MiR-221/-222 differentiate prognostic groups in advanced breast cancers and influence cell invasion

Background:

MiR-221/-222 are frequently overexpressed in breast cancer and are associated with increased malignancy. The specific modification of microRNAs (miRNAs) expression could be a promising strategy in breast cancer therapy, leading to the suppression of tumourigenic processes in tumour cells.

Methods:

MiR-221/-222 expressions were analysed in 86 breast cancer tissues by quantitative RT–PCR and tested for correlation with immunohistochemistry data and clinical follow-up. In vitro assays were conducted using human breast cancer cell lines with lentiviral overexpression of miR-221/-222.

Results:

In tumour tissues, miR-221/-222 were associated with the occurrence of distant metastases. In particular, high levels of miR-221 were revealed to have a high prognostic impact for the identification of significantly different groups with advanced tumours. MiR-221/-222 overexpression strongly increased cell proliferation and invasion in vitro. Following miR-221/-222 overexpression an increased uPAR expression and cell invasion were observed.

Conclusion:

This study demonstrates a significant role for highly expressed miR-221/-222 in advanced breast cancers allowing for the identification of significantly different prognostic groups, particularly for HER2-positive and lymph-node-positive breast cancers. Considering that miR-221/-222 are strongly involved in cell invasion, these miRNAs may be promising markers for breast cancer prognosis and therapy.     

Deregulated microRNAs in gastric cancer tissue-derived mesenchymal stem cells: novel biomarkers and a mechanism for gastric cancer

Background:

MicroRNAs (miRNAs) are involved in gastric cancer development and progression. However, the expression and role of miRNAs in gastric cancer stromal cells are still unclear.

Methods:

The miRNAs differentially expressed in gastric cancer tissue-derived mesenchymal stem cells (GC-MSCs) relative to adjacent non-cancerous tissue-derived MSCs (GCN-MSCs) and in cancer tissues relative to adjacent non-cancerous tissues were screened using miRNA microarray and validated by quantitative RT–PCR. The impact of GC-MSCs on HGC-27 cells was observed in vitro using colony formation and transwell assays, and these cells were subcutaneously co-injected into mice to assess tumour growth in vivo. Exogenous downregulation of miR-221 expression in cells was achieved using an miRNA inhibitor.

Results:

miR-214, miR-221 and miR-222 were found to be commonly upregulated in GC-MSCs and cancer tissues. Their levels were tightly associated with lymph node metastasis, venous invasion and the TNM stage. Gastric cancer tissue-derived mesenchymal stem cells significantly promoted HGC-27 growth and migration and increased the expression of miR-221 via paracrine secretion, and the targeted inhibition of miR-221 in GC-MSCs could block its tumour-supporting role. GC-MSC-derived exosomes were found to deliver miR-221 to HGC-27 cells and promoted their proliferation and migration.

Conclusions:

Gastric cancer tissue-derived mesenchymal stem cells favour gastric cancer progression by transferring exosomal miRNAs to gastric cancer cells, thus providing a novel mechanism for the role of GC-MSCs and new biomarkers for gastric cancer.     

MicroRNA expression signature of castration-resistant prostate cancer: the microRNA-221/222 cluster functions as a tumour suppressor and disease progression marker

Background:

Our present study of the microRNA (miRNA) expression signature in castration-resistant prostate cancer (CRPC) revealed that the clustered miRNAs microRNA-221 (miR-221) and microRNA-222 (miR-222) are significantly downregulated in cancer tissues. The aim of this study was to investigate the functional roles of miR-221 and miR-222 in prostate cancer (PCa) cells.

Methods:

A CRPC miRNA signature was constructed by PCR-based array methods. Functional studies of differentially expressed miRNAs were analysed using PCa cells. The association between miRNA expression and overall survival was estimated by the Kaplan–Meier method. In silico database and genome-wide gene expression analyses were performed to identify molecular targets regulated by the miR-221/222 cluster.

Results:

miR-221 and miR-222 were significantly downregulated in PCa and CRPC specimens. Kaplan–Meier survival curves showed that low expression of miR-222 predicted a short duration of progression to CRPC. Restoration of miR-221 or miR-222 in cancer cells revealed that both miRNAs significantly inhibited cancer cell migration and invasion. Ecm29 was directly regulated by the miR-221/222 cluster in PCa cells.

