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.     

MicroRNA-576-3p Inhibits Proliferation in Bladder Cancer Cells by Targeting Cyclin D1

MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3′-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3′-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.     

miR-490-3p通过靶向调控TGFBR1抑制非小细胞肺癌细胞的增殖和侵袭

目的:以非小细胞肺癌A549细胞为模型,探讨miR-490-3p在肺癌发生发展过程中的作用及其调控机制。方法:通过miRBase数据库获得miR-490-3p序列,设计miR-490-3pmimics并转染A549细胞,CCK8、细胞划痕及Transwell实验分别检测miR-490-3p过表达对A549细胞增殖、迁移和侵袭能力的影响;使用miRwalk在线工具预测miR-490-3p可能的调控基因,通过实时荧光定量PCR及Western印迹对候选调控基因进行筛选,最后通过双萤光素酶报告基因实验验证miR-490-3p与调控基因之间的靶向关系。结果:过表达miR-490-3p可显著抑制A549细胞的增殖、侵袭和迁移能力;在预测的miR-490-3p候选靶基因中,选择与细胞增殖、迁移等表型相关的RASAL2、TGFBR1、PAPPA、HMGA2、TGFA靶基因进行实时荧光定量PCR及Western印迹筛选,结果仅TGFBR1基因在mRNA和蛋白水平的表达与miR-490-3p水平呈负相关,且双萤光素酶报告实验证实miR-490-3p可直接与TGFBR1的3'-UTR结合并抑制其表达。结论:miR-490-3p通过靶向调控TGFBR1的表达抑制非小细胞肺癌A549细胞的增殖和侵袭。     

Decreased miR-204 in H. pylori-Associated Gastric Cancer Promotes Cancer Cell Proliferation and Invasion by Targeting SOX4

Background

The molecular mechanism between Helicobacter pylori (H. pylori) infection and gastric cancer remained largely unknown. In this study, we determined the role of miRNA in H. pylori induced gastric cancer.

Methods and Results

We found that miR-204 was decreased in H. pylori positive tissues by qRT-PCR. Knockdown of miR-204 enhanced the invasion and proliferation ability of gastric cancer cells in vitro. Luciferase assay revealed that SOX4 was target gene of miR-204, which was found up-regulated in H. pylori positive tissues. Down-regulation of miR-204 and over-expression of SOX4 promoted epithelial-mesenchymal transition process.

Conclusion

Taken together, our findings demonstrated that miR-204 may act as a tumor suppressor in H. pylori induced gastric cancer by targeting SOX4.     

MicroRNA-181a Suppresses Mouse Granulosa Cell Proliferation by Targeting Activin Receptor IIA

Activin, a member of the transforming growth factor-β superfamily, promotes the growth of preantral follicles and the proliferation of granulosa cells. However, little is known about the role of microRNAs in activin-mediated granulosa cell proliferation. Here, we reported a dose- and time-dependent suppression of microRNA-181a (miR-181a) expression by activin A in mouse granulosa cells (mGC). Overexpression of miR-181a in mGC suppressed activin receptor IIA (acvr2a) expression by binding to its 3′-untranslated region (3′-UTR), resulting in down-regulation of cyclin D2 and proliferating cell nuclear antigen expression, leading to inhibition of the cellular proliferation, while overexpression of acvr2a attenuated the suppressive effect of miR-181a on mGC proliferation. Consistent with the inhibition of acvr2a expression, miR-181a prevented the phosphorylation of the activin intracellular signal transducer, mothers against decapentaplegic homolog 2 (Smad2), leading to the inactivation of activin signaling pathway. Interestingly, we found that miR-181a expression decreased in ovaries of mice at age of 8, 12, and 21 days, as compared with that in ovaries of 3-day old mice, and its level was reduced in preantral and antral follicles of mice compared with that in primary ones. Moreover, the level of miR-181a in the blood of patients with premature ovarian failure was significantly increased compared with that in normal females. This study identifies an interplay between miR-181a and acvr2a, and reveals an important role of miR-181a in regulating granulosa cell proliferation and ovarian follicle development.     

