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Extracellular microRNAs (miRNAs) play a critical role in horizontal gene regulation. Uptake of extracellular miRNAs by recipient cells and their intracellular transport, however, remains elusive. Here RNA phase separation is shown as a novel pathway of miRNA uptake. In the presence of serum, synthetic miRNAs rapidly self-assembly into ≈110 nm discrete nanoparticles, which enable miRNAs' entry into different cells. Depleting serum cationic proteins prevents the formation of such nanoparticles and thus blocks miRNA uptake. Different from lipofectamine-mediated miRNA transfection in which majority of miRNAs are accumulated in lysosomes of transfected cells, nanoparticles-mediated miRNA uptake predominantly delivers miRNAs into mitochondria in a polyribonucleotide nucleotidyltransferase 1(PNPT1)-dependent manner. Functional assays further show that the internalized miR-21 via miRNA phase separation enhances mitochondrial translation of cytochrome b (CYB), leading to increase in adenosine triphosphate (ATP) and reactive oxygen species (ROS) reduction in HEK293T cells. The findings thus reveal a previously unrecognized mechanism for uptake and delivery functional extracellular miRNAs into mitochondria., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Patients with hormone receptor (HR)-positive tumors breast cancer usually experience a relatively low pathological complete response (pCR) to neoadjuvant chemotherapy (NAC). Here, we derived a 10-microRNA risk score (10-miRNA RS)-based model with better performance in the prediction of pCR and validated its relation with the disease-free survival (DFS) in 755 HR-positive breast cancer patients (273, 265, and 217 in the training, internal, and external validation sets, respectively). This model, presented as a nomogram, included four parameters: the 10-miRNA RS found in our previous study, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, and volume transfer constant (K trans ). Favorable calibration and discrimination of 10-miRNA RS-based model with areas under the curve (AUC) of 0.865, 0.811, and 0.804 were shown in the training, internal, and external validation sets, respectively. Patients who have higher nomogram score (>92.2) with NAC treatment would have longer DFS (hazard ratio=0.57; 95%CI: 0.39-0.83; P=0.004). In summary, our data showed the 10-miRNA RS-based model could precisely identify more patients who can attain pCR to NAC, which may help clinicians formulate the personalized initial treatment strategy and consequently achieves better clinical prognosis for patients with HR-positive breast cancer., (© 2022. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Development of rapid and accurate detection of miRNAs in complex samples is of great significance for potential early diagnosis of disease. Herein, we report a magnetic relaxation switching (MRS)-based strategy for direct detection of miRNAs in complex samples via the assistance of signal amplification system of CRISPR/Cas13a which has the ability to specifically recognize target RNA. In the designed strategy, 30 nm-magnetic nanoparticles (MB 30 ) and 1000 nm-magnetic particles (MM 1000 ) linked by single-strand RNA 1 complexes (MB 30 -RNA 1 - MM 1000 ) were employed as signal probe. After the target miRNAs (taking miR-21 as model) recognition by CRISPR/Cas13a system, the resulted trans-cleavage degrades the MB 30 -RNA 1 -MM 1000 , releasing MB 30 which caused transverse relaxation time (T 2 ) signal change. The combination of CRISPR/Cas13a assisted signal amplification and the MRS assay achieved direct detection of miR-21 in the serum sample without extracting within 60min, with a detection limit of 0.22 pM. Moreover, the detection accuracy is confirmed by performing the detection of miR-21 using qRT-PCR. The CRISPR/Cas13a system assisted MRS assay successfully achieved accurate, simple, and rapid detection of miRNAs in complex samples, showing great potential for detection miRNAs in potential clinical applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Although both circular RNAs (circRNAs) and autophagy are associated with the function of breast cancer (BC), whether circRNAs regulate BC progression via autophagy remains unknown. In this study, we aim to explore the regulatory mechanisms and the clinical significance of autophagy-associated circRNAs in BC., Methods: Autophagy associated circRNAs were screened by circRNAs deep sequencing and validated by qRT-PCR in BC tissues with high- and low- autophagic level. The biological function of autophagy associated circRNAs were assessed by plate colony formation, cell viability, transwells, flow cytometry and orthotopic animal models. For mechanistic study, RNA immunoprecipitation, circRNAs pull-down, Dual luciferase report assay, Western Blot, Immunofluorescence and Immunohistochemical staining were performed., Results: An autophagy associated circRNA circCDYL was elevated by 3.2 folds in BC tissues as compared with the adjacent non-cancerous tissues, and circCDYL promoted autophagic level in BC cells via the miR-1275-ATG7/ULK1 axis; Moreover, circCDYL enhanced the malignant progression of BC cells in vitro and in vivo. Clinically, increased circCDYL in the tumor tissues and serum of BC patients was associated with higher tumor burden, shorter survival and poorer clinical response to therapy., Conclusions: circCDYL promotes BC progression via the miR-1275-ATG7/ULK1-autophagic axis and circCDYL could act as a potential prognostic and predictive molecule for breast cancer patients.

