MiR-133b Is Down-Regulated in Human Osteosarcoma and Inhibits Osteosarcoma Cells Proliferation,Migration and Invasion,and Promotes Apoptosis

MicroRNAs (miRNAs) decrease the expression of specific target oncogenes or tumor suppressor genes and thereby play crucial roles in tumorigenesis and tumor growth. To date, the potential miRNAs regulating osteosarcoma growth and progression are not fully identified yet. In this study, the miRNA microarray assay and hierarchical clustering analysis were performed in human osteosarcoma samples. In comparison with normal human skeletal muscle, 43 miRNAs were significantly differentially expressed in human osteosarcomas (fold change ≥2 and p≤0.05). Among these miRNAs, miR-133a and miR-133b expression was decreased by 135 folds and 47 folds respectively and the decreased expression was confirmed in both frozen and paraffin-embedded osteosarcoma samples. The miR-133b precursor expression vector was then transfected into osteosarcoma cell lines U2-OS and MG-63, and the stable transfectants were selected by puromycin. We found that stable over-expression of miR-133b in osteosarcoma cell lines U2-OS and MG-63 inhibited cell proliferation, invasion and migration, and induced apoptosis. Further, over-expression of miR-133b decreased the expression of predicted target genes BCL2L2, MCL-1, IGF1R and MET, as well as the expression of phospho-Akt and FAK. This study provides a new insight into miRNAs dysregulation in osteosarcoma, and indicates that miR-133b may play as a tumor suppressor gene in osteosarcoma.     

The Rescue of miR-148a Expression in Pancreatic Cancer: An Inappropriate Therapeutic Tool

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.     

Dependence of Intracellular and Exosomal microRNAs on Viral E6/E7 Oncogene Expression in HPV-positive Tumor Cells

Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous E6/E7 oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that E6/E7 silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of E6/E7 silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The E6/E7-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained E6/E7 expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative p21 gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon E6/E7 silencing. Several of the E6/E7-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the E6/E7-dependent growth of HPV-positive cancer cells.     

Differential MicroRNAs Expression in Serum of Patients with Lung Cancer, Pulmonary Tuberculosis, and Pneumonia

MicroRNAs (miRNAs) play critical regulatory roles in the physiological and pathological processes. The high stability of miRNAs in human serum represents attractive novel diagnostic biomarkers of clinical conditions. Several studies have shown that aberrant expression of miRNAs in human cancer including lung cancer, but little is known about their effects on some infectious lung diseases such as pulmonary tuberculosis (TB) and pneumonia. In this study, we investigated miRNA expression pattern in serum of Egyptian patients with lung cancer, TB, and pneumonia compared with matched healthy controls. Using microarray-based expression profiling followed by real-time quantitative polymerase chain reaction validation, we compared the levels of a series of circulating miRNAs (miR-21, miR-155, miR-182, and miR-197) in serum from patients with lung cancer (n = 65), pulmonary tuberculosis (n = 29), pneumonia (n = 29), and transudate (n = 16) compared with matched healthy controls (n = 37). MiRNA SNORD68 was the housekeeping endogenous control. We found that the serum levels of miR-21, miR-155, and miR-197 were significantly elevated in the patients with lung cancer and pneumonia whereas miR-182 and miR-197 levels were increased only in patients with lung cancer and TB, respectively, compared with controls. Receiver operating characteristic analysis revealed that miR-182, miR-155, and miR-197 have superior diagnostic potential in discriminating patients with lung cancer, pneumonia, and TB, respectively, from controls. Our results conclude that the differential expression of the four studied miRNAs can be potential non-invasive biomarkers for patients with lung cancer, TB and pneumonia.     

MicroRNA-650 targets ING4 to promote gastric cancer tumorigenicity

MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of target mRNAs. Altered expression of specific miRNAs in human gastric cancer progression has been reported; however, the role of miR-650 in gastric cancer is poorly understood. In this study, we show that miR-650 is involved in lymphatic and distant metastasis in human gastric cancer, and we find that ectopic expression of miR-650 promotes tumorigenesis and proliferation of gastric cancer cells. A luciferase reporter assay demonstrates that Inhibitor of Growth 4 (ING4) is a direct target of miR-650. Collectively, our study demonstrates that over-expression of miR-650 in gastric cancer may promote proliferation and growth of cancer cells, at least partially through directly targeting ING4. These findings help clarify the molecular mechanisms involved in gastric carcinogenesis and indicate that miR-650 modulation may be a bona fide miRNA-based treatment of gastric cancer.     

miR-186 and 326 Predict the Prognosis of Pancreatic Ductal Adenocarcinoma and Affect the Proliferation and Migration of Cancer Cells