MicroRNA-137 Contributes to Dampened Tumorigenesis in Human Gastric Cancer by Targeting AKT2

MiRNAs play important roles in tumorigenesis. This study focused on exploring the effects and regulation mechanism of miRNA-137 on the biological behaviors of gastric cancer. Total RNA was extracted from tissues of 100 patients with gastric cancer and from four gastric cancer cell lines. Expression of miR-137 was detected by real-time PCR from 100 patients. The effects of miR-137 overexpression on gastric cancer cells’ proliferation, apoptosis, migration and invasion ability were investigated in vitro and in vivo. The target gene of miR-137 was predicted by Targetscan on line software, screened by dual luciferase reporter gene assay and demonstrated by western blot. As a result, the expression of miR-137 was significant reduced in gastric cancer cell line HGC-27, HGC-803, SGC-7901 and MKN-45 as well as in gastric cancer tissues compared with GES-1 cell or matched adjacent non-neoplastic tissues (p<0.001). The re-introduction of miR-137 into gastric cancer cells was able to inhibit cell proliferation, migration and invasion. The in vivo experiments demonstrated that the miR-137 overexpression can reduce the gastric cancer cell proliferation and metastasis. Bioinformatic and western blot analysis indicated that the miR-137 acted as tumor suppressor roles on gastric cancer cells through targeting AKT2 and further affecting the Bad and GSK-3β. In conclusion, the miR-137 which is frequently down-regulated in gastric cancer is potentially involved in gastric cancer tumorigenesis and metastasis by regulating AKT2 related signal pathways.     

miR-143-3p通过靶向TAK1抑制肺癌增殖和侵袭

目的:分析miR-143-3p在肺癌细胞中的表达情况以及对肺癌进展的作用。方法:通过starBase数据库和肺癌病例组织检测分析miR-143-3p在肺癌组织和正常对照组织中的表达差异;分析miR-143-3p在肺癌细胞HCC27、H1975、A549和肺上皮细胞BEAS-2B中的mRNA水平差异;采用CCK-8法检测miR-143-3p对肺癌细胞增殖活性的影响;采用Transwell实验检测miR-143-3p对肺癌细胞迁移和侵袭的影响;通过qRT-PCR检测miR-143-3p对整合素α6(ITGA6)、锚蛋白重复及PH结构域3(ASAP3)、黑色素瘤相关抗原A9(MAGE-A9)和转化生长因子β激活激酶1(TAK1)表达的影响;通过Western印迹检测miR-143-3p对TAK1蛋白表达的影响。结果:starBase分析和肺癌病例组织检测结果显示miR-143-3p在肺癌组织中低表达,同样地,miR-143-3p在肺癌细胞中的表达也显著低于正常肺上皮细胞;过表达miR-143-3p抑制了肺癌细胞的增殖活性、迁移和侵袭能力;过表达miR-143-3p显著抑制TAK1的表达。结论:miR-143-3p在肺癌中通过靶向TAK1抑制肺癌的增殖和侵袭,miR-143-3p在肺癌进展中详细的分子作用机制和信号通路仍须进一步探讨。     

Luteolin Suppresses Cancer Cell Proliferation by Targeting Vaccinia-Related Kinase 1

Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.     

MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA

The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.     

MicroRNA-135a Inhibits Cell Proliferation by Targeting Bmi1 in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid tumor due to the lack of reliable early detection markers and effective therapies. MicroRNAs (miRNAs), noncoding RNAs that regulate gene expression, are involved in tumorigenesis and have a remarkable potential for the diagnosis and treatment of malignancy. In this study, we investigated aberrantly expressed miRNAs involved in PDAC by comparing miRNA expression profiles in PDAC cell lines with a normal pancreas cell line and found that miR-135a was significantly down-regulated in the PDAC cell lines. The microarray results were validated by qRT-PCR in PDAC tissues, paired adjacent normal pancreatic tissues, PDAC cell lines, and a normal pancreas cell line. We then defined the tumor-suppressing significance and function of miR-135a by constructing a lentiviral vector to express miR-135a. The overexpression of miR-135a in PDAC cells decreased cell proliferation and clonogenicity and also induced G1 arrest and apoptosis. We predicted Bmi1 may be a target of miR-135a using bioinformatics tools and found that Bmi1 expression was markedly up-regulated in PDAC. Its expression was inversely correlated with miR-135a expression in PDAC. Furthermore, a luciferase activity assay revealed that miR-135a could directly target the 3''-untranslated region (3''-UTR) of Bmi1. Taken together, these results demonstrate that miR-135a targets Bmi1 in PDAC and functions as a tumor suppressor. miR-135a may offer a new perspective for the development of effective miRNA-based therapy for PDAC.     

