MiR-128-3p靶向抑制HOXA5调控多形性胶质母细胞瘤的增殖、侵袭与凋亡

目的 探索HOXA5在胶质母细胞瘤（GBM）中高表达的原因及miR-128-3p调控胶质母细胞瘤进展的分子机制。方法 通过慢病毒转染上调或下调U87细胞中miR-128-3p的表达水平,再利用蛋白质免疫印迹法检测HOXA5的表达水平的变化来探索miR-128-3p与HOXA5在GBM中表达的相关性。利用双荧光素报告基因实验验证miR-128-3p对HOXA5的靶向抑制关系。利用miR-128-3p与HOXA5过表达的质粒转染U87细胞进行拯救实验,通过CCK-8、Transwell、流式细胞学分析与裸鼠体内实验验证miR-128-3p调控GBM增殖、侵袭及凋亡方面的分子机制。结果 上调U87细胞中miR-128-3p表达后HOXA5的表达水平显著下降,下调U87细胞中miR-128-3p表达后HOXA5表达水平明显升高（P<0.05）,两者表达呈显著负相关。miR-128-3p可靶向结合HOXA5基因的3’UTR区并抑制HOXA5表达。miR-128-3p+Control组U87细胞增殖、侵袭及抗凋亡能力显著下降。结论 miR-128-3p可通过靶向抑制HOXA5负向调控GBM细胞的增殖、侵袭及抗凋亡能力,HOXA5在GBM中呈高表达与miR-128-3p表达水平降低有关。     

Mature miR-184 as Potential Oncogenic microRNA of Squamous Cell Carcinoma of Tongue.

PURPOSE: The aim of this study was to evaluate the microRNA expression patterns in squamous cell carcinoma (SCC) of the tongue. EXPERIMENTAL DESIGN: Expression levels of 156 human mature microRNAs were examined using real-time quantitative PCR (Taq Man MicroRNA Assays; Human Panel) on laser microdissected cells of 4 tongue carcinomas and paired normal tissues. Expression of mature miR-184 was further validated in 20 paired tongue SCC and the normal tissues. Potential oncogenic functions of miR-184 were evaluated in tongue SCC cell lines (Cal27, HN21B, and HN96) with miR-184 inhibitor. Plasma miR-184 levels were evaluated using real-time quantitative PCR. RESULTS: Using 3-fold expression difference as a cutoff level, we identified 24 up-regulated mature miRNAs including miR-184, miR-34c, miR-137, miR-372, miR-124a, miR-21, miR-124b, miR-31, miR-128a, miR-34b, miR-154, miR-197, miR-132, miR-147, miR-325, miR-181c, miR-198, miR-155, miR-30a-3p, miR-338, miR-17-5p, miR-104, miR-134, and miR-213; and 13 down-regulated mature miRNAs including miR-133a, miR-99a, miR-194, miR-133b, miR-219, miR-100, miR-125b, miR-26b, miR-138, miR-149, miR-195, miR-107, and miR-139. Overexpression of miR-184 was further validated in 20 paired tongue SCC and normal tissues (P = 0.002). Inhibition of miR-184 in tongue SCC cell lines could reduce cell proliferation rate. Down-regulation of c-Myc was observed in two cell lines in response to miR-184 inhibitor. Suppressing miR-184 could induce apoptosis in all three cell lines. Plasma miR-184 levels were significantly higher in tongue SCC patients in comparison with normal individuals, and the levels were significantly reduced after surgical removal of the primary tumors. CONCLUSIONS: Overexpression of miR-184 might play an oncogenic role in the antiapoptotic and proliferative processes of tongue SCC. In addition, plasma miR-184 levels were associated with the presence of primary tumor. Further studies on the aberrantly expressed miRNAs in tongue SCC as well as using plasma miRNAs as novel tumor markers are warranted.     

miR-331-3p regulates expression of neuropilin-2 in glioblastoma

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of high-grade gliomas. However, the precise mechanistic role of many miRNAs in this disease remains unclear. Here, we investigate the functional role of miR-331-3p in glioblastoma multiforme (GBM). We found that miR-331-3p expression in GBM cell lines is significantly lower than in normal brain, and that transient overexpression of miR-331-3p inhibits GBM cell line proliferation and clonogenic growth, suggesting a possible tumor suppressor role for miR-331-3p in this system. Bioinformatics analysis identified neuropilin-2 (NRP-2) as a putative target of miR-331-3p. Using transfection studies, we validated NRP-2 mRNA as a target of miR-331-3p in GBM cell lines, and show that NRP-2 expression is regulated by miR-331-3p. RNA interference (RNAi) to inhibit NRP-2 expression in vitro decreased the growth and clonogenic growth of GBM cell lines, providing further support for an oncogenic role for NRP-2 in high-grade gliomas. We also show that miR-331-3p inhibits GBM cell migration, an effect due in part to reduced NRP-2 expression. Finally, we identified a significant inverse correlation between miR-331-3p and NRP-2 expression in The Cancer Genome Atlas GBM cohort of 491 patients. Together, our results suggest that a loss of miR-331-3p expression contributes to GBM development and progression, at least in part via upregulating NRP-2 expression and increasing cell proliferation and clonogenic growth.     