Conclusions:

Loss of the tumour-suppressive miR-221/222 cluster enhanced migration and invasion in PCa cells. Our data describing targets regulated by the tumour-suppressive miR-221/222 cluster provide insights into the mechanisms of PCa and CRPC progression.     

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.     

PUMA is a novel target of miR-221/222 in human epithelial cancers

miR-221 and miR-222 (miR-221/222) are frequently up-regulated in human epithelial cancers. However, the mechanism of miR-221/222 action involved in carcinogenesis has not been extensively studied. Here, we found that reduction of miR-221/222 inhibited cell proliferation and induced mitochondrial-mediated apoptosis in human epithelial cancer cells (A549 lung cancer and MCF-7 breast cancer cells). Bioinformatics and luciferase reporter assays showed that miR-221/222 co-modulated the p53 upregulated modulator of apoptosis (PUMA) expression by directly targeting the binding site within the 3'UTR. Together, these findings suggest that PUMA is a direct target of miR-221/222 that functions as an endogenous apoptosis regulator in these epithelial cancers.     

反义microRNA-221与反义microRNA-222抑制人脑胶质瘤细胞的体外与体内生长

目的 探讨敲低microRNA(miR)-221和miR-222表达以抑制人脑胶质瘤U251细胞生长的作用及其机制.方法 脂质体介导转染反义寡聚核苷酸(AS-miR-221和AS-miR-222)于人脑胶质瘤细胞U251.采用Northern blot鉴定转染后U251细胞的miR-221和miR-222表达水平;四甲基偶氮唑蓝(MTT)法评价AS-miR-221和AS-miR-222抑制U251细胞生长的作用;Transwell实验检测细胞侵袭能力;流式细胞术检测细胞周期的分布和凋亡;Western blot检测转染后U251细胞相关蛋白表达的变化,并用AS-miR-221和AS-miR-222治疗裸鼠皮下移植瘤,观察其在活体内对肿瘤生长的抑制作用.结果 Northern blot检测结果 显示,AS-miR-221和AS-miR-222共转染后,肿瘤细胞miR-221和miR-222表达明显下降,细胞生长速度降低,细胞穿过率为14.5%,细胞周期出现G0/G1期阻滞,凋亡率(13.7%)增高,并可见connexin43、p27、PUMA、caspase-3、PTEN、TIMP3和Bax等相关蛋白表达增高,而bcl-2表达降低,p53无明显变化.经AS-miR-221和AS-miR-222治疗后,裸鼠皮下移植瘤生长明显受抑.结论 AS-miR-221和AS-miR-222共转染可抑制U251细胞的增殖与侵袭,miR-221和miR-222可以作为人脑胶质瘤基因治疗的侯选靶点.     

miR-221和miR-222在胃癌中的表达及临床意义

[目的]探讨miR-221和miR-222在胃癌中的表达及与临床病理特征的关系。[方法]应用茎环RT-PCR方法检测32例胃癌及癌旁胃黏膜组织中miR-221和miR-222表达,分析miR-221和miR-222表达与胃癌临床病理指标的关系。[结果]胃癌组织miR-221和miR-222表达明显高于对应的正常胃黏膜组织（P〈0.05）,且胃癌与正常胃黏膜组织中miR-221和miR-222比值也有显著性差异（P=0.05）。miR-222表达与胃癌分化程度相关（P=0.046）;miR-221表达与淋巴结转移相关（P=0.013）。[结论]miR-221和miR-222在胃癌中表达明显上调是胃癌的重要标志,但两者在胃癌发生发展的不同阶段,可能发挥不同的效应。     