MicroRNA-542-3p Suppresses Tumor Cell Invasion via Targeting AKT Pathway in Human Astrocytoma

The molecular mechanism underlying constitutive activation of AKT signaling, which plays essential roles in astrocytoma progression, is not fully characterized. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of astrocytoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we found that microRNA (miR)-542-3p expression was decreased in glioblastoma cell lines and astrocytoma tissues, and reduced levels of miR-542-3p expression correlated with high histopathological grades and poor prognosis of astrocytoma patients. Exogenous miR-542-3p suppressed glioblastoma cell invasion through not only targeting AKT1 itself but also directly down-regulating its two important upstream regulators, namely, integrin-linked kinase and PIK3R1. Notably, overexpressing miR-542-3p decreased AKT1 phosphorylation and directly and indirectly repressed nuclear translocation and transactivation activity of β-catenin to exert its anti-invasive effect. Furthermore, the miR-542-3p expression level negatively correlated with AKT activity as well as levels of integrin-linked kinase and PIK3R1 in human astrocytoma specimens. These findings suggest that miR-542-3p acts as a negative regulator in astrocytoma progression and that miR-542-3p down-regulation contributes to aberrant activation of AKT signaling, leaving open the possibility that miR-542-3p may be a potential therapeutic target for high grade astrocytoma.     

MicroRNA-149 Inhibits Proliferation and Cell Cycle Progression through the Targeting of ZBTB2 in Human Gastric Cancer

An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNA''s 3′UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.     

MicroRNA-338 Inhibits Growth,Invasion and Metastasis of Gastric Cancer by Targeting NRP1 Expression

NRP1 as multifunctional non-tyrosine-kinase receptors play critical roles in tumor progression. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, particularly cancer. It remains unclear whether miRNAs can regulate the expression of NRP1. The goal of this study was to identify miRNAs that could inhibit the growth, invasion and metastasis of gastric cancer by targeting NRP1 expression. We found that miR-338 expression was reduced in gastric cancer cell lines and in gastric cancer tissues. Moreover, we found that miR-338 inhibited gastric cancer cell migration, invasion, proliferation and promoted apoptosis by targeting NRP1 expression. As an upstream regulator of NRP1, miR-338 directly targets NRP1. The forced expression of miR-338 inhibited the phosphorylation of Erk1/2, P38 MAPK and Akt; however, the expression of phosphorylated Erk1/2, P38 MAPK and Akt was restored by the overexpression of NRP1. In AGS cells infected with miR-338 or transfected with SiNRP1, the protein levels of fibronectin, vimentin, N-cadherin and SNAIL were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-338-expressing cells was restored to normal levels by the restoration of NRP1 expression. In vivo, miR-338 also decreased tumor growth and suppressed D-MVA by targeting NRP1. Therefore, we conclude that miR-338 acts as a novel tumor suppressor gene in gastric cancer. miR-338 can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as gastric cancer EMT, by attenuating the expression of NRP1.     

MicroRNA-214 Suppresses Oncogenesis and Exerts Impact on Prognosis by Targeting PDRG1 in Bladder Cancer

MicroRNA-214 (miR-214) has been reported to be dysregulated in human bladder cancer tissues. We aimed to investigate the clinical correlation, biological significance and molecular network of miR-214 in bladder cancer. Our results showed miR-214 was down-regulated in bladder cancer tissues and significantly associated with tumor stage, lymph node status, grade, multifocality, history of non-muscle-invasive bladder cancer (NMIBC). Moreover, miR-214 could serve as an independent factor of recurrence-free survival (RFS) and overall survival (OS) for patients with muscle-invasive bladder cancer (MIBC). Restoration of miR-214 expression in bladder cancer cell lines inhibited cell proliferation, migration, invasion and markedly promoted apoptosis. Dual-luciferase reporter assay recognized PDRG1 as direct downstream target gene of miR-214. PDRG1 was significantly increased in tumors low of miR-214 and knockdown of PDRG1 mimicked the effects of miR-214 overexpression. Our findings manifest that miR-214 could exert tumor-suppressive effects in bladder cancer by directly down-regulating oncogene PDRG1 and suggest an appealing novel indicator for prognostic and therapeutic intervention of bladder cancer.     

MicroRNA-302a Suppresses Tumor Cell Proliferation by Inhibiting AKT in Prostate Cancer

Micro (mi) RNAs are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in prostate cancer (PCa). MiRNA-302a expression was detected in 44 PCa tissues and 10 normal prostate tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on PCa cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal prostate tissues, miRNA-302a expression was downregulated in PCa tissues, and was even lower in PCa tissues with a Gleason score ≥8. Overexpression of miRNA-302a induced G1/S cell cycle arrest in PCa cells, and suppressed PCa cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibits AKT expression by directly binding to its 3΄ untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 and AKT-p27^Kip1 pathway. These results reveal miRNA-302a as a tumor suppressor in PCa, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in PCa.