Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1

Multidrug resistance-associated protein (MRP-1/ABCC1) transports a wide range of therapeutic agents and may play a critical role in the development of multidrug resistance (MDR) in tumor cells. However, the regulation of MRP-1 remains controversial. To explore whether miRNAs are involved in the regulation of MRP-1 expression and modulate the sensitivity of tumor cells to chemotherapeutic agents, we analyzed miRNA expression levels in VP-16-resistant MDR cell line, MCF-7/VP, in comparison with its parent cell line, MCF-7, using a miRNA microarray. MCF-7/VP overexpressed MRP-1 mRNA and protein not MDR-1 and BCRP. miR-326 was downregulated in MCF-7/VP compared to MCF-7. Additionally, miR-326 was downregulated in a panel of advanced breast cancer tissues and consistent reversely with expression levels of MRP-1. Furthermore, the elevated levels of miR-326 in the mimics-transfected VP-16-resistant cell line, MCF-7/VP, downregulated MRP-1 expression and sensitized these cells to VP-16 and doxorubicin. These findings demonstrate for the first time the involvement of miRNAs in multidrug resistance mediated by MRP-1 and suggest that miR-326 may be an efficient agent for preventing and reversing MDR in tumor cells.     

Epirubicin-mediated expression of miR-302b is involved in osteosarcoma apoptosis and cell cycle regulation

Epirubicin is widely used in osteosarcoma chemotherapy. Growing evidence indicates that the microRNA (miRNA) expression levels which are induced by chemotherapeutic agents play an important role in osteosarcoma development and progression. In this study we investigate the alterations of miRNA expression in the osteosarcoma cells after epirubicin treatment and whether miRNAs can enhance its anti-osteosarcoma effect. After epirubicin exposure, microarray shows 40 miRNAs are differentially expressed in osteosarcoma cells including 24 down-regulated miRNAs. Notably, miR-302b, which is stably low-expressed in osteosarcoma, could be induced by the epirubicin. Furthermore, we find that miR-302b can inhibit the osteosarcoma cell proliferation, promote cell apoptosis and cell cycle arrest MiR-302b can activate caspase-3 and regulate the Akt/pAkt, Bcl-2, Bim expression to increase the cell apoptosis. Meanwhile, miR-302b also attenuates cyclin D1 and CDKs expression to induce cell cycle arrest. Therefore, our results suggest miR-302b can play an essential role in osteosarcoma treatment as a potential tumor suppressor.     

Genomic instability and mouse microRNAs

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23–28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.     

The changes of miRNA expression in rat hippocampus following chronic lead exposure

miRNAs have been found to contribute to normal brain functions, nervous system diseases, as well as neurotoxicities induced by external agents. However, whether they are involved in lead-induced neurotoxicities is still not clear. To identify that, a lead-induced chronic neurotoxicity model of rats was built. Both miRNA microarray analysis and qRT-PCR were performed to determine the change of miRNA expression in hippocampus. Then 3 bioinformatics databases were used to analyze the relative target genes of these miRNA, which were further confirmed by qRT-PCR and Western blot. In the present study, lead exposure resulted in the changed expression of 7 miRNAs: miR-204, miR-211, miR-448, miR-449a, miR-34b, and miR-34c were greatly up-regulated while miR-494 was greatly down-regulated. Bioinformatics analysis results showed that the target genes of 6 up-regulated miRNAs were related to neural injury and neurodegeration, axon and synapse function, neural development and regeneration. Correspondingly, the expression levels of mature mRNAs and proteins of three target genes (Bcl-2, Itpr1, and Map2k1) were greatly repressed, verifying the results of bioinformatics analysis. Taken together, our results showed that the expression of several miRNAs reported to be associated with neurophysiological pathways and neurodegenerative diseases changed in rat hippocampus following chronic lead exposure. These miRNAs may play important roles in lead-induced neurotoxicity.     

Genomic instability and mouse microRNAs

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23-28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.     

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e.g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e.g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity.     

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1?mmol/kg/day by gavage) for 7?days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.     

Genome-wide miRNA expression profiling of human lymphoblastoid cell lines identifies tentative SSRI antidepressant response biomarkers

Aim: Over 30% of patients with major depression do not respond well to first-line treatment with selective serotonin reuptake inhibitors (SSRIs). Using genome-wide expression profiling of human lymphoblastoid cell lines (LCLs) CHL1 was identified as a tentative SSRI sensitivity biomarker. This study reports on miRNAs implicated in SSRI sensitivity of LCLs. Methods: Eighty LCLs were screened from healthy adult female individuals for growth inhibition by paroxetine. Eight LCLs exhibiting high or low sensitivities to paroxetine were chosen for genome-wide expression profiling with miRNA microarrays. Results: The miRNA miR-151-3p had 6.7-fold higher basal expression in paroxetine-sensitive LCLs. This corresponds with lower expression of CHL1, a target of miR-151-3p. The additional miRNAs miR-212, miR-132, miR-30b*, let-7b and let-7c also differed by >1.5-fold (p < 0.05) between the two LCL groups. Conclusion: The potential value of these miRNAs as tentative SSRI response biomarkers awaits validation with lymphocyte samples of major depression patients. Original submitted 28 March 2012; Revision submitted 21 May 2012.     

Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells

MicroRNAs are short non-coding RNA molecules able to affect stability and/or translation of mRNA, thereby regulating the expression of genes involved in many biological processes. We report here that microRNAs miR-27a and miR-451 are involved in activating the expression of P-glycoprotein, the MDR1 gene product that confers cancer cell resistance to a broad range of chemotherapeutics. We showed that expressions of miR-27a and miR-451 were up-regulated in multidrug resistant (MDR) cancer cell lines A2780DX5 and KB-V1, as compared with their parental lines A2780 and KB-3-1. Treatment of A2780DX5 cells with the antagomirs of miR-27a or miR-451 decreased the expression of P-glycoprotein and MDR1 mRNA. In contrast, the mimics of miR-27a and miR-451 increased MDR1 expression in the parental cells A2780. The sensitivity to and intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein were enhanced by the treatment with the antagomirs of miR-27a or miR-451. Our results demonstrate for the first time the roles of microRNAs in the regulation of drug resistance mediated by MDR1/P-glycoprotein, and suggest the potential for targeting miR-27a and miR-451 as a therapeutic strategy for modulating MDR in cancer cells.     

Involvement of miR-133a and miR-326 in ADM resistance of HepG2 through modulating expression of ABCC1

Recent studies have shown that a class of small, functional RNAs, named microRNAs, may regulate multidrug resistance-associated protein 1 (ABCC1). Since ABCC1 is an important efflux transporter responsible for cellular drug disposition, the discovery of microRNAs (miRNA) brings an idea that there may be some other unknown multidrug resistance (MDR) mechanisms exist. Using computational programs, we predicted that the 3′untranslated region (3′UTR) of ABCC1 contains a potential miRNA binding site for miR-133a and also two other for miR-326. These binding sites were confirmed by luciferase reporter assay. ABCC1 mRNA degradation was accelerated dramatically in cells transfected with miR-133a or miR-326 mimics using qRT-PCR, Furthermore, western blot analysis indicated that ABCC1 protein expression was significantly down-regulated in hepatocellular carcinoma cells line HepG2 after transfection with miR-133a or miR-326 mimics, suggesting the involvement of mRNA degradation and protein expression mechanism. The effects of the two miRNAs on adriamycin (ADM) sensitivity to HepG2 cells were determined by MTT assay. Compared with mock transfection, miR-133a or miR-326 mimics transfection sensitized these cells to ADM. These findings for the first time demonstrated that the involvement of miR-133a and miR-326 in MDR is mediated by ABCC1 in hepatocellular carcinoma cell line HepG2 and suggested that miR-133a and miR-326 may be efficient agents for preventing and reversing ADM resistance in cancer cells.     

MicroRNA expression in BRAF-mutated and wild-type metastatic melanoma and its correlation with response duration to BRAF inhibitors

Objective: Currently, the treatment of BRAF V600-mutated metastatic melanoma with BRAF inhibitors gives a response rate of ～ 50% with a progression-free survival of ～ 6 – 7 months. In order to identify predictive biomarkers capable of stratifying BRAF-mutated patients at high risk of shorter response duration to anti-BRAF therapy, the authors analyzed the expression of 15 microRNAs (miRNAs) targeting crucial genes involved in melanoma biology and drug response.

Research design and methods: A total of 15 miRNAs and target gene expression were investigated in 43 patients (30 BRAF-mutated, and 13 BRAF wild-type). Moreover, 20 BRAF-mutated patients treated with vemurafenib were analyzed for miRNA expression in respect to time-to-progression.

Results: All miRNAs except miR-192 showed low expression in BRAF-mutated as compared with BRAF wild-type patients. In particular, miR-101, miR-221, miR-21, miR-338-3p and miR-191 resulted in significant downregulation in BRAF-mutated patients. Moreover, high expression of miR-192 and miR-193b* and low expression of miR-132 resulted in significant association with shorter progression.

Conclusion: Three miRNAs were significantly associated with clinical outcome in metastatic melanoma patients. An increased understanding of the molecular assessment of BRAF-mutated melanomas could allow development of specific molecular tests able to predict response duration.     

MiR-199a-5p and miR-375 affect colon cancer cell sensitivity to cetuximab by targeting PHLPP1

Objectives: We aimed to analyze the differentially-expressed miRNAs in colon cancer cells in order to identify novel potential biomarkers involved in cancer cell resistance.

Design and methods: We investigated the miRNA expression profile of GEO human colon carcinoma cells, sensitive to the EGFR inhibitor Cetuximab (CTX) and their CTX-resistant counterpart (GEO CR) by using a miRNA chip.

