及转移,并能够抑制U251细胞中ZEB2的蛋白表达;ZEB2过表达有效阻断了miR-153抑制U251细胞侵袭及转移的作用。结论:miR-153能够靶向下调ZEB2,进而抑制胶质瘤U251细胞上皮间质转化及细胞侵袭转移。     

MicroRNA-107 inhibits glioma cell migration and invasion by modulating Notch2 expression

MicroRNAs (miRNAs), small non-protein-coding RNA molecules, modulate target gene expression by binding to 3′untranslated regions (UTR) of target mRNA. These molecules are aberrantly expressed in many human cancers, and can function either as tumor suppressors or oncogenes. In the current study, we show that miR-107 is down-regulated in glioma tissues and cell lines, and its overexpression leads to inhibition of the migratory and invasive ability of glioma cells via direct targeting of Notch2, which is known to transactivate Tenascin-C and Cox-2. Experiments with Notch2 siRNA further suggest that miR-107 may exerts its anti-invasive activity through Notch2 signaling pathways. Our findings collectively indicate that miR-107 is involved in glioma cell migration and invasion, and support its utility as a potential target for glioma treatment.     

MicroRNA expression signature in gastric cancer

Objective: To identify the miRNA specific signature as novel diagnostic and prognostic tools for gastric cancer. Methods: miRNAs expression profiling of 3 normal gastric tissues, 24 malignant tissues, gastric cancer cell SGC7901 and normal gastric cell GES-1 were detected using microarray technology. The hierarchical clustering algorithm of the Cluster software was used to analyse the miRNAs expression of all samples. The expression levels of miR-433 and miR-9 which were significantly down-regulated in gastric cancer tissues and SGC7901 cells by microarray technology were validated by quantitative Real-time PCR (qRT-PCR).Results: Differential expressions of 26 individual miRNAs between normal samples (including 3 normal gastric tissues and GES-1 cells) and carcinomas (including 24 malignant tissues and SGC7901 cells) were discovered,19 of them showing down-regulation and 7 up-regulation in carcinoma samples. Hierarchical clustering of the cancer samples by their miRNA expression accurately separated the carcinomas from normal samples and further their histotypes of carcinomas. The expression levels of miR-433 and miR-9 were significantly down-regulated in gastric cancer tissues and SGC7901cells Conclusion: The differential expression of miR-433 and miR-9 may be used as a novel diagnostic tool for gastric cancer.     

Human glioma growth is controlled by microRNA-10b

MicroRNA (miRNA) expression profiling studies revealed a number of miRNAs dysregulated in the malignant brain tumor glioblastoma. Molecular functions of these miRNAs in gliomagenesis are mainly unknown. We show that inhibition of miR-10b, a miRNA not expressed in human brain and strongly upregulated in both low-grade and high-grade gliomas, reduces glioma cell growth by cell-cycle arrest and apoptosis. These cellular responses are mediated by augmented expression of the direct targets of miR-10b, including BCL2L11/Bim, TFAP2C/AP-2γ, CDKN1A/p21, and CDKN2A/p16, which normally protect cells from uncontrolled growth. Analysis of The Cancer Genome Atlas expression data set reveals a strong positive correlation between numerous genes sustaining cellular growth and miR-10b levels in human glioblastomas, while proapoptotic genes anticorrelate with the expression of miR-10b. Furthermore, survival of glioblastoma patients expressing high levels of miR-10 family members is significantly reduced in comparison to patients with low miR-10 levels, indicating that miR-10 may contribute to glioma growth in vivo. Finally, inhibition of miR-10b in a mouse model of human glioma results in significant reduction of tumor growth. Altogether, our experiments validate an important role of miR-10b in gliomagenesis, reveal a novel mechanism of miR-10b-mediated regulation, and suggest the possibility of its future use as a therapeutic target in gliomas.     

MicroRNA-27a distinguishes glioblastoma multiforme from diffuse and anaplastic astrocytomas and has prognostic value

MicroRNAs (miRNAs) are a class of small noncoding RNAs that bind to 3’-untranslated (UTR) regions of target messenger RNAs to regulate protein synthesis. Reports have suggested that a set of specific miRNAs may be used as diagnostic and/or prognostic markers for astrocytoma grading. However, there are few studies of the specific miRNAs differentially expressed in each astrocytoma grade. MiRNA-containing total RNA was isolated from archived formalin-fixed, paraffin-embedded (FFPE) samples from WHO grade II-IV astrocytoma patients. The RNA was labeled and hybridized to Affymetrix miRNA 2.0 arrays. Statistical analysis identified several miRNAs differentially expressed in each astrocytoma grade. In particular, miR-27a, miR-210, and miR-1225-5p expression levels were able to differentiate grade IV from grade II and III astrocytomas as confirmed by real-time PCR. Kaplan-Meier survival analysis showed that disease progression occurred faster for Glioblastoma Multiforme (GBM) patients with a lower miR-27a expression level. Transfection of CRL-1690 GBM human cancer cells with a miR-27a oligonucleotide inhibitor followed by Real-time PCR identified six potential miR-27a target genes. Furthermore, the miR-27a oligonucleotide inhibitor induced CRL-1690 cell apoptosis. Taken together, our results provide additional miRNA signatures for distinguishing GBM from lower astrocytoma grades and suggest miR-27a as a prognostic and therapeutic target for GBM.     