MicroRNAs can function as key tumor suppressors or oncogenes and act as biomarkers for cancer diagnosis or prognosis. Although high-throughput assays have revealed many miRNA biomarkers for pancreatic ductal adenocarcinoma (PDAC), only a few have been validated in independent populations or investigated for functional significance in PDAC pathogenesis. In this study, we correlated the expression of 36 potentially prognostic miRNAs within PDAC tissue with clinico-pathological features and survival in 151 Chinese patients. We then analyzed the functional roles and target genes of two miRNAs in PDAC development. We found that high expression of miR-186 and miR-326 predict poor and improved survival, respectively. miR-186 was over-expressed in PDAC patients compared with controls, especially in patients with large tumors (>2 cm), lymph node metastasis, or short-term survival (< 24 months). In contrast, miR-326 was down-regulated in patients compared with controls and displayed relatively increased expression in the patients with long-term survival or without venous invasion. Functional experiments revealed that PDAC cell proliferation and migration was decreased following inhibition and enhanced following over-expression of miR-186. In contrast, it was enhanced following inhibition and decreased after over-expression of miR-326. A luciferase assay indicated that miR-186 can bind directly to the 3′-UTR of NR5A2 to repress gene expression. These findings suggest that miR-186 over-expression contributes to the invasive potential of PDAC, likely via suppression of NR5A2, thereby leading to a poor prognosis; high miR-326 expression prolongs survival likely via the decreasing invasive potential of PDAC cells. These two miRNAs can be used as markers for clinical diagnosis and prognosis, and they represent therapeutic targets for PDAC.     

MicroRNA-181b promotes ovarian cancer cell growth and invasion by targeting LATS2

MicroRNAs (miRNAs) are strongly implicated in tumorigenesis and metastasis. In this study, we showed significant upregulation of miR-181b in ovarian cancer tissues, compared with the normal ovarian counterparts. Forced expression of miR-181b led to remarkably enhanced proliferation and invasion of ovarian cancer cells while its knockdown induced significant suppression of these cellular events. The tumor suppressor gene, LATS2 (large tumor suppressor 2), was further identified as a novel direct target of miR-181b. Specifically, miR-181b bound directly to the 3′-untranslated region (UTR) of LATS2 and suppressed its expression. Restoration of LATS2 expression partially reversed the oncogenic effects of miR-181b. Our results indicate that miR-181b promotes proliferation and invasion by targeting LATS2 in ovarian cancer cells. These findings support the utility of miR-181b as a potential diagnostic and therapeutic target for ovarian cancer.     

MiR-199a Regulates Cell Proliferation and Survival by Targeting FZD7

A growing amount of evidence indicates that miRNAs are important regulators of multiple cellular processes and, when expressed aberrantly in different types of cancer such as hepatocellular carcinoma (HCC), play significant roles in tumorigenesis and progression. Aberrant expression of miR-199a-5p (also called miR-199a) was found to contribute to carcinogenesis in different types of cancer, including HCC. However, the precise molecular mechanism is not yet fully understood. The present study showed that miR-199a is frequently down-regulated in HCC tissues and cells. Importantly, lower expression of miR-199a was significantly correlated with the malignant potential and poor prognosis of HCC, and restoration of miR-199a in HCC cells led to inhibition of the cell proliferation and cell cycle in vitro and in vivo. Furthermore, Frizzled type 7 receptor (FZD7), the most important Wnt receptor involved in cancer development and progression, was identified as a functional target of miR-199a. In addition, these findings were further strengthened by results showing that expression of FZD7 was inversely correlated with miR-199a in both HCC tissues and cells and that over-expression of miR-199a could significantly down-regulate the expression of genes downstream of FZD7, including β-catenin, Jun, Cyclin D1 and Myc. In conclusion, these findings not only help us to better elucidate the molecular mechanisms of hepatocarcinogenesis from a fresh perspective but also provide a new theoretical basis to further investigate miR-199a as a potential biomarker and a promising approach for HCC treatment.     

miR-146a and miR-150 promote the differentiation of CD133+ cells into T-lymphoid lineage

MicroRNAs control the genes involved in hematopoietic stem cell (HSCs) survival, proliferation and differentiation. The over-expression of miR-146 and miR-150 has been reported during differentiation of HSCs into T-lymphoid lineage. Therefore, in this study we evaluated the effect of their over-expression on CD133⁺ cells differentiation to T cells. miR-146a and miR-150 were separately and jointly transduced to human cord blood derived CD133⁺ cells (>97 % purity). We used qRT-PCR to assess the expression of CD2, CD3ε, CD4, CD8, CD25, T cell receptor alpha (TCR-α) and Ikaros genes in differentiated cells 4 and 8 days after transduction of the miRNAs. Following the over-expression of miR-146a, significant up-regulation of CD2, CD4, CD25 and Ikaros genes were observed (P < 0.01). On the other hand, over-expression of miR-150 caused an increase in the expression of Ikaros, CD4, CD25 and TCR-α. To evaluate the combinatorial effect of miR-146a and miR-150, transduction of both miRNAs was concurrently performed which led to increase in the expression of Ikaros, CD4 and CD3 genes. In conclusion, it seems that the effect of miR-150 and miR-146a on the promotion of T cell differentiation is time-dependant. Moreover, miRNAs could be used either as substitutes or complements of the conventional differentiation protocols for higher efficiency.     

