Up-regulation of microRNA-21 correlates with lower kidney cancer survival

Background

MicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.

Methods and Results

We measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.

Conclusions

The current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21.     

MicroRNA-1280 Inhibits Invasion and Metastasis by Targeting ROCK1 in Bladder Cancer

MicroRNAs (miRNAs) are non-protein-coding sequences that can function as oncogenes or tumor suppressor genes. This study documents the tumor suppressor role of miR-1280 in bladder cancer. Quantitative real-time PCR and in situ hybridization analyses showed that miR-1280 is significantly down-regulated in bladder cancer cell lines and tumors compared to a non-malignant cell line or normal tissue samples. To decipher the functional significance of miR-1280 in bladder cancer, we ectopically over-expressed miR-1280 in bladder cancer cell lines. Over-expression of miR-1280 had antiproliferative effects and impaired colony formation of bladder cancer cell lines. FACS (fluorescence activated cell sorting) analysis revealed that re-expression of miR-1280 in bladder cancer cells induced G2-M cell cycle arrest and apoptosis. Our results demonstrate that miR-1280 inhibited migration and invasion of bladder cancer cell lines. miR-1280 also attenuated ROCK1 and RhoC protein expression. Luciferase reporter assays demonstrated that oncogene ROCK1 is a direct target of miR-1280 in bladder cancer. This study also indicates that miR-1280 may be of diagnostic and prognostic importance in bladder cancer. For instance, ROC analysis showed that miR-1280 expression can distinguish between malignant and normal bladder cancer cases and Kaplan-Meier analysis revealed that patients with miR-1280 high expression had higher overall survival compared to those with low miR-1280 expression. In conclusion, this is the first study to document that miR-1280 functions as a tumor suppressor by targeting oncogene ROCK1 to invasion/migration and metastasis. Various compounds are currently being used as ROCK1 inhibitors; therefore restoration of tumor suppressor miR-1280 might be therapeutically useful either alone or in combination with these compounds in the treatment of bladder cancer.     

MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20

MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, its function and molecular mechanism in cervical squamous carcinoma have not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the tumor differentiation and nodal status by ISH. Furthermore, we demonstrated that miR-21 regulates proliferation, apoptosis, and migration of HPV16-positive cervical squamous cells. In order to identify candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assays, we confirmed that CCL20 is one of its target genes, which is related to the HPV16 E6 and E7 oncogenes. Our results suggest that miR-21 may be involved in cervical squamous cell tumorigenesis.     

miR-124和miR-520b下调Eps8表达并抑制HeLa细胞增殖

Eps8是一个多功能的信号分子,参与肌动蛋白重排、受体内吞和肿瘤的发生发展. 为了寻找靶向Eps8的microRNAs (miRNAs)并研究其在宫颈癌中的调控作用,本文采用软件预测获得4个可能调控Eps8表达的miRNAs. 利用双荧光素酶报告系统和Western印迹研究发现,miR-124 和miR-520b结合到人EPS8 mRNA的3′非翻译区(untranslated region, UTR)并有效抑制Eps8蛋白的表达. 进一步细胞存活检测、MTT法和克隆形成实验分析显示,miR-124和miR-520b过表达显著抑制HeLa细胞的生长和增殖.而且,miRNA调节的Eps8下调能提高HeLa细胞对化疗药物顺铂的敏感性. 同时证明,miR-124和miR-520b激活肿瘤抑制基因p53与下游基因p21报告基因转录活性,也相应地上调了p53与p21的蛋白表达. 这些结果提示,miR-124和miR-520b下调癌基因EPS8表达,从而抑制HeLa细胞增殖,负调控宫颈癌细胞生长.     

A conjugate of a tumor-targeting ligand and a T cell costimulatory antibody to treat brain tumors

T cell immunotherapy is a potential strategy for the treatment of brain tumors because it offers a high degree of specificity, the ability to extravasate into solid tumors, and the potential for eliciting a long-term protective immune response. Various approaches have been developed to overcome T cell immune tolerance to cancer, including the use of cytokines and bispecific antibodies. T cell stimulation with the proinflammatory cytokine IL-12 can elicit antitumor immunity. T cell activation can be increased using bispecific antibodies against activating molecules on the surface of T cells and a tumor antigen. We studied the effects of systemic IL-12 administration in combination with a conjugate of an anti-CD28 antibody and a ligand for the folate receptor. The high affinity folate receptor is expressed on endogenously arising choroid plexus tumors of SV11 mice, which are transgenic for large T antigen under the control of the SV40 promoter. SV11 mice are immunocompetent, yet immunologically tolerant to large T antigen expressed by choroid plexus tumors. MRI analysis showed that the administration of IL-12 and anti-CD28 Fab/folate significantly slowed tumor growth. Proliferating CD8(+) T cells were found in choroid plexus tumors of treated animals. Treatment of animals with IL-12 + anti-CD28 Fab/folate prolonged survival compared to IL-12 alone. Cytokine treatment combined with tumor-targeted costimulation may be a useful adjunct treatment.     