Results: We found 27 upregulated and 10 downregulated miRNAs in GEO CR compared with GEO cells with a fold change ≥ 2. Among the upregulated miRNAs, we focused on miR-199a-5p and miR-375. We report that their enforced expression promotes CTX resistance, whereas their silencing sensitizes to the same drug. The ability of miR-199a-5p and miR-375 to target PHLPP1 (PH domain and leucine-rich repeat protein phosphatase 1), a tumor suppressor that negatively regulates the AKT pathway, accounts, at least in part, for their drug-resistance activity. Indeed, restoration of PHLPP1 increases sensitivity of the GEO cells to CTX and reverts the resistance-promoting effect of miR-199a-5p and miR-375.

Conclusion: This study proposes miR-199a-5p and miR-375 as contributors to CTX resistance in colon cancer and suggests a novel approach based on miRNAs as tools for the therapy of this tumor.     

MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression

Oral squamous cell carcinoma, the most frequently occurring malignant head and neck tumour, generally exhibits poor prognosis and metastases are the main cause of death. The discovery of reliable prognostic indicators of tumour progression could greatly improve clinical practice. MicroRNAs are involved in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. Since miRNAs have been shown to be abnormally expressed in different tumours their importance as potential cancer prognostic indicators is increasing. To define the role of miRNA in OSCC tumours we investigated the expression profile of 15 OSCC (8 without metastasis and 7 with lymph node metastasis) using microarray analysis. Thirteen miRNA were significantly overexpressed (miR-489, miR-129, miR-23a, miR-214, miR-23b, miR-92, miR-25, miR-210, miR-212, miR-515, miR-146b, miR-21, miR-338) and 6 miRNA were underexpressed (miR-520h, miR-197, miR-378, miR-135b, miR-224, miR-34a) in oral tumours. Underexpression of mir-155, let-7i, mir-146a was found to characterize progression to metastastatic tumours. Further investigations will elucidate whether differentially expressed miRNAs will help to better classify OSCCs, thus improving diagnoses and patient care.     

Aberrant expression of miR-638 contributes to benzo(a)pyrene-induced human cell transformation

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.     

MiR-497对人胃癌细胞株顺铂耐药性的影响和机制

目的:通过建立人胃癌细胞SGC-7901的顺铂耐药细胞株SGC-7901/DDP,探讨miR-497对SGC-7901/DDP耐药性的影响及其机制。方法:体外研究采用顺铂体外逐步加量诱导法建立人胃癌细胞SGC-7901耐药株,并通过检测药物半抑制浓度和耐药基因MDR1、BCRP、MRP1的表达以鉴定耐药细胞株;检测在亲本及耐药细胞中miR-497、MDR1、MRP1、BCRP和凋亡相关基因Bax、Bcl-2的表达水平;miR-497模拟物分别转染SGC-7901/DDP细胞株,利用SRB法和流式细胞术检测转染miR-497模拟物后细胞对顺铂醇的敏感程度和细胞的周期、凋亡的变化,并检测耐药基因和凋亡相关基因的表达。结果:成功建立人胃癌SGC-7901/DDP耐药细胞株,耐药细胞株中耐药基因MDR1、BCRP、MRP1表达均升高,抗凋亡基因Bcl-2升高,凋亡基因Bax下降,miR-497表达下降(P<0.05);miR-497模拟物提高耐药细胞株对顺铂的药物敏感性,凋亡水平增加,细胞周期G₀/G₁期细胞增多(P<0.05),并可抑制耐药细胞中耐药基因的表达,降低Bcl-2/Bax比值(P<0.05)。结论:人胃癌耐药细胞株SGC-7901/DDP的miR-497表达下调;上调miR-497的表达可逆转人胃癌SGC-7901/DDP细胞株对化疗药物顺铂的耐药性。     

Paraquat inhibited differentiation in human neural progenitor cells (hNPCs) and down regulated miR-200a expression by targeting CTNNB1

Paraquat (PQ) exposure influences central nervous system and results in serious neurotoxicity in vitro and in vivo. However, the role of PQ exposure in the development of CNS remains unclear. In present study, we investigated microRNAs (miRNAs) expression profiling and cell differential status following PQ treatment in human neural progenitor cells (hNPCs) as well as involved mechanism. Microarray profiling of miRNAs expression of PQ treated cell line and their corresponding control was determined. Differentially expression miRNAs were confirmed by quantitative real time PCR. Neural cell differentiation was performed with immunocytochemical analysis. Predicated target of miRNA was identified with luciferase reports and quantitatively analyzed using western blotting. Our results found PQ dramatically suppressed neural cell differentiation ability. 43 differentially expressed miRNAs were identified in PQ treated cells. The expression levels were over expressed in 25 miRNAs, whereas 18 miRNAs were suppressed. More importantly, we observed that miR-200a expression level to be lower in PQ treated cells. Luciferase assay and protein expression results confirmed the direct binding effect between CTNNB1 and miR-200a following PQ exposure. Collectively, our data suggested that down regulation of miR-200a in the PQ treated neural stem cell significantly participated in the differentiation processes and subsequently resulting in decreased cell viability, increased epithelial-mesenchymal transition process and the inhibited differential through CTNNB1 pathway.