Glioblastoma (GBM) is the most common and deadly form of malignant brain tumor in the United States, and current therapies fail to provide significant improvement in survival. Local delivery of nanoparticles is a promising therapeutic strategy that bypasses the blood-brain barrier, minimizes systemic toxicity, and enhances intracranial drug distribution and retention. Here, we developed nanoparticles loaded with agents that inhibit miR-21, an oncogenic microRNA (miRNA) that is strongly overexpressed in GBM compared to normal brain tissue. We synthesized, engineered, and characterized two different delivery systems. One was designed around an anti-miR-21 composed of RNA and employed a cationic poly(amine-co-ester) (PACE). The other was designed around an anti-miR-21 composed of peptide nucleic acid (PNA) and employed a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG). We show that both nanoparticle products facilitate efficient intracellular delivery and miR-21 suppression that leads to PTEN upregulation and apoptosis of human GBM cells. Further, when administered by convection-enhanced delivery (CED) to animals with intracranial gliomas, they both induced significant miR-21 knockdown and provided chemosensitization, resulting in improved survival when combined with chemotherapy. The challenges involved in optimizing the two delivery systems differed, and despite offering distinct advantages and limitations, results showed significant therapeutic efficacy with both methods of treatment. This study demonstrates the feasibility and promise of local administration of miR-21 inhibiting nanoparticles as an adjuvant therapy for GBM., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Non-small cell lung cancer (NSCLC) remains a major cause of death worldwide. As metastatic disease is primarily responsible for the poor clinical outcome of NSCLC, it is important to understand the process, and its underlying molecular mechanism as well, via which NSCLC cells disseminate. In this study, we identified a new competing endogenous RNA (ceRNA), namely, the MYEOV transcript, and found that it is upregulated in NSCLC and associated with a poor prognosis of the disease. We further uncovered that the MYEOV ceRNA plays a critical role in the invasion and metastasis of NSCLC cells. Intriguingly, the MYEOV ceRNA exerted its pro-metastatic function independent of its protein-coding capacity, but in a miR-30c-2-3p binding-dependent manner. Further experiments demonstrated that the MYEOV ceRNA sequestered miR-30c-2-3p from binding its targets TGFBR2 and USP15 mRNAs, which in turn leading to constitutive activation of TGF-β signaling and tumor progression in NSCLC. By identifying a new layer of regulatory modality for TGF-β signaling, our findings extend the current understanding on the molecular mechanism mediating NSCLC progression and highlight a potential role of MYEOV transcript to serve as the therapeutic target.

Glaucoma is a progressive optic neuropathy and is one of the leading causes of blindness in the industrialized countries. The involvement of microRNAs (miRs) has been implicated in regulating the complex biological responses to changes in intraocular pressure. However, the therapeutic role of miR-200a on glaucoma has not been well studied yet. In this study, we confirmed the role of miR-200a in glaucoma progression and identified the related mechanism. Microarray expression profiles were used to screen the glaucoma-related genes. The relationship between miR-200a and FGF7 was validated by bioinformatics analysis and dual-luciferase reporter gene assay. Glaucoma-related parameters including the expression of CD11b and iNOS, activation of Muller cells, and apoptosis of retinal ganglion cells (RGCs) in the mouse model were measured by immunohistochemistry, MTT assay and TUNEL assay, respectively. miR-200a was reduced in glaucoma, whereas FGF7 was robustly induced. Thereby, we speculated that FGF7 was negatively regulated by miR-200a. Downregulated miR-200a could activate the MAPK signaling pathway following elevations in ERK, JNK, p38 and Bax expression and reduction in Bcl-2 expression. In the mouse model, downregulated miR-200a increased the expression of CD11b and iNOS and the apoptosis of RGCs, but stimulated the inactivation of Muller cells. However, the above-mentioned alternations induced by downregulated miR-200a were reversed after FGF7 repression. miR-200a can inhibit the FGF7-mediated MAPK signaling pathway and play a protective role on improving the glaucoma-induced optical nerve injury., (Copyright © 2018. Published by Elsevier Inc.)

Increasing evidence suggests circular RNAs (circRNAs) exert critical functions in tumor progression via sponging miRNAs (microRNAs). However, the role of circRNAs in breast cancer remains unclear. Here we systematically analyzed the circular RNAs in breast cancer based on their characteristic in sponging disease-specific miRNAs and identified hsa_circ_001783 as a top ranked circRNA in our computation and verified its high expression in both breast cancer cells and cancer tissue. A higher level of hsa_circ_001783 was significantly correlated with heavier tumor burden and poorer prognosis of patients with breast cancer. Knockdown of this circRNA remarkably inhibited the proliferation and invasion of breast cancer cells. Importantly, hsa_circ_001783 promoted progression of breast cancer cells via sponging miR-200c-3p. Taken together, hsa_circ_001783 may serve as a novel prognostic and therapeutic target for breast cancer.

Trastuzumab is a standard treatment for HER2-positive (HER2 + ) breast cancer, but some patients are refractory to the therapy. MicroRNAs (miRNAs) have been used to predict therapeutic effects for various cancers, but whether miRNAs can serve as biomarkers for HER2 + metastatic breast cancer (MBC) patients remains unclear. Using miRNA microarray, we identify 13 differentially expressed miRNAs in the serum of HER2 + MBC patients with distinct response to trastuzumab, and four miRNAs are selected to construct a signature to predict survival using LASSO model. Further, our data show that miR-940 is mainly released from the tumor cells and miR-451a, miR-16-5p and miR-17-3p are mainly from the immune cells. All these four miRNAs directly target signaling molecules that play crucial roles in regulating trastuzumab resistance. In summary, we develop a serum-based miRNA signature that potentially predicts the therapeutic benefit of trastuzumab for HER2 + MBC patients and warrants future validation in prospective clinical trials.

Cancer chemoresistance and metastasis are tightly associated features. However, whether they share common molecular mechanisms and thus can be targeted with one common strategy remain unclear in non-small cell lung cancer (NSCLC). Here, we report that high levels of microRNA-128-3p (miR-128-3p) is key to concomitant development of chemoresistance and metastasis in residual NSCLC cells having survived repeated chemotherapy and correlates with chemoresistance, aggressiveness and poor prognosis in NSCLC patients. Mechanistically, miR-128-3p induces mesenchymal and stemness-like properties through downregulating multiple inhibitors of Wnt/β-catenin and TGF-β pathways, leading to their overactivation. Importantly, antagonism of miR-128-3p potently reverses metastasis and chemoresistance of highly malignant NSCLC cells, which could be completely reversed by restoring Wnt/β-catenin and TGF-β activities. Notably, correlations among miR-128-3p levels, activated β-catenin and TGF-β signalling, and pro-epithelial-to-mesenchymal transition/pro-metastatic protein levels are validated in NSCLC patient specimens. These findings suggest that miR-128-3p might be a potential target against both metastasis and chemoresistance in NSCLC.