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3'-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility.     

miR-154 suppresses colorectal cancer cell growth and motility by targeting TLR2

MicroRNAs play critical roles in the development and progression of colorectal cancer (CRC). miR-154 acts as a tumor suppressor in several tumors; however, its role in CRC is poorly understood. Herein, we found that miR-154 was decreased in CRC tissues and cell lines. Ectopic expression of miR-154 remarkably suppressed cell proliferation and colony formation, migration and invasion in CRC cells. The toll-like receptor 2 (TLR2) was found to be a direct target of miR-154 in CRC cells. Inhibition of TLR2 performed similar effects with miR-154 overexpression on CRC cells, and overexpression of TLR2 could significantly reverse the tumor suppressive effects of miR-154 on CRC cells. This study suggests an essential role for miR-154 in CRC.     

Up-regulation of miR-21 mediates resistance to trastuzumab therapy for breast cancer

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.     

MicroRNA-212 displays tumor-promoting properties in non-small cell lung cancer cells and targets the hedgehog pathway receptor PTCH1

Dysexpression of microRNAs has been found in many tumors, including lung cancer. The hedgehog (Hh) signaling pathway plays an important role during normal development, and the abnormal regulation of its members has also been related to many tumors. However, little is known about the relationship between microRNA and the Hh pathway. In this paper, we report microRNA-212 (miR-212) playing a role in non-small cell lung cancer (NSCLC) and targeting PTCH1, a receptor of the Hh pathway. We found that miR-212 was up-regulated when cells were treated with 4ß-12-O-tetradecanoylphorbol-13-acetate (TPA). We ectopically expressed miR-212 in NSCLC cell lines to examine the influence of miR-212 overexpression. The results showed that overexpression of miR-212 in NSCLC cells promoted cell cycle progression and cell proliferation, migration, and invasion. The promoting effects of miR-212 on cell proliferation, migration, and invasion were partially reversed by the miR-212 inhibitor anti-miR-212. These results suggested that miR-212 might have tumor-promoting properties. Potential targets of miR-212 were predicted, and we showed tumor suppressor PTCH1 was a functional target of miR-212. PTCH1 may be responsible for the effect of miR-212 on cell proliferation. Altogether, our results indicated that miR-212 was involved in tumorigenesis, and the oncogenic activity of miR-212 in NSCLC cells was due, in part, to suppression of PTCH1.     

miR-9-5p靶向Ambra1促进舌癌细胞增殖

miRNAs在肿瘤中异常表达,且与肿瘤的发生发展密切相关。目前发现,miR-9-5p在肿瘤中可能发挥原癌或抑癌效应,功能尚未完全阐述清楚。本文拟探讨miR-9-5p在舌癌中的作用。前期研究中收集10例舌癌组织及配对的癌旁组织,实时荧光定量PCR技术检测后发现,miR-9-5p在舌癌组织中的表达量显著高于癌旁组织,且其在舌癌细胞中的表达量也明显高于正常舌上皮细胞。此外,在舌癌细胞Tca8113中过表达miR-9-5p显著增加细胞的增殖能力。生物信息学预测及双荧光素酶报告基因实验证实,miR-9-5p可直接结合在自噬/苄氯素1调节因子1（activating molecule in beclin1-regulated autophagy, Ambra1）的 3′-UTR区域,靶向抑制Ambra1表达。Western印迹结果证实过表达miR-9-5p降低Ambra1的表达,反之亦然。Ambra1在舌癌细胞中的表达量显著低于正常舌上皮细胞。BrdU实验证实在舌癌细胞SCC-25中过表达Ambra1可显著抑制其增殖能力;相反,使用siRNA技术沉默Ambra1能够显著促进Tca8113细胞的增殖。在干预miR-9-5p的细胞中同时干预Ambra1的表达,结果发现Ambra1可显著逆转miR-9-5p对舌癌细胞增殖的促进作用。总之,miR-9-5p在舌癌中可能发挥原癌基因样作用,通过直接靶向抑制Ambra1表达进而促进舌癌细胞发生增殖。     