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-195, miR-455-3p and miR-10a are implicated in acquired temozolomide resistance in glioblastoma multiforme cells

To identify microRNAs (miRNAs) specifically involved in the acquisition of temozolomide (TMZ) resistance in glioblastoma multiforme (GBM), we first established a resistant variant, U251R cells from TMZ-sensitive GBM cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251R and parental cells using miRNA microarrays. miR-195, miR-455-3p and miR-10a^∗ were the three most up-regulated miRNAs in the resistant cells. To investigate the functional role of these miRNAs in TMZ resistance, U251R cells were transfected with miRNA inhibitors consisting of DNA/LNA hybrid oligonucleotides. Suppression of miR-455-3p or miR-10a^∗ had no effect on cell growth, but showed modest cell killing effect in the presence of TMZ. On the other hand, knockdown of miR-195 alone displayed moderate cell killing effect, and combination with TMZ strongly enhanced the effect. In addition, using in silico analysis combined with cDNA microarray experiment, we present possible mRNA targets of these miRNAs. In conclusion, our findings suggest that those miRNAs may play a role in acquired TMZ resistance and could be a novel target for recurrent GBM treatment.     

MiR-219-5p对人脑成胶质细胞瘤恶性表型的影响

目的:探讨miR-219-5p对人脑成胶质细胞瘤(glioblastoma,GBM)的恶性表型——增殖、凋亡和侵袭的影响,初步寻找与miR-219-5p发挥抑癌作用有关的靶点基因.方法:在60例GBM组织中分析miR-219-5p表达水平及mRNA表达谱,筛选与miR-219-5p表达量呈负相关的mRNA;选取前50个相关性最大的mRNA,用基因功能分析软件DAVID从中筛选出与GBM恶性进展相关的2组mRNA;最后用4个在线靶点预测网站从这2组中预测miR-219-5p的潜在靶点.采用MTT法、流式细胞术和Transwell实验初步验证miR-219-5p对GBM细胞增殖、凋亡和侵袭的影响.结果:60例GBM组织中筛出14个与增殖和凋亡相关的基因和5个与侵袭相关的基因(P＜0.001);在这19个候选基因中预测到4个基因(TWIST1、MYO1B、WEE1和SPRED2)是miR-219-5p的潜在靶点.实验初步证明,miR-219-5p能够抑制GBM细胞的增殖和侵袭以及促进细胞凋亡(P＜0.05).结论:MiR-219-5p可能通过作用于多个靶点对GBM的恶性表型起到抑制作用.     

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。      

miR-124调控SOS1表达及对U87胶质瘤细胞增殖的影响

目的:探讨miR-124对胶质瘤细胞增殖的抑制作用及作用靶点.方法:应用生物信息整合分析,SOS1是miR-124负向调节胶质瘤细胞的靶点.应用细胞转染和荧光素酶检测分析证实miR-124对SOS1的作用关系.结果:SOS1是miR-124作用靶点,miR-124直接作用于SOS1 mRNA 3'端非编码区(UTR),但不作用于突变型的3(UTR).miR-124通过作用于靶点SOS1抑制了体外胶质瘤细胞的增殖.结论:SOS1在恶性胶质瘤细胞中呈正向调控,miR-124的含量上升可导致SOS1含量下降,miR-124通过调节SOS1/Raf/ERK信号通路在抑制细胞生长中起重要作用.     

MiR-124-5p inhibits the growth of high-grade gliomas through posttranscriptional regulation of LAMB1

Background

Glioblastoma multiforme (GBM) is the most aggressive form of human brain tumor. It was previously shown that high levels of laminin-8 expression were a predictor of tumor recurrence and patient survival. It is thus important to elucidate the mechanism by which laminin-8 expression is regulated and determine how this contributes to glioma progression. This study investigated the mechanism of regulation of LAMB1, which encodes the β1 chain of laminin-8, in glioma cells lines and in a mouse model of GBM.

Methods

The expression levels of LAMB1 and miR-124-5p were examined in glioma cell lines (U87 and U251) and GBM tissue samples by quantitative PCR and Western blotting. The potential regulation of LAMB1 by miR-124-5p was investigated by assessing the effects of restored miR-124-5p expression on cell proliferation, colony formation, and tumor growth and angiogenesis. The effects of inhibiting LAMB1 on tumor growth and angiogenesis were also assessed.