Purpose: Breast cancer patients with high proportion of cancer stem cells (BCSCs) have unfavorable clinical outcomes. MicroRNAs (miRNAs) regulate key features of BCSCs. We hypothesized that a biology-driven model based on BCSC-associated miRNAs could predict prognosis for the most common subtype, hormone receptor (HR)-positive, HER2-negative breast cancer patients., Patients and Methods: After screening candidate miRNAs based on literature review and a pilot study, we built a miRNA-based classifier using LASSO Cox regression method in the training group (n=202) and validated its prognostic accuracy in an internal (n=101) and two external validation groups (n=308)., Results: In this multicenter study, a 10-miRNA classifier incorporating miR-21, miR-30c, miR-181a, miR-181c, miR-125b, miR-7, miR-200a, miR-135b, miR-22 and miR-200c was developed to predict distant relapse free survival (DRFS). With this classifier, HR+HER2- patients were scored and classified into high-risk and low-risk disease recurrence, which was significantly associated with 5-year DRFS of the patients. Moreover, this classifier outperformed traditional clinicopathological risk factors, IHC4 scoring and 21-gene Recurrence Score (RS). The patients with high-risk recurrence determined by this classifier benefit more from chemotherapy., Conclusions: Our 10-miRNA-based classifier provides a reliable prognostic model for disease recurrence in HR+HER2- breast cancer patients. This model may facilitate personalized therapy-decision making for HR+HER2- individuals., (Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.)

The world of noncoding RNAs (ncRNAs) has gained widespread attention in recent years due to their novel and crucial potency of biological regulation. Noncoding RNAs play essential regulatory roles in a broad range of developmental processes and diseases, notably human cancers. Regulatory ncRNAs represent multiple levels of structurally and functionally distinct RNAs, including the best-known microRNAs (miRNAs), the complicated long ncRNAs (lncRNAs), and the newly identified circular RNAs (circRNAs). However, the mechanisms by which they act remain elusive. In this chapter, we will review the current knowledge of the ncRNA field, discussing the genomic context, biological functions, and mechanisms of action of miRNAs, lncRNAs, and circRNAs. We also highlight the implications of the biogenesis and gene expression dysregulation of different ncRNA subtypes in the initiation and development of human malignancies.

About 50-70% of breast cancers are estrogen receptor α (ERα) positive and most of them are sensitive to endocrine therapy including tamoxifen. However, one third of these patients will eventually develop resistance and relapse. We found that the expression of miR-15a and miR-16 were significantly decreased in tamoxifen resistant ER positive breast cancer cell lines. Exogenous expression of miR-15a/16 mimics re-sensitized resistant cells to tamoxifen by inhibiting Cyclin E1 and B cell lymphoma-2 (Bcl-2) to induce cell growth arrest and apoptosis respectively. Further, we identified that a repressive member of E2F family, E2F7, was responsible for the suppression of miR-15a/16 cluster by competing with E2F1 for E2F binding site at the promoter of their host gene DLEU2. Moreover, high expression of E2F7 is correlated with high risk of relapse and poor prognosis in breast cancer patients receiving tamoxifen treatment. Together, our results suggest that overexpression of E2F7 represses miR-15a/16 and then increases Cyclin E1 and Bcl-2 that result in tamoxifen resistance. E2F7 may be a valuable prognostic marker and a therapeutic target of tamoxifen resistance in breast cancer.

Macrophages play a pivotal role in tissue fibrogenesis, which underlies the pathogenesis of many end-stage chronic inflammatory diseases. MicroRNAs are key regulators of immune cell functions, but their roles in macrophage's fibrogenesis have not been characterized. Here we show that IL-4 and IL-13 induce miR-142-5p and downregulate miR-130a-3p in macrophages; these changes sustain the profibrogenic effect of macrophages. In vitro, miR-142-5p mimic prolongs STAT6 phosphorylation by targeting its negative regulator, SOCS1. Blocking miR-130a relieves its inhibition of PPARγ, which coordinates STAT6 signalling. In vivo, inhibiting miR-142-5p and increasing miR-130a-3p expression with locked nucleic acid-modified oligonucleotides inhibits CCL4-induced liver fibrosis and bleomycin-induced lung fibrosis in mice. Furthermore, macrophages from the tissue samples of patients with liver cirrhosis and idiopathic pulmonary fibrosis display increased miR-142-5p and decreased miR-130a-3p expression. Therefore, miR-142-5p and miR-130a-3p regulate macrophage profibrogenic gene expression in chronic inflammation.

Our previous work has indicated that CCL18 secreted by tumor-associated macrophages (TAMs) promotes breast cancer metastasis, which is associated with poor patient prognosis. However, it remains unclear whether microRNAs (miRNAs), which may modulate multiple cellular pathways, are involved in the regulation of CCL18 signaling and the ensuing metastasis of breast cancer. In this study, we demonstrated that CCL18 reduces miR98 and miR27b expression via the N-Ras/ERK/PI3K/NFκB/Lin28b signaling pathway, while down-regulation of these mRNAs feedbacks to increase N-Ras and Lin28b levels. This cascade of events forms a positive feedback loop that sustains the activation of CCL18 signaling. More importantly, reduction in miR98 and miR27b enhances the epithelial-mesenchymal transition (EMT) of breast cancer cells, and thus promotes breast cancer metastasis. These findings suggest that down-regulation of miR98 and miR27b promotes CCL18-mediated invasion and migration of breast cancer cells.

