MiR-5047在乳腺癌细胞增殖迁移中的作用及表达调控*

探讨miR-5047在乳腺癌细胞中的表达及其在乳腺癌细胞增殖和迁移中的作用,并明确地西他滨在miR-5047表达调控中的作用。通过实时荧光定量PCR(qRT-PCR)检测人乳腺癌细胞系和正常乳腺上皮细胞MCF10A中miR-5047的表达水平;将miR-5047模拟物(mimic),阴性对照(NC)分别转染至MDA-MB-231和MCF7细胞,经平板克隆实验、MTT实验、划痕愈合实验检测乳腺癌细胞的增殖和迁移能力,通过qRT-PCR和Western blot检测相关基因表达及蛋白水平。使用浓度5 μmol/L和10 μmol/L的地西他滨分别处理MDA-MB-231和MCF-7细胞,经qRT-PCR检测不同浓度和处理时间条件下地西他滨对miR-5047表达的影响。同时,通过形态观察和Western blot检测地西他滨对乳腺癌细胞上皮间质转化的影响。与正常乳腺上皮细胞MCF-10A相比,miR-5047在乳腺癌细胞中表达均显著下调。miR-5047过表达可显著抑制乳腺癌细胞的增殖和迁移,促进上皮细胞标志物E-cadherin的表达,抑制间质细胞标志物Vimentin的表达。不同浓度地西他滨处理MDA-MB-231和MCF7细胞后,miR-5047表达均增强,且10 μmol/L作用48 h效果最显著。地西他滨可诱导MDA-MB-231细胞向上皮样转变。miR-5047在乳腺癌细胞系中表达显著下调,过表达miR-5047可抑制乳腺癌细胞的增殖和迁移,地西他滨可促进乳腺癌细胞中miR-5047的表达,并诱导细胞向上皮样转变。     

miR-30a suppresses breast cancer cell proliferation and migration by targeting Eya2

Eye absent (Eya) proteins are involved in cell fate determination in a broad spectrum of cells and tissues. Aberrant expression of Eya2 has been documented in a variety of cancers and correlates with clinical outcome. However, whether microRNAs (miRNAs) can regulate Eya2 expression remains unknown. Here, we show that miR-30a represses Eya2 expression by binding to the 3′-untranslated region of Eya2. Overexpression of Eya2 in miR-30a-transfected breast cancer cells effectively rescued the inhibition of cell proliferation and migration caused by miR-30a. Knockdown of Eya2 by small-interfering RNA (siRNA) in breast cancer cells mimicked the effect induced by miR-30a and abolished the ability of miR-30a to regulate breast cancer cell proliferation and migration. The miR-30a/Eya2 axis could regulate G1/S cell cycle progression, accompanied by the modulation of expression of cell cycle-related proteins, including cyclin A, cyclin D1, cyclin E, and c-Myc. Moreover, miR-30a expression was downregulated in breast cancer patients, and negatively correlated with Eya2, which was upregulated in breast cancer patients. These data suggest that the miR-30a/Eya2 axis may play an important role in breast cancer development and progression and that miR-30a activation or Eya2 inhibition may be a useful strategy for cancer treatment.     

M2 macrophages contribute to cell proliferation and migration of breast cancer

Breast cancer is a kind of malignant tumor that severely threatens women's lives and health worldwide. Tumor-associated macrophages (TAMs) have been reported to mediate tumor progression, while the mechanism still needs further identification. In this study, we found that M2 macrophages promoted increased cell proliferation and migration as well as reduced expression of interferon regulatory factor 7 (IRF7) and increased the expression of miR-1587 in breast cancer cells. Overexpression of IRF7 or miR-1587 knockdown reversed M2 macrophage-induced cell proliferation and migration as well as tumor growth in vivo. Mechanistically, miR-1587 targeted the 3ʹ-untranslated region (3ʹ-UTR) of IRF7 mRNA to regulate its protein expression leading to tumor progression. Collectively, this study revealed that the miR-1587/IRF7 axis mediates M2 macrophage-induced breast cancer progression, and this sheds light on further clinical therapy for breast cancer by targeting TAMs as well as the miR-1587/IRF7 axis.     