LncRNA AL133467.1作为miR-661的ceRNA抑制乳腺癌细胞增殖和侵袭

长非编码RNAs((long non-coding RNAs,lncRNAs)在多种肿瘤中异常表达并参与肿瘤的发生发展.然而,众多lncRNAs在肿瘤的表达及功能尚未完全阐明.本文通过分析TCGT数据库113例正常乳腺组织和1109例乳腺癌组织发现,LneRNA AL133467.1在乳腺癌组织中低表达,并与乳腺癌患者...     

MEG3 interacted with miR-494 to repress bladder cancer progression through targeting PTEN

The long noncoding RNA MEG3 is a significant tumor-suppressive gene in various tumors. But its biological role in bladder cancer remains uninvestigated. Herein, the biological mechanism of MEG3 in bladder cancer pathogenesis was explored. First, the expression of MEG3 in bladder cancer cells was examined, and we found that it was significantly reduced. In addition, in bladder cancer cells, we observed htat miR-494 was increased. Then, MEG3 was overexpressed in UMUC3 and SW780 cells and it could negatively modulate miR-494 expression. Bladder cancer cell proliferation was repressed, cell apoptosis was triggered and meanwhile, the cell cycle was remarkably arrested by the overexpression of MEG3. Moreover, the increase of MEG3 suppressed bladder cancer cell migration and invasion capacity. MEG3 can sponge miR-494 and the binding sites between them were confirmed by carrying out a series of functional assays. Furthermore, PTEN was speculated as a putative target of miR-494. Meanwhile, we found that miR-494 inhibitors induced PTEN. Finally, in vivo assays were conducted to prove that MEG3 can restrain bladder tumor growth by modulating miR-494 and PTEN. In conclusion, it was suggested MEG3 can interact with miR-494 to regulate PTEN in bladder cancer development.     

Long noncoding RNA LINC01234 promoted cell proliferation and invasion via miR-1284/TRAF6 axis in colorectal cancer

Colorectal cancer is one of the most common and leading malignancies globally. Long noncoding RNAs (lncRNAs) function as potentially critical regulator in colorectal cancer. LINC01234, a novel lncRNA in tumor biology, regulates the progression of various tumors. However, the tumorigenic mechanism of LINC01234 in colorectal cancer is still unclear. This study was performed with the aim to prospectively investigate clinical significance, effect, and mechanism of lncRNA LINC01234 in colorectal cancer. First, we found that LINC01234, localized in the cytoplasm, was increased in both colorectal cancer cell lines and tissues. Subsequent functional assays suggested LINC01234 knockdown suppressed cell proliferation, migration, and invasion of colorectal cancer cells, while blocked cell cycle and induced cell apoptosis. Moreover, we identified that miR-1284 was target of LINC01234, we further demonstrated a negative correlation with LINC01234 in colorectal cancer tissues and cells. Furthermore, miR-1284 targeted and suppressed tumor necrosis factor receptor–associated factor 6 (TRAF6). Loss-of-function assay revealed that LINC01234 silencing suppressed colorectal cancer progression through inhibition of miR-1284. In vivo subcutaneous xenotransplanted tumor model indicated LINC01234 knockdown inhibited in vivo tumorigenic ability of colorectal cancer via downregulation of TRAF6. Collectively, this study clarified the biological significance of LINC01234/miR-1284/TRAF6 axis in colorectal cancer progression, providing insights into LINC01234 as novel potential therapeutic target for colorectal cancer therapeutic from bench to clinic.     

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Select changes in microRNA (miRNA) expression correlate with estrogen receptor α (ERα) expression in breast tumors. miR-21 is higher in ERα positive than negative tumors, but no one has examined how estradiol (E₂) regulates miR-21 in breast cancer cells. Here we report that E₂ inhibits miR-21 expression in MCF-7 human breast cancer cells. The E₂-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ERα indicating that the suppression is ERα-mediated. ERα and ERβ agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E₂ increased luciferase activity from reporters containing the miR-21 recognition elements from the 3′-UTRs of miR-21 target genes, corroborating that E₂ represses miR-21 expression resulting in a loss of target gene suppression. The E₂-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ERα blocked the E₂-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E₂-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E₂ represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.