Cisplatin has been widely employed as a cornerstone chemotherapy treatment for a wide spectrum of solid neoplasms; increasing tumor responsiveness to cisplatin has been a topic of interest for the past 30 years. Strong evidence has indicated that mitochondrial fission participates in the regulation of apoptosis in many diseases; however, whether mitochondrial fission regulates cisplatin sensitivity remains poorly understood. Here, we show that MFF mediated mitochondrial fission and apoptosis in tongue squamous cell carcinoma (TSCC) cells after cisplatin treatment and that miR-593-5p was downregulated in this process. miR-593-5p attenuated mitochondrial fission and cisplatin sensitivity by targeting the 3' untranslated region sequence of MFF and inhibiting its translation. In exploring the underlying mechanism of miR-593-5p downregulation, we observed that BRCA1 transactivated miR-593-5p expression and attenuated cisplatin sensitivity in vitro. The BRCA1-miR-593-5p-MFF axis also affected cisplatin sensitivity in vivo. Importantly, in a retrospective analysis of multiple centers, we further found that the BRCA1-miR-593-5p-MFF axis was significantly associated with cisplatin sensitivity and the survival of patients with TSCC. Together, our data reveal a model for mitochondrial fission regulation at the transcriptional and post-transcriptional levels; we also reveal a new pathway for BRCA1 in determining cisplatin sensitivity through the mitochondrial fission program.

Diphenyl difluoroketone (EF24), a curcumin analog, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. However, the efficacy and modes of action of EF24 on melanoma metastasis remain elusive. In this study, we found that at non-cytotoxic concentrations, EF24 suppressed cell motility and epithelial-to-mesenchymal Transition (EMT) of melanoma cell lines, Lu1205 and A375. EF24 also suppressed HMGA2 expression at mRNA and protein levels. miR-33b directly bound to HMGA2 3' untranslated region (3'-UTR) to suppress its expression as measured by dual-luciferase assay. EF24 increased expression of E-cadherin and decreased STAT3 phosphorylation and expression of the mesenchymal markers, vimentin and N-cadherin. miR-33b inhibition or HMGA2 overexpression reverted EF24-mediated suppression of EMT phenotypes. In addition, EF24 modulated the HMGA2-dependent actin stress fiber formation, focal adhesion assembly and FAK, Src and RhoA activation by targeting miR-33b. Thus, the results suggest that EF24 suppresses melanoma metastasis via upregulating miR-33b and concomitantly reducing HMGA2 expression. The observed activities of EF24 support its further evaluation as an anti-metastatic agent in melanoma therapy., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Background: Salivary Adenoid cystic carcinoma (SACC) patients with local invasion and lung metastasis are often resistant to conventional therapy such as operation, chemotherapy and radiotherapy. To explore the underling mechanisms, we studied the roles of miRNA in regulating invasiveness of SACC cells., Methods: MicroRNA profiling was done in SACC cells with microarray. MiRNA mimics or antisense oligonucleotide was transfected and invasiveness of SACC cells was evaluated by adhesion assay and transwell assay. The target gene of miRNA was identified by luciferase reporter assay and "rescue" experiment. Tumor metastasis was evaluated by BALB/c-nu mice xenografts. MiRNA and its target gene expression were identified by in-situ hybridization and immunohistochemistry respectively, in 302 patients from affiliated hospitals of Sun Yat-sen University and in 148 patients from affiliated hospitals of Central South University, and correlated to the clinicopathological status of the patients., Results: MiR-320a was down-regulated in high lung metastatic ACCM and SACC-LM cells compared with the corresponding low metastatic ACC2 and SACC-83 cells, and inhibited adhesion, invasion and migration of SACC cells by targeting integrin beta 3 (ITGB3). In vivo, enforced miR-320a expression suppressed metastasis of SACC xenografts. In the two independent sets, miR-320a was downregulated in primary SACCs with metastasis compared to those without metastasis, and low expression of this miRNA predicts poor patient survival and rapid metastasis. Multivariate analysis showed that miR-320a expression was an independent indicator of lung metastasis., Conclusions: MiR-320a inhibits metastasis in SACCs by targeting ITGB3 and may serve as a therapeutic target and prognostic marker in salivary cancers.

TGF-β has paradoxical effects on cancer cell proliferation, as it suppresses proliferation of normal epithelial and low-invasive cancer cells, but enhances that of high-invasive cancer cells. However, how cancer cells acquire the ability to evade the tumor-suppressing effects of TGF-β, yet still take advantage of its tumor-promoting effects, remains elusive. Here, we identified miR-106b as a molecular switch to determine TGF-β effects on cell proliferation. TGF-β1 enhances the transcription of miR-106b via a promoter independent of its host gene MCM7 by activating c-jun. In high-invasive breast cancer cells, miR-106b is upregulated by TGF-β1 at a much higher level than that in normal or low-invasive cancer cells. Accumulation of miR-106b counterbalances TGF-β growth-inhibiting effects by eliminating activated retinoblastoma (RB) and results in enhanced proliferation. Furthermore, miR-106b mediates TGF-β effects on tumor growth and metastasis in breast cancer xenografts. In addition, miR-106b expression is elevated in higher stage tumors and correlated with tumor progression in breast cancer patients. These findings suggest that high level of miR-106b induced by TGF-β determines the tumor-promoting effects of TGF-β in breast cancer.

Malignant melanoma, the most deadly form of skin cancer, has a high propensity for metastatic spread and is notoriously chemotherapy-resistant. Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials. However, the mechanisms of anti-metastatic effects of cordycepin at cellular levels remain elusive. We analyzed the effect of cordycepin on human melanoma miRNA expression profiles by miRNAarray and found that miR-33b was upregulated in highly-metastatic melanoma cell lines following cordycepin exposure. Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3'-UTR to suppress their expression. The negative correlations between miR-33b levels and HMGA2, Twist1 or ZEB1 expression were detected in 72 patient melanoma tissue samples. By targeting HMGA2 and Twist1, miR-33b attenuated melanoma migration and invasiveness upon cordycepin exposure. miR-33b knockdown or ZEB1 overexpression reverted cordycepin-mediated mesenchymal-epithelial transition (MET), triggering the expression of N-cadherin. In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth. We showed for the first time that targeting miRNA by cordycepin indicates a new mechanism of cordycepin-induced suppression of tumor metastasis and miR-33b/HMGA2/Twist1/ZEB1 axis plays critical roles in regulating melanoma dissemination.