miR-32-5p通过靶向Dickkopf相关蛋白3的表达调控乳腺癌MDA-MB-231 细胞的生物学行为

目的：通过生物信息学手段筛选乳腺癌中差异表达的关键miRNA及其靶基因,干预其在乳腺癌细胞中的表达并观察对乳腺癌细胞功能的影响。方法：利用GEO数据库筛选在乳腺癌中差异表达的miRNA,ENCORI数据库验证差异miRNA的表达,以选定最显著的差异表达 miRNA 为研究对象;利用 Starbase、miRDB 和 miRWalk 数据库预测 miR-32-5p 的靶基因,利用DAVID数据库对靶基因进行GO分析和KEGG分析,利用String数据库联合Cytoscape3.6.2软件进行PPI网络分析及核心基因的筛选,从核心基因中选择相互联系紧密“度值”最显著的Dickkopf相关蛋白3（DDK3）基因进行后续实验。qPCR检测miR-32-5p在人正常乳腺细胞 MCF10A和人乳腺癌细胞MCF7、MDA-MB-231、MDA-MB-453细胞中的表达。向MDA-MB-231细胞中转染miR-32-5p mimic、miR-32-5p inhibitor及各自的对照（NC）序列,分别用CCK-8法、流式细胞术和Transwell实验检测过表达或抑制miR-32-5p对细胞增殖、凋亡和侵袭的影响。结果：从GEO数据库中获取的两个数据集共识别出两个差异miRNA,ENCORI数据库验证差异miRNA的表达发现miR-32-5p的表达水平与GEO数据库的结果一致,故选择其进行研究;预测得到198个miR-32-5p 潜在的靶基因并鉴定出 10 个核心基因（DKK3、WNT2B、SFRP5、SFRP2、SFRP1、LRP6、WNT6、KREMEN1、NEDD4L、TRIP12）,其中DKK3的度值最大可能在乳腺癌中较为重要,于是选择miR-32-5p/DKK3轴进行后续研究。miR-32-5p在3种乳腺癌细胞中的表达水平显著高于正常乳腺细胞（均P<0.01）,其中以MDA-MB-231细胞中表达最高。双荧光素酶基因报告实验验证了miR-32-5p与DKK3基因的靶向结合及其对后者表达的负向调控。转染miR-32-5p mimic、miR-32-5p inhibitor后成功提高或抑制了MDA-MB-231细胞中miR-32-5p的表达。与对照组相比,过表达miR-32-5p可抑制MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭（P<0.05或P<0.01）,敲低miR-32-5p则起相反的作用（均P<0.01）。结论：miR-32-5p/DKK3轴可能是影响乳腺癌发生发展的关键通路,过表达miR-32-5p能够抑制乳腺癌MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭。     

MiR-145 functions as a tumor suppressor by directly targeting histone deacetylase 2 in liver cancer

Aberrant regulation of histone deacetylase 2 (HDAC2) plays a pivotal role in the development of hepatocellular carcinoma (HCC), but, the underlying mechanism leading to HDAC2 overexpression is not well understood. We performed microRNA (miRNA) profiling analysis in a subset of HCCs, and identified four down-regulated miRNAs that may target HDAC2 in HCC. Ectopic expression of miRNA mimics evidenced that miR-145 suppresses HDAC2 expression in HCC cells. This treatment repressed cancer cell growth and recapitulated HDAC2 knockdown effects on HCC cells. In conclusion, we suggest that loss or suppression of miR-145 may cause aberrant overexpression of HDAC2 and promote HCC tumorigenesis.     

MiR224-3p inhibits hypoxia-induced autophagy by targeting autophagy-related genes in human glioblastoma cells

Human glioblastoma multiforme (GBM) is a malignant solid tumor characterized by severe hypoxia. Autophagy plays a protective role in cancer cells under hypoxia. However, the microRNA (miRNA)-related molecular mechanisms underlying hypoxia-reduced autophagy remain poorly understood in GBM. In this study, we performed a miRNA microarray analysis on GBM cells and found that numerous miRNAs were differentially expressed under hypoxic conditions. Further research showed that miR224-3p, one of the significantly down-regulated miRNAs, was involved in regulating hypoxia-induced autophagy in GBM cells. Overexpression of miR224-3p abolished hypoxia-induced autophagy, whereas knocking down endogenous miR224-3p increased autophagic activity under normoxia. In addition, we demonstrated that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, in vitro, miR224-3p attenuated cell proliferation and promoted hypoxia-induced apoptosis, and in vivo, overexpression of miR224-3p inhibited tumorigenesis of GBM cells. Collectively, our study identified a novel hypoxia-down-regulated miRNA, miR224-3p, as a key modulator of autophagy by inhibiting ATGs in GBM cells.     