Results

The upregulation of LAMB1 expression was highly correlated with the downregulation of miR-124-5p. LAMB1 protein expression was suppressed by miR-124-5p. The restoration of miR-124-5p expression suppressed glioma growth by inhibiting angiogenesis, effects that were also observed upon LAMB1 knockdown.

Conclusions

The findings indicate that miR-124-5p functions as a tumor suppressor and could serve as a molecular marker for glioma diagnosis and as a potential therapeutic target in GBM treatment.     

MiR-218 reverses high invasiveness of glioblastoma cells by targeting the oncogenic transcription factor LEF1

The invasive behavior of glioblastoma multiforme (GBM) cells is one of the most important reasons for the poor prognosis of this cancer. For invasion, tumor cells must acquire an ability to digest the extracellular matrix and infiltrate the normal tissue bordering the tumor. Preventing this by altering effector molecules can significantly improve a patient's prognosis. Accumulating evidence suggests that miRNAs are involved in multiple biological functions, including cell invasion, by altering the expression of multiple target genes. The expression levels of miR-218 correlate with the invasive potential of GBM cells. In this study, we found that miR-218 expression was low in glioma tissues, especially in GBM. The data showed an inverse correlation in 60 GBM tissues between the levels of miR-218 and MMP mRNAs (MMP-2, -7 and -9). Additionally, ectopic expression of miR-218 suppressed the invasion of GBM cells whereas inhibition of miR-218 expression enhanced the invasive ability. Numerous members of the MMP family are downstream effectors of the Wnt/LEF1 pathway. Target prediction databases and luciferase data showed that LEF1 is a new direct target of miR-218. Importantly, western blot assays demonstrated that miR-218 can reduce protein levels of LEF1 and MMP-9. We, therefore, hypothesize that miR-218 directly targets LEF1, resulting in reduced synthesis of MMP-9. Results suggest that miR-218 is involved in the invasive behavior of GBM cells and by targeting LEF1 and blocking the invasive axis, miR-218-LEF1-MMPs, it may be useful for developing potential clinical strategies.     

Co-inhibition of microRNA-10b and microRNA-21 exerts synergistic inhibition on the proliferation and invasion of human glioma cells

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative gene regulators. Alterations in the expression of miRNAs have been implicated in the pathogenesis and development of most human malignancies. Recent data indicate that microRNA-21 and microRNA-10b are significantly elevated in glioblastoma multiforme (GBM) suggesting their role in the regulation of multiple genes associated with cancer. In this study, U87MG human glioblastoma cells were treated with miRNA inhibitors targeting miR-10b and miR-21, alone or in combination. The results showed that the miR-21 inhibitor additively interacted with miR-10b inhibitor on U87MG cells. The 50% inhibitory concentration values were dramatically decreased in cells treated with the combination of miR-10b and miR-21 inhibitors. Furthermore, inhibitors synergistically combined, enhanced apoptosis significantly and reduced invasion ability assessed by flow cytometry and Transwell migration assay. Thus, the miR-21 inhibitor may interrupt the activity of EGFR pathways, increasing PDCD4 and TPM1 expression and reducing MMP activities, independently of PTEN status. Meanwhile, miR-10b inhibitor reduced by Twist proceeds to inhibit translation of the mRNA encoding HOXD10 leading to the increase of the expression of the well-characterized pro-metastatic gene RHOC. Taken together, these data strongly suggest that a combination of miR-21 inhibitor and miR-10b inhibitor could be an effective therapeutic strategy for controlling the growth of GBM by inhibiting oncogene expression and overexpressing tumor suppressor genes. Moreover, a regulatory strategy based on the combination of miRNA inhibitors may provide insights into the mechanisms of the modulation of signaling genes involved in tumor cell apoptosis and invasiveness.     

MicroRNA-34a targets notch1 and inhibits cell proliferation in glioblastoma multiforme

Aberrant expression of microRNAs (miRNAs) has been implicated in cancer initiation and progression. In this study, we found that microRNA-34a (miR-34a) is significantly downregulated in glioblastoma multiforme (GBM) specimens compared with normal brain tissues. Growth curve and colony formation assays revealed that miR-34a suppresses proliferation of U373MG and SHG44 glioblastoma cells. Overexpression of miR-34a could induce apoptosis of glioblastoma cells. Also, we identified notch1 as a direct target gene of miR-34a. Knockdown of notch1 showed similar cellular functions as overexpression of miR-34a both in vitro and in vivo. Collectively, our findings show that miR-34a is downregulated in GBM cells and inhibits GBM growth by targeting notch1.