Human breast cancers include cancer stem cell populations as well as nontumorigenic cancer cells. Breast cancer stem cells have self-renewal capability and are resistant to conventional chemotherapy. miRNAs regulate the expression of many target genes; therefore, dysregulation of miRNAs has been associated with the pathogenesis of human diseases, including cancer. However, a role for miRNA dysregulation in stemness and drug resistance has yet to be identified. Members of the miR34 family are reportedly tumor-suppressor miRNAs and are associated with various human cancers. Our results confirm that miR34a expression was downregulated in MCF7/ADR cells compared with MCF7 cells. We hypothesized that this reduction was due to the p53 (TP53) mutation in MCF7/ADR cells. In this study, we found that primary and mature miR34a were suppressed by treatment with p53 RNAi or the dominant-negative p53 mutant in MCF7 cells. Ectopic miR34a expression reduced cancer stem cell properties and increased sensitivity to doxorubicin treatment by directly targeting NOTCH1. Furthermore, tumors from nude mice treated with miR34a were significantly smaller compared with those of mice treated with control lentivirus. Our research suggests that the ectopic expression of miR34a represents a novel therapeutic approach in chemoresistant breast cancer treatment., (©2014 American Association for Cancer Research.)

miRNAs are essential for self-renewal and differentiation of normal and malignant stem cells by regulating the expression of key stem cell regulatory genes. Here, we report evidence implicating the miR100 in self-renewal of cancer stem-like cells (CSC). We found that miR100 expression levels relate to the cellular differentiation state, with lowest expression in cells displaying stem cell markers. Utilizing a tetracycline-inducible lentivirus to elevate expression of miR100 in human cells, we found that increasing miR100 levels decreased the production of breast CSCs. This effect was correlated with an inhibition of cancer cell proliferation in vitro and in mouse tumor xenografts due to attenuated expression of the CSC regulatory genes SMARCA5, SMARCD1, and BMPR2. Furthermore, miR100 induction in breast CSCs immediately upon their orthotopic implantation or intracardiac injection completely blocked tumor growth and metastasis formation. Clinically, we observed a significant association between miR100 expression in breast cancer specimens and patient survival. Our results suggest that miR100 is required to direct CSC self-renewal and differentiation., (©2014 American Association for Cancer Research.)

Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myofibroblast markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and stromal cell-derived factor-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-21 was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-21 expression was primarily localized to α-SMA-positive myofibroblasts. More importantly, α-SMA and miR-21 are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-21 mimics promoted, whereas miR-21 antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-21 to induce myofibroblast differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-21 accelerated tumor growth, induced myofibroblast differentiation, and promoted metastasis. This study suggests an important role of myofibroblast differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-21., (©2014 American Association for Cancer Research.)

Breast cancer stem-like cells (BCSC) have been implicated in tumor growth, metastasis, drug resistance, and relapse but druggable targets in appropriate subsets of this cell population have yet to be identified. Here we identify a fundamental role for the prolyl isomerase Pin1 in driving BCSC expansion, invasiveness, and tumorigenicity, defining it as a key target of miR200c, which is known to be a critical regulator in BCSC. Pin1 overexpression expanded the growth and tumorigenicity of BCSC and triggered epithelial-mesenchymal transition. Conversely, genetic or pharmacological inhibition of Pin1 reduced the abundance and self-renewal activity of BCSC. Moreover, moderate overexpression of miR200c-resistant Pin1 rescued the BCSC defect in miR200c-expressing cells. Genetic deletion of Pin1 also decreased the abundance and repopulating capability of normal mouse mammary stem cells. In human cells, freshly isolated from reduction mammoplasty tissues, Pin1 overexpression endowed BCSC traits to normal breast epithelial cells, expanding both luminal and basal/myoepithelial lineages in these cells. In contrast, Pin1 silencing in primary breast cancer cells freshly isolated from clinical samples inhibited the expansion, self-renewal activity, and tumorigenesis of BCSC in vitro and in vivo. Overall, our work demonstrated that Pin1 is a pivotal regulator acting downstream of miR200c to drive BCSC and breast tumorigenicity, highlighting a new therapeutic target to eradicate BCSC., (©2014 American Association for Cancer Research.)

Recent studies suggested that cancer stem cells (CSCs) are capable of differentiating into endothelial cells and tumor endothelium may be derived from CSCs. But the mechanism remains unclear. We showed that vascular endothelial growth factor (VEGF) induced the expression of endothelial markers in breast cancer stem like cells (BCSLCs). In addition, the VEGF-treated BCSLCs formed capillary structure in matrigel and released vWF upon histamine treatment. The miR-27a expression was significantly increased in VEGF-treated BCSLCs. Antagonizing miR-27a by miR-27a anti-sense oligos (ASOs) in VEGF-treated BCSLCs led to decreased endothelial markers and function, while increasing miR-27a in BCSLCs resulted in enhanced endothelial properties. VEGF enhanced the transcription of miR-27a by increasing RUNX1 binding to miR-27a promoter. Increased miR-27a paralleled the reduced expression of ZBTB10, a known miR-27a target. Both expression of miR-27a and knockdown of ZBTB10 in BCSLCs promoted in vivo angiogenesis and tumor metastasis. Further, we demonstrated that VEGF-treated BCSLCs secreted more endogenous VEGF compared with undifferentiated BCSLCs. Thus, miR-27a promotes angiogenesis by mediating endothelial differentiation of BCSLCs and it may be a new target for anti-angiogenesis cancer therapy.