Secreted uPAR isoform 2 (uPAR7b) is a novel direct target of miR-221

miR-221/-222 and components of the urokinase-type plasminogen activator system (uPAS) are associated with metastasis and poor prognosis in breast cancer, including the triple-negative subtype (TNBC). Modification of components of uPAS and involved miRNAs may contribute to targeted therapy for breast cancer patients. miR-221−/−222-overexpressing or miR-221-depleted cells were employed for qRT-PCR and Western blots to show associations of uPAR with miR-221/-222. To substantiate direct targeting of miR-221/-222 within 3′ UTR of the uPAR isoform 2, in silico analysesand in vitro assays were conducted. Significant associations between miR-221 and uPAR isoform 2 expressions were observed at the mRNA and protein levels in breast cancer cells representing TNBC. For the first time, the uPAR isoform 2 was demonstrated as direct target for miR-221/-222. Inhibition of miR-221 reduced uPAR protein expression and expression of the tumor cell invasion markers vimentin and RHOC. These results demonstrate a direct and positive regulation of the secreted uPAR isoform 2 by miR-221, increasing its protein expression, a prerequisite for malignancy, while the other uPAR isoforms (1, 3 and 4) are indirectly regulated through miR-10b and miR-221/-222. By targeting uPAR isoforms and/or miRNA-221/-222, the diagnosis and therapy of breast cancer, in particular in TNBC, could be significantly improved.     

In Vitro and in Vivo Anti-tumor Activity of miR-221/222 Inhibitors in Multiple Myeloma

A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.     

Downregulation of miR-221/222 sensitizes glioma cells to temozolomide by regulating apoptosis independently of p53 status

A previous study showed that miR-221/222 can regulate cell apoptosis. p53 is a well known tumor suppressor which can influence the chemosensitivity of glioma cells. However, the effect of miR-221/222 in gliomas with different p53 status is unknown. Here, we demostrate that knockdown of miR-221/222 increases apoptosis in human gliomas of different p53 types (U251 cells, p53 mutant-type; LN308 cells, p53 null-type; and U87 cells, p53 wild-type). Furthermore, the effect of miR-221/22 caused no change of p53 expression in the glioma cells studied. In addition, when a specific siRNA against p53 was employed in U87 cells, no attenuation of apoptosis was found after knockdown of miR-221/222. Importantly, we found that As-miR-221/222-treated cells increased expression of Bax, cytochrome c, Apaf-1 and cleaved-caspase-3. Our results showed that low expression of miR-221/222 sensitized glioma cells to temozolomide (TMZ); in addition, ectopic expression of PUMA by pcDNA-PUMA had a similar effect. Taken together, our study indicates that downregulated miR-221/222 can sensitize glioma cells to TMZ by regulating apoptosis independently of p53 status.     

miR-130a targets MET and induces TRAIL-sensitivity in NSCLC by downregulating miR-221 and 222

Non-small cell lung cancer (NSCLC) accounts for ～80% of all lung cancers. Although some advances in lung cancer therapy have been made, patient survival is still quite poor. Two microRNAs, miR-221 and miR-222, upregulated by the MET proto-oncogene, have been already described to enhance cell survival and to induce TNF-related apoptosis-inducing ligand (TRAIL) resistance in NSCLC cell lines, through the downregulation of p27(kip1), PTEN and TIMP3. Here, we further investigated this pathway and showed that miR-130a, expressed at low level in lung cancer cell lines, by targeting MET was able to reduce TRAIL resistance in NSCLC cells through the c-Jun-mediated downregulation of miR-221 and miR-222. Moreover, we found that miR-130a reduced migratory capacity of NSCLC. A better understanding of MET-miR-221 and 222 axis regulation in drug resistance is the key in developing new strategies in NSCLC therapy.     

MicroRNA-21 inhibitor sensitizes human glioblastoma cells U251 (PTEN-mutant) and LN229 (PTEN-wild type) to taxol

Background  

Substantial data indicate that the oncogene microRNA 21 (miR-21) is significantly elevated in glioblastoma multiforme (GBM) and regulates multiple genes associated with cancer cell proliferation, apoptosis, and invasiveness. Thus, miR-21 can theoretically become a target to enhance the chemotherapeutic effect in cancer therapy. So far, the effect of downregulating miR-21 to enhance the chemotherapeutic effect to taxol has not been studied in human GBM.     