Identification of miR-7 as an oncogene in renal cell carcinoma

MicroRNA-7 (miR-7) has been described as a tumor suppressor in several human cancers, but the results of a study to identify miRNAs associated with metastatic capability in breast cancer suggested that miR-7 may be characterized as an oncogene. The present study was to determine the expression and function of miR-7 in renal cell carcinoma. Quantitative real-time polymerase chain reaction was used to validate the expressions of miR-7 in 48 paired renal cell carcinomas (RCC) and normal tissues, based on the preliminary sequencing results of miRNAs. Furthermore, the impacts of miR-7 on cell migration, proliferation and apoptosis were analyzed using wound scratch assay, MTT and flow cytometry, respectively. The results demonstrated that miR-7 was up-regulated in RCC compared with normal tissues (p = 0.001). Down-regulation of miR-7 with synthesized inhibitor inhibited cell migration in vitro, suppressed cell proliferation and induced renal cancer cell apoptosis, prompting that miR-7 could be characterized as an oncogene in RCC. The present study was the first to reveal that miR-7 was up-regulated in RCC and it played an important role in RCC by affecting cellular migration, proliferation and apoptosis. Further researches should be conducted to explore the roles and target genes of miR-7 in RCC and other cancers.     

Integrated microRNA–mRNA analysis of coronary artery disease

Although patients with coronary artery disease (CAD) have a high mortality rate, the pathogenesis of CAD is still poorly understood. The purpose of this study was to explore the underlying molecular mechanisms and potential target molecules for CAD. The platelet miRNA (GSE28858) and blood mRNA (GSE42148) expression profiles of patients with CAD and healthy controls were downloaded from Gene Expression Omnibus. Differentially expressed miRNAs and genes (DEGs) were identified by significant analysis of microarray algorithm after data preprocessing. Furthermore, the miRNA-target gene regulatory network was constructed based on miRecords database. The spearman correlation coefficients (ρ) between miRNAs and their target genes were calculated. Six up- (miR-340, miR-545, miR-451, miR454-5p, miR-624 and miR-585) and four down-regulated (miR-199a, miR-17-3p, miR-154 and miR-339) miRNAs were screened. Total 295 target genes of miR-545, miR-451, miR-585 and miR-154 were predicted. Among these 295 target genes, 7 genes were DEGs. Further analysis showed miR-545-TFEC and miR-585-SPOCK1 were highly positively correlated (ρ = 0.808091264; ρ = 0.874680776) in CAD samples. Therefore, differentially expressed miRNAs might participate in the pathogenesis of CAD by regulating their target genes.     

microRNA-1298 inhibits the malignant behaviors of breast cancer cells via targeting ADAM9

MicroRNAs (miRNAs) regulate the progression of human malignancy by targeting oncogenes or tumor suppressors, which are 12 promising targets for cancer treatment. Increasing evidence has suggested the aberrant expression and tumor-suppressive function of miR-1298 in cancers, however, the regulatory mechanism of miR-1298 in breast cancer (BC) remains unclear. Here, our findings showed that miR-1298 was down-regulated in BC tissues and cell lines. Lower level of miR-1298 was significantly correlated with the advanced progression of BC patients. Experimental study showed that overexpression of miR-1298 inhibited the proliferation, induced apoptosis and cell cycle arrest in BC cells. The in vivo xenograft mice model showed that highly expressed miR-1298 significantly reduced the tumor growth and metastasis. Further mechanism analysis revealed that miR-1298 bound the 3′-untranslated region (UTR) of a disintegrin and metalloproteinase 9 domain (ADAM9) and suppressed the expression of ADAM9 in BC cells. ADAM9 was overexpressed in BC tissues and inversely correlated with miR-1298. Down-regulation of ADAM9 induced apoptosis and cell cycle arrest of BC cells. Moreover, ectopic expression of ADAM9 by transiently transfecting with vector encoding the full coding sequence of ADAM9 attenuated the inhibitory effects of miR-1298 on the proliferation and cell cycle progression of BC cells. Collectively, our results illustrated that miR-1298 played a suppressive role in regulating the phenotype of BC cells through directly repressing ADAM9, suggesting the potential application of miR-1298 in the therapy of BC.