MicroRNAs are involved in regulating the biology of cancer cells, but their involvement in chemoresistance is not fully understood. We found that miR-663 was up-regulated in our induced multidrug-resistant MDA-MB-231/ADM cell line and that this up-regulation was closely related to chemosensitivity. In the present study, we aimed to clarify the role of miR-663 in regulating the chemoresistance of breast cancer. MicroRNA microarray and quantitative RT-PCR assays were used to identify differentially expressed microRNAs. Cell apoptosis was evaluated by annexin V/propidium iodide staining, TUNEL, and reactive oxygen species generation analysis. The expression of miR-663 and HSPG2 in breast cancer tissues was detected by in situ hybridization and immunohistochemistry. The potential targets of miR-663 were defined by a luciferase reporter assay. Bisulfite sequencing PCR was used to analyze the methylation status. We found that miR-663 was significantly elevated in MDA-MB-231/ADM cells, and the down-regulation of miR-663 sensitized MDA-MB-231/ADM cells to both cyclophosphamide and docetaxel. The overexpression of miR-663 in breast tumor tissues was associated with chemoresistance; in MDA-MB-231 cells, this chemoresistance was accompanied by the down-regulation of HSPG2, which was identified as a target of miR-663. MDA-MB-231/ADM contained fewer methylated CpG sites than its parental cell line, and miR-663 expression in MDA-MB-231 cells was reactivated by 5-aza-29-deoxycytidine treatment, indicating that DNA methylation may play a functional role in the expression of miR-663. Our findings suggest that the overexpression of hypomethylated miR-663 induced chemoresistance in breast cancer cells by down-regulating HSPG2, thus providing a potential target for the development of an microRNA-based approach for breast cancer therapy.

Context: There are no known effective and reliable biomarkers to distinguish benign thyroid nodules from papillary thyroid carcinomas (PTC). Previous studies have indicated that serum microRNA (miRNA) profiles may be diagnostic and/or prognostic markers for numerous other cancers., Objective: We studied circulating miRNA profiles in patients with PTC or benign nodules and healthy controls to identify serum miRNA that may be useful as markers for PTC., Design, Setting, and Participants: Genome-wide serum miRNA expression profiles were determined using Solexa sequencing followed by extensive quantitative RT-PCR validation in 245 subjects (106 patients with PTC, 95 patients with benign nodules, and 44 healthy controls). A panel of miRNA was used to assess the expression of specific miRNA in the sera and thyroid tissues of patients with PTC or benign nodules., Results: The expression of serum let-7e, miR-151-5p, and miR-222 was significantly increased in PTC cases relative to benign cases and healthy controls. Receiver operating characteristic curve analyses indicated that use of these three miRNA had a high diagnostic sensitivity and specificity for PTC. Serum let-7e, miR-151-5p, and miR-222 levels were found to be well correlated with certain clinicopathological variables, such as nodal status, tumor size, multifocal lesion status, and Tumor-Node-Metastasis stage. Expression of serum miR-151-5p and miR-222 in a subset of PTC patients decreased significantly after tumor excision. Increased expression of miR-151-5p and miR-222 was also found in the tissue of PTC patients., Conclusions: Our study demonstrates that serum miRNA profiles may be used as novel and minimally invasive diagnostic markers for PTC.

Chemotherapy has been reported to induce epithelial-mesenchymal transition (EMT) in tumor cells, which is a critical step in the process of metastasis leading to cancer spreading and treatment failure. However, the underlying mechanisms of chemotherapy-induced EMT remain unclear, and the involvement of microRNAs (miRNA) in this process is poorly understood. To address these questions, we established stable chemotherapy-resistant tongue squamous cell carcinoma (TSCC) cell lines CAL27-res and SCC25-res by exposing the parental CAL27 and SCC25 lines to escalating concentrations of cisplatin for 6 months. CAL27-res and SCC25-res cells displayed mesenchymal features with enhanced invasiveness and motility. MiRNA microarray illustrated that miR-200b and miR-15b were the most significantly downregulated microRNAs in CAL27-res cells. Ectopic expression of miR-200b and miR-15b with miRNA mimics effectively reversed the phenotype of EMT in CAL27-res and SCC25-res cells, and sensitized them to chemotherapy, but inhibition of miR-200b and miR-15b in the sensitive lines with anti-sense oligonucleotides induced EMT and conferred chemoresistance. Retrieving the expression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a target for miR-200b and miR-15b, in the presence of the miRNA mimics by transfecting CAL27-res cells with pcDNA3.1-BMI1-carrying mutated seed sequences of miR-200b or miR-15b at its 3'-UTR recapitulated chemotherapy-induced EMT. In vivo, enforced miR-200b or miR-15b expression suppressed metastasis of TSCC xenografts established by CAL27-res cells. Clinically, reduced miR-200b or miR-15b expression was associated with chemotherapeutic resistance in TSCCs and poor patient survival. Our data suggest that reduced expression of miR-200b and miR-15b underscores the mechanisms of chemotherapy-induced EMT in TSCC, and may serve as therapeutic targets to reverse chemotherapy resistance in tongue cancers.

Tumor-initiating cells (T-ICs), a subpopulation of cancer cells with stem cell-like properties, are related to tumor relapse and metastasis. Our previous studies identified a distinct profile of microRNA (miRNA) expression in breast T-ICs (BT-ICs), and the dysregulated miRNAs contribute to the self-renewal and tumorigenesis of these cells. However, the underlying mechanisms for miRNA dysregulation in BT-ICs remain obscure. In the present study, we demonstrated that the expression and function of miR-34c were reduced in the BT-ICs of MCF-7 and SK-3rd cells, a breast cancer cell line enriched for BT-ICs. Ectopic expression of miR-34c reduced the self-renewal of BT-ICs, inhibited epithelial-mesenchymal transition, and suppressed migration of the tumor cells via silencing target gene Notch4. Furthermore, we identified a single hypermethylated CpG site in the promoter region of miR-34c gene that contributed to transcriptional repression of miR-34c in BT-ICs by reducing DNA binding activities of Sp1. Therefore, miR-34c reduction in BT-ICs induced by a single hypermethylated CpG site in the promoter region promotes self-renewal and epithelial-mesenchymal transition of BT-ICs.