miR-221/222 is the regulator of Cx43 expression in human glioblastoma cells

The miR-221/222 cluster is significantly upregulated in malignant glioma cells and regulates the expression of multiple genes associated with glioma cell proliferation, invasion and apoptosis, which was shown in our previous studies. Cx43 has been identified as a tumor suppressor and major component for the establishment of gap junction intercellular communication (GJIC) in glial cells, which is frequently reduced or deleted in high-grade gliomas. According to bioinformatic analysis, connexin 43 (Cx43) may be one of the target genes of miR-221/222. The aim of the present study was to validate Cx43 as a target gene of miR-221/222 and to determine whether overexpression of miR-221/222 is one of the molecular mechanisms for the reduced expression of Cx43 in malignant gliomas. We transfected miR-221/222 antisense oligonucleotides (AS-miR-221/222) into U251 human glioblastoma cells using a lipofectamine method. Northern blot analysis was conducted to detect the expression of the miR-221/222 cluster. Luciferase reporter assays were exploited to confirm Cx43 as a target gene of miR-221/222. Cx43 expression was assessed by western blotting and immunofluorescence staining. Scrape loading and dye transfer (SLDT) assays were used for examination of GJIC. Proliferation and invasion of U251 cells were evaluated by MTT and transwell assays, respectively. Cell cycle kinetics and apoptosis were determined with flow cytometry. We found that expression of the miR-221/222 cluster was significantly reduced while Cx43 expression was upregulated in U251 cells transfected with AS-miR-221/222, and the GJIC deficiency in parental U251 cells was re-established. Moreover, the luciferase activity determined by the luciferase reporter assay was enhanced in AS-miR-221/222-treated cells, and cell proliferation and invasion were suppressed while apoptosis was induced. We conclude that miR-221/222 function as oncogenic microRNAs in human gliomas, at least in part, by targeting Cx43.     

MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer

To define novel pathways that regulate susceptibility to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in non-small cell lung cancer (NSCLC), we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant NSCLC cells, levels of different miRs are increased, and in particular, miR-221 and -222. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. Indeed, transfection with anti-miR-221 and -222 rendered CALU-1-resistant cells sensitive to TRAIL. Conversely, H460-sensitive cells treated with -221 and -222 pre-miRs become resistant to TRAIL. miR-221 and -222 target the 3'-UTR of Kit and p27(kip1) mRNAs, but interfere with TRAIL signaling mainly through p27(kip1). In conclusion, we show that high expression levels of miR-221 and -222 are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets or diagnostic tool for TRAIL resistance in NSCLC.     

Exosomal miR-221/222 enhances tamoxifen resistance in recipient ER-positive breast cancer cells

Recent studies have demonstrated that specific miRNAs, such as miR-221/222, may be responsible for tamoxifen resistance in breast cancer. Secreted miRNAs enclosed in exosomes can act as intercellular bio-messengers. Our objective is to investigate the role of secreted miR-221/222 in tamoxifen resistance of ER-positive breast cancer cells. Transmission electron microscopy analysis and nanoparticle tracking analysis were performed to determine the exosomes difference between MCF-7^TamR (tamoxifen resistant) and MCF-7^wt (tamoxifen sensitive) cells. PKH67 fluorescent labeling assay was used to detect exosomes derived from MCF-7^TamR cells entering into MCF-7^wt cells. The potential function of exosomes on tamoxifen resistance transmission was analyzed with cell viability, apoptosis ,and colony formation. MiRNA microarrays and qPCR were used to detect and compare the miRNAs expression levels in the two cells and exosomes. As the targets of miR-221/222, p27 and ERα were analyzed with western blot and qPCR. Compared with the MCF-7^wt exosomes, there were significant differences in the concentration and size distribution of MCF-7^TamR exosomes. MCF-7^wt cells had an increased amount of exosomal RNA and proteins compared with MCF-7^TamR cells. MCF-7^TamR exosomes could enter into MCF-7^wt cells, and then released miR-221/222. And the elevated miR-221/222 effectively reduced the target genes expression of P27 and ERα, which enhanced tamoxifen resistance in recipient cells. Our results are the first to show that secreted miR-221/222 serves as signaling molecules to mediate communication of tamoxifen resistance.