Modulation of MicroRNA-194 and cell migration by HER2-targeting trastuzumab in breast cancer

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of the HER2 oncoprotein, can effectively target HER2-positive breast cancer through several mechanisms. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis and apoptosis have been investigated in depth, the effect of trastuzumab on microRNA (miRNA) has not been extensively studied. We have performed miRNA microarray profiling before and after trastuzumab treatment in SKBr3 and BT474 human breast cancer cells that overexpress HER2. We found that trastuzumab treatment of SKBr3 cells significantly decreased five miRNAs and increased three others, whereas treatment of BT474 cells significantly decreased two miRNAs and increased nine. The only change in miRNA expression observed in both cell lines following trastuzumab treatment was upregulation of miRNA-194 (miR-194) that was further validated in vitro and in vivo. Forced expression of miR-194 in breast cancer cells that overexpress HER2 produced no effect on apoptosis, modest inhibition of proliferation, significant inhibition of cell migration/invasion in vitro and significant inhibition of xenograft growth in vivo. Conversely, knockdown of miR-194 promoted cell migration. Increased miR-194 expression markedly reduced levels of the cytoskeletal protein talin2 and specifically inhibited luciferase reporter activity of a talin2 wild-type 3'-untranslated region, but not that of a mutant reporter, indicating that talin2 is a direct downstream target of miR-194. Trastuzumab treatment inhibited breast cancer cell migration and reduced talin2 expression in vitro and in vivo. Knockdown of talin2 inhibited cell migration/invasion. Knockdown of trastuzumab-induced miR-194 expression with a miR-194 inhibitor compromised trastuzumab-inhibited cell migration in HER2-overexpressing breast cancer cells. Consequently, trastuzumab treatment upregulates miR-194 expression and may exert its cell migration-inhibitory effect through miR-194-mediated downregulation of cytoskeleton protein talin2 in HER2-overexpressing human breast cancer cells.     

Downregulation of microRNA-92b-3p suppresses proliferation,migration, and invasion of gastric cancer SGC-7901 cells by targeting Homeobox D10

To investigate the effect and mechanism of microRNA-92b-3p (miR-92b-3p) targeting Homeobox D10 (HOXD10) on proliferation, migration, and invasion of gastric cancer, we detected t he expression of miR-92b-3p and HOXD10 in SGC-7901 cells. The effects of miR-92b-3p or HOXD10 on proliferation, migration, invasion, and matrix metalloproteinase (MMP)-2/9 expression in SGC-7901 cells were measured by the Cell Counting Kit-8 assay, Transwell assay, and Western blot, respectively. The results showed that miR-92b-3p expression was increased, and HOXD10 expression was decreased in SGC-7901 cells, compared with human normal gastric epithelial cells GES-1. Functional experiments demonstrated that cell proliferation, migration, invasion, and expression of MMP-2/9 in SGC-7901 cells were significantly inhibited by miR-92b-3p silencing and HOXD10 overexpression. Moreover, HOXD10 was a potential target gene of miR-92b-3p as evidenced by the TargetScan software and double luciferase reporter assay. In the rescue experiment, knockdown of HOXD10, accompanied by higher expression of MMP-2/9, could significantly eliminate the inhibitory effects of miR-92b-3p silencing on cell proliferation, migration, and invasion. In conclusion, miR-92b-3p is highly expressed in gastric cancer SGC-7901 cells, and interfering with its expression might inhibit SGC-7901 cell proliferation, migration, and invasion via downregulating MMP-2/9 expression and targeting HOXD10.     

MicroRNA-335 suppresses the proliferation,migration, and invasion of breast cancer cells by targeting EphA4

MicroRNAs (miRNAs) are small noncoding RNAs that exert their functions by targeting specific mRNA sequences. Many studies have demonstrated that miRNAs are crucial for cancer progression, during which they can act as either oncogenes or tumor suppressors. Previous research has shown that miR-335 is downregulated in breast cancer, and it has been shown to be a breast cancer suppressor. In addition, emerging evidence indicates that erythropoietin-producing hepatocellular A4 (EphA4) is implicated in cancer cell proliferation, migration, and invasion. However, little is known about the relationship between miR-335 and EphA4 in breast cancer. In the present study, we used bioinformatic and biochemical analyses to demonstrate that EphA4 is a direct downstream target of miR-335 in human breast cancer MCF-7 and MDA-MB-23 cells and revealed that miR-335 negatively regulates the expression of EphA4 in these cells. Further investigation revealed that miR-335 overexpression inhibits MCF-7 and MDA-MB-231 cell proliferation and that this inhibition is attenuated by EphA4 coexpression. Similarly, miR-335 overexpression also inhibited growth and downregulated EphA4 expression in tumors in nude mice. Moreover, our results demonstrated that miR-335 overexpression suppresses migration and invasion in MCF-7 and MDA-MB-231 cells, an effect that was reversed by EphA4 overexpression. These findings confirmed that EphA4 is a direct target gene of miR-335 and that miR-335 suppresses breast cancer cell proliferation and motility in part by directly inhibiting EphA4 expression.     

MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer

Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25 nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28-fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3′UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.     

MiR-487a Promotes TGF-β1-induced EMT,the Migration and Invasion of Breast Cancer Cells by Directly Targeting MAGI2

Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subsequently, we found that the transfection of miR-487a inhibitor significantly decreased the expression of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epithelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demonstrated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) significantly increased the miR-487a promoter activity and expression, and TGF-β1 induced the increased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

miR-125b targets ARID3B in breast cancer cells

Mounting evidence suggests involvement of deregulated microRNA (miRNA) expression during the complex events of tumorigenesis. Among such deregulated miRNAs in cancer, miR-125b expression is reported to be consistently low in breast cancers. In this study, we screened a panel of breast cancer cell lines (BCCLs) for miR-125b expression and detected decreased expression in 14 of 19 BCCLs. Due to the heterogeneity of breast cancers, MCF7 cells were chosen as a model system for ERBB2 independent breast cancers to restore miR-125b expression (MCF7-125b) to investigate the phenotypical and related functional changes. Earlier, miR-125b was shown to regulate cell motility by targeting ERBB2 in ERBB2 overexpressing breast cancer cells. Here we showed decreased motility and migration in miR-125b expressing MCF7 cells, independent of ERBB2. MCF7-125b cells demonstrated profoundly decreased cytoplasmic protrusions detected by phalloidin staining of filamentous actin along with decreased motility and migration behaviors detected by in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Among possible numerous targets of miR-125b, we showed ARID3B (AT-rich interactive domain 3B) to be a novel target with roles in cell motility in breast cancer cells. When ARID3B was transiently silenced, the decreased cell migration was also observed. In light of these findings, miR-125b continues to emerge as an interesting regulator of cancer related phenotypes.     

MicroRNA-188-5p promotes apoptosis and inhibits cell proliferation of breast cancer cells via the MAPK signaling pathway by targeting Rap2c

Breast cancer is a common malignancy that is highly lethal with poor survival rates and immature therapeutics that urgently needs more effective and efficient therapies. MicroRNAs are intrinsically involved in different cancer remedies, but their mechanism in breast cancer has not been elucidated for prospective treatment. The function and mechanism of microRNA-188-5p (miR-188) have not been thoroughly investigated in breast cancer. In our study, we found that the expression of miR-188 in breast cancer tissues was obviously reduced. Our findings also revealed the abnormal overexpression of miR-188 in 4T1 and MCF-7 cells significantly suppressed cell proliferation and migration and also enhanced apoptosis. miR-188 induced cell cycle arrest in the G1 phase. To illuminate the molecular mechanism of miR-188, Rap2c was screened as a single target gene by bioinformatics database analysis and was further confirmed by dual-luciferase assay. Moreover, Rap2c was found to be a vital molecular switch for the mitogen-activated protein kinase signaling pathway in tumor progression by decreasing apoptosis and promoting proliferation and migration. In conclusion, our results revealed that miR-188 is a cancer progression suppressor and a promising future target for breast cancer therapy.     

MicroRNA-630 inhibits breast cancer progression by directly targeting BMI1

MicroRNAs (miRNAs) play critical roles in breast cancer cell biological processes, including proliferation and apoptosis by inhibiting the expression of their target genes. Herein, we reported that miR-630 overexpression initiates apoptosis, blocks cell cycle progression and suppresses cell proliferation in breast cancer cells. Furthermore, BMI1, a member of polycomb group family, was identified as a direct target of miR-630, and there was a negative correlation between the expression levels of BMI1 and miR-630 in human breast cancer samples. With a series of biology approaches, subsequently, we proved that BMI1 was a functional downstream target of miR-630 and mediated the property of miR-630-dependent inhibition of breast cancer progression. Taken together, these findings provide further evidence on the tumor-suppression function of miR-630 in breast cancer, and clarify BMI1 as a novel functional target gene of miR-630.