Metastasis is a multistep process involving modification of morphology to suit migration, reduction of tumor cell adhesion to the extracellular matrix, increase of cell mobility, tumor cell resistance to anoikis, and other steps. MicroRNAs are well-suited to regulate tumor metastasis due to their capacity to repress numerous target genes in a coordinated manner, thereby enabling their intervention at multiple steps of the invasion-metastasis cascade. In this study, we identified a microRNA exemplifying these attributes, miR-124, whose expression was reduced in aggressive MDA-MB-231 and SK-3rd breast cancer cells. Down-regulation of miR-124 expression in highly aggressive breast cancer cells contributed in part to DNA hypermethylation around the promoters of the three genes encoding miR-124. Ectopic expression of miR-124 in MDA-MB-231 cells suppressed metastasis-related traits including formation of spindle-like morphology, migratory capacity, adhesion to fibronectin, and anoikis. These findings indicate that miR-124 suppresses multiple steps of metastasis by diverse mechanisms in breast cancer cells and suggest a potential application of miR-124 in breast cancer treatment.

Purpose: Tumor-initiating cells are resistant to chemotherapy, but how microRNAs play a role in regulating drug resistance of breast tumor-initiating cells (BT-IC) needs to be clarified., Experimental Design: Lentivirus-mediated miR-128 transduction was done in BT-ICs, enriched by mammosphere cultures or CD44(+)CD24(-) fluorescence-activated cell sorting. Apoptosis and DNA damage were determined upon treatment with doxorubicin. Expression of miR-128 in breast cancer tissues was examined by in situ hybridization and correlated with breast tumor response to neoadjuvant chemotherapy and patient survival., Results: MiR-128 was significantly reduced in chemoresistant BT-ICs enriched from breast cancer cell lines and primary breast tumors (P < 0.01), accompanied by an overexpression of Bmi-1 and ABCC5, which were identified as targets of miR-128. Ectopic expression of miR-128 reduced the protein levels of Bmi-1 and ABCC5 in BT-ICs, along with decreased cell viability (P < 0.001) and increased apoptosis (P < 0.001) and DNA damage (P < 0.001) in the presence of doxorubicin. Reduced miR-128 expression in breast tumor tissues was associated with chemotherapeutic resistance (P < 0.001) and poor survival of breast cancer patients (P < 0.05; n = 57)., Conclusions: Reduction in miR-128 leading to Bmi-1 and ABCC5 overexpression is a stem cell-like feature of BT-ICs, which contributes to chemotherapeutic resistance in breast cancers. Ectopic expression of miR-128 sensitizes BT-ICs to the proapoptotic and DNA-damaging effects of doxorubicin, indicating therapeutic potential.

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Background: Tumor-associated macrophages (TAMs) are alternatively activated cells induced by interleukin-4 (IL-4)-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs) circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells., Results: We utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO) that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway., Conclusions: We conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.

Background: Hepatitis B virus X protein (HBx) is involved in the initiation and progression of hepatocellular carcinoma (HCC) by regulating the host protein-coding genes. In this study, we showed that HBx altered the expression of microRNAs (miRNAs) to promote proliferation and transformation in malignant hepatocytes in vitro., Methods: miRNA microarray and quantitative reverse-transcription polymerase chain reactions (qRT-PCRs) were performed to identify miRNAs that were differentially regulated by HBx in HCC cells. Protein, mRNA, and miRNA expression analyses; cell cycle and apoptosis analyses; loss/gain-of-function analysis; and luciferase reporter assays were performed to delineate the consequences of miR-16 family repression in HepG2 cells., Results: Hepatitis B virus X protein induced widespread deregulation of miRNAs in HepG2 cells, and the downregulation of the miR-16 family was reproducible in HepG2, SK-HEP-1, and Huh7 cells. CCND1, a target of the miR-16 family, was derepressed by HBx in HepG2 cells. c-Myc mediated the HBx-induced repression of miR-15a/16 in HepG2 cells. Ectopically expressed miR-15a/16 suppressed the proliferation, clonogenicity, and anchorage-independent growth of HBx-expressing HepG2 cells by arresting them in the G1 phase and inducing apoptosis, whereas reduced expression of miR-16 accelerated the growth and cell-cycle progression of HepG2 cells., Conclusions: Hepatitis B virus X protein altered the in vitro expression of miRNAs in host malignant hepatocytes, particularly downregulating the miR-16 family. Repression of miR-15a/16 is c-Myc mediated and is required for the HBx-induced transformation of HepG2 cells in vitro. Therefore, miR-16 family may serve as a therapeutic target for hepatitis B virus (HBV)-associated HCC.

Trastuzumab resistance emerges to be a major issue in anti-human epidermal growth factor receptor 2 (HER2) therapy for breast cancers. Here, we demonstrated that miR-21 expression was up-regulated and its function was elevated in HER2(+) BT474, SKBR3, and MDA-MB-453 breast cancer cells that are induced to acquire trastuzumab resistance by long-term exposure to the antibody, whereas protein expression of the PTEN gene, a miR-21 target, was reduced. Blocking the action of miR-21 with antisense oligonucleotides re-sensitized the resistant cells to the therapeutic activities of trastuzumab by inducing growth arrest, proliferation inhibition, and G(1)-S cell cycle checking in the presence of the antibody. Ectopic expression of miR-21 in HER2(+) breast cancer cells confers resistance to trastuzumab. Rescuing PTEN expression with a p3XFLAG-PTEN-mut construct with deleted miR-21 targeting sequence at its 3' UTR restored the growth inhibition of trastuzumab in the resistant cells by inducing PTEN activation and AKT inhibition. In vivo, administering miR-21 antisense oligonucleotides restored trastuzumab sensitivity in the resistant breast cancer xenografts by inducing PTEN expression, whereas injection of miR-21 mimics conferred trastuzumab resistant in the sensitive breast tumors via PTEN silence. Up-regulatin of miR-21 in tumor biopsies obtained from patients receiving pre-operative trastuzumab therapy was associated with poor trastuzumab response. Therefore, miR-21 overexpression contributes to trastuzumab resistance in HER2(+) breast cancers and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to anti-HER2 treatment.