miR-221/222 overexpession in human glioblastoma increases invasiveness by targeting the protein phosphate PTPμ

Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. In order to identify microRNAs (miRs) involved in glioma tumorigenesis, we evaluated, by a miRarray, differential expression of miRs in the tumorigenic glioma LN-18, LN-229 and U87MG cells compared with the non-tumorigenic T98G cells. Among different miRs we focused our attention on miR-221 and -222. We demonstrated the presence of a binding site for these two miRs in the 3' untranslated region of the protein tyrosine phosphatase μ (PTPμ). Previous studies indicated that PTPμ suppresses cell migration and is downregulated in glioblastoma. Significantly, we found that miR-221 and -222 overexpression induced a downregulation of PTPμ as analyzed by both western blot and real-time PCR. Furthermore, miR-222 and -221 induced an increase in cell migration and growth in soft agar in glioma cells. Interestingly, the re-expression of PTPμ gene was able to revert the miR-222 and -221 effects on cell migration. Furthermore, we found an inverse correlation between miR-221 and -222 and PTPμ in human glioma cancer samples. In conclusion, our results suggest that miR-221 and -222 regulate glioma tumorigenesis at least in part through the control of PTPμ protein expression.     

Mesenchymal stem cells deliver synthetic microRNA mimics to glioma cells and glioma stem cells and inhibit their cell migration and self-renewal

MicroRNAs (miRNAs) have emerged as potential cancer therapeutics; however, their clinical use is hindered by lack of effective delivery mechanisms to tumor sites. Mesenchymal stem cells (MSCs) have been shown to migrate to experimental glioma and to exert anti-tumor effects by delivering cytotoxic compounds. Here, we examined the ability of MSCs derived from bone marrow, adipose tissue, placenta and umbilical cord to deliver synthetic miRNA mimics to glioma cells and glioma stem cells (GSCs). We examined the delivery of miR-124 and miR-145 mimics as glioma cells and GSCs express very low levels of these miRNAs. Using fluorescently labeled miRNA mimics and in situ hybridization, we demonstrated that all the MSCs examined delivered miR-124 and miR-145 mimics to co-cultured glioma cells and GSCs via gap junction–dependent and independent processes. The delivered miR-124 and miR-145 mimics significantly decreased the luciferase activity of their respected reporter target genes, SCP-1 and Sox2, and decreased the migration of glioma cells and the self-renewal of GSCs. Moreover, MSCs delivered Cy3-miR-124 mimic to glioma xenografts when administered intracranially. These results suggest that MSCs can deliver synthetic exogenous miRNA mimics to glioma cells and GSCs and may provide an efficient route of therapeutic miRNA delivery in vivo.     

血清microRNA 在多形性胶质母细胞瘤术预后评估中的研究

目的:筛选多形性胶质母细胞瘤（glioblastoma multiform,GBM）患者术前、术后血清中差异表达的microRNAs（miRNAs）,并探讨差异表达的miRNAs与患者术后预后的相关性。方法:收集2006年1 月至2009年6 月48例北京天坛医院经临床病理诊断为GBM患者的术前术后血清样本。采用Solexa 测序的方法初步筛选出术前术后表达量有差异的 miRNA ,用实时荧光定量PCR（quantitative real-time PCR ,RT-qPCR）的方法对每个样本进行逐一验证,应用t 检验的方法筛选出满足条件的miRNA（两组之间的平均值差异在2 倍以上,且P < 0.05）,对48例患者进行随访,统计生存时间,根据48例患者中位生存时间494 d,将所有标本分为长生存期组和短生存期组,应用Kaplan-Meier 法和Log-rank 检验,研究患者术后血清miRNAs的表达量与患者生存时间之间是否存在统计学意义的相关性。结果:Solexa 结果显示,有63个miRNA 表达量存在差异,基于本研究先前的研究成果和其他文献的报道,从中选出4 个miRNA（miR-26b,miR-30e ,miR-129- 3p,miR-206）进行逐一验证并进行统计学分析,结果只有1 个miRNAs（miR-30e）在术后患者血清中的表达水平有明显上调现象（术前与术后表达水平平均值差异≥ 2 倍且P < 0.05）,随访结果显示,生存时间> 494 d,患者术后血清miR-30e 的表达水平有降低的趋势（P < 0.05）,但生存分析显示,患者术后血清中miR-30e 的表达量与患者总生存时间之间差异无统计学意义（P = 0.101）。 结论:GBM患者术前术后血清中差异表达的miRNA 只有miR-30e ,且术后患者血清中的miR-30e 水平与肿瘤负荷成负相关关系。生存分析结果显示,术后患者血清miR-30e 的表达水平与患者的预后没有明显的相关性。