Accumulating evidence indicates that a sub-population of cancer cells with stem-like properties, termed tumor-initiating cells (T-ICs), exist in many different kinds of malignancies, which have a pivotal role in tumorigenesis, tumor progression, metastasis and post-treatment relapse. However, how the stem-like properties of T-ICs are regulated remains obscure. Our previous study showed that reduction of let-7 microRNA (miRNA) in breast tumor-initiating cells (BT-ICs) contributes to the maintenance of their self-renewal capacity and undifferentiated status. In this study we show the effect of mir-30 reduction on the stem-like features of BT-ICs. Similar to let-7, mir-30 is reduced in BT-ICs, and the protein level of Ubc9 (ubiquitin-conjugating enzyme 9) and ITGB3 (integrin beta3), the target genes of mir-30, is markedly upregulated. Enforced constitutive expression of mir-30 in BT-ICs inhibits their self-renewal capacity by reducing Ubc9, and induces apoptosis through silencing ITGB3. On the contrary, blocking the miRNA with a specific antisense oligonucleotide (ASO) in differentiated breast cancer cells revived their self-renewal capacity. Furthermore, ectopic expression of mir-30 in BT-IC xenografts reduces tumorigenesis and lung metastasis in nonobese diabetic/severe combined immunodeficient mice, whereas blocking mir-30 expression enhances tumorigenesis and metastasis. Together, our data suggest mir-30 as one of the important miRNAs in regulating the stem-like features of T-ICs.

Background: The development of metastases involves the dissociation of cells from the primary tumor to penetrate the basement membrane, invade and then exit the vasculature to seed, and colonize distant tissues. The last step, establishment of macroscopic tumors at distant sites, is the least well understood. Four isogenic mouse breast cancer cell lines (67NR, 168FARN, 4TO7, and 4T1) that differ in their ability to metastasize when implanted into the mammary fat pad are used to model the steps of metastasis. Only 4T1 forms macroscopic lung and liver metastases. Because some miRNAs are dysregulated in cancer and affect cellular transformation, tumor formation, and metastasis, we examined whether changes in miRNA expression might explain the differences in metastasis of these cells., Methodology/principal Findings: miRNA expression was analyzed by miRNA microarray and quantitative RT-PCR in isogenic mouse breast cancer cells with distinct metastatic capabilities. 4T1 cells that form macroscopic metastases had elevated expression of miR-200 family miRNAs compared to related cells that invade distant tissues, but are unable to colonize. Moreover, over-expressing miR-200 in 4TO7 cells enabled them to metastasize to lung and liver. These findings are surprising since the miR-200 family was previously shown to promote epithelial characteristics by inhibiting the transcriptional repressor Zeb2 and thereby enhancing E-cadherin expression. We confirmed these findings in these cells. The most metastatic 4T1 cells acquired epithelial properties (high expression of E-cadherin and cytokeratin-18) compared to the less metastatic cells., Conclusions/significance: Expression of miR-200, which promotes a mesenchymal to epithelial cell transition (MET) by inhibiting Zeb2 expression, unexpectedly enhances macroscopic metastases in mouse breast cancer cell lines. These results suggest that for some tumors, tumor colonization at metastatic sites might be enhanced by MET. Therefore the epithelial nature of a tumor does not predict metastatic outcome.

Purpose: We aim to examine miR-21 expression in tongue squamous cell carcinomas (TSCC) and correlate it with patient clinical status, and to investigate its contribution to TSCC cell growth, apoptosis, and tumorigenesis., Experimental Design: MicroRNA profiling was done in 10 cases of TSCC with microarray. MiR-21 overexpression was quantitated with quantitative reverse transcription-PCR in 103 patients, and correlated to the pathoclinical status of the patients. Immunohistochemistry was used to examine the expression of TPM1 and PTEN, and terminal deoxynucleotidyl transferase-mediated dUTP labeling to evaluate apoptosis. Moreover, miR-21 antisense oligonucleotide (ASO) was transfected in SCC-15 and CAL27 cell lines, and tumor cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adherent colony formation, and soft agar assay, whereas apoptosis was determined by Annexin V assay, cytochrome c release, and caspase 3 assay. Tumorigenesis was evaluated by xenografting SCC-15 cells in nude mice., Results: MiR-21 is overexpressed in TSCC relative to adjacent normal tissues. The level of miR-21 is reversely correlated with TPM1 and PTEN expression and apoptosis of cancer cells. Multivariate analysis showed that miR-21 expression is an independent prognostic factor indicating poor survival. Inhibiting miR-21 with ASO in TSCC cell lines reduces survival and anchorage-independent growth, and induces apoptosis in TSCC cell lines. Simultaneous silencing of TPM1 with siRNA only partially recapitulates the effect of miR-21 ASO. Furthermore, repeated injection of miR-21 ASO suppresses tumor formation in nude mice by reducing cell proliferation and inducing apoptosis., Conclusions: miR-21 is an independent prognostic indicator for TSCC, and may play a role in TSCC development by inhibiting cancer cell apoptosis partly via TPM1 silencing.

Cancers may arise from rare self-renewing tumor-initiating cells (T-IC). However, how T-IC self renewal, multipotent differentiation, and tumorigenicity are maintained remains obscure. Because miRNAs can regulate cell-fate decisions, we compared miRNA expression in self-renewing and differentiated cells from breast cancer lines and in breast T-IC (BT-IC) and non-BT-IC from 1 degrees breast cancers. let-7 miRNAs were markedly reduced in BT-IC and increased with differentiation. Infecting BT-IC with let-7-lentivirus reduced proliferation, mammosphere formation, and the proportion of undifferentiated cells in vitro and tumor formation and metastasis in NOD/SCID mice, while antagonizing let-7 by antisense oligonucleotides enhanced in vitro self renewal of non-T-IC. Increased let-7 paralleled reduced H-RAS and HMGA2, known let-7 targets. Silencing H-RAS in a BT-IC-enriched cell line reduced self renewal but had no effect on differentiation, while silencing HMGA2 enhanced differentiation but did not affect self renewal. Therefore let-7 regulates multiple BT-IC stem cell-like properties by silencing more than one target.