Hsa_Circ_0007843 Acts as a mIR-518c-5p Sponge to Regulate the Migration and Invasion of Colon Cancer SW480 Cells.

Circular RNA (circRNA), a type of RNA that is widely expressed in mammalian cells, is considered to be essential in tumorigenesis. CircRNA can regulate target gene expression by interacting with the corresponding microRNA (miRNA). Our preliminary results showed that the expression levels of 1,817 circRNAs were significantly different in colon cancer tissue compared with paracancerous tissue, of which 1,236 were upregulated and 581 were downregulated. By using RT-PCR, we confirmed that the expression of hsa_circ_0007843, hsa_circ_0010575, hsa_circ_0007331, and hsa_circ_0001615 was significantly higher in colon cancer tissue than in normal colonic tissue; however, the expression levels of hsa_circ_0014879 and hsa_circRNA_401801 were not significantly different between normal and neoplastic colonic tissue. Among the circRNAs that were confirmed to be upregulated in colon cancer tissue, hsa_circ_0007843 was also found to be highly expressed in colon cancer SW480 cells. Overexpression of hsa_circ_0007843 promoted the invasion and migration of SW480 cells, whereas its downregulation suppressed their invasion and migration. Overexpression of hsa_circ_0007843 promoted tumor growth, whereas its downregulation inhibited tumor growth. We found that hsa_circ_0007843 interacted with miR-518c-5p and suppressed its expression, and miR-518c-5p interacted with matrix metallopeptidase 2 (MMP2) and promoted its expression and translation. Taken together, this study demonstrated that hsa_circ_0007843 acted as an miRNA sponge to regulate MMP2 expression by removing the inhibitory effect of miR-518c-5p on MMP2 gene translation, which further affected the invasive capability of SW480 cells.

The CircRNA-ACAP2/Hsa-miR-21-5p/ Tiam1 Regulatory Feedback Circuit Affects the Proliferation, Migration, and Invasion of Colon Cancer SW480 Cells.

BACKGROUND/AIMS: Circular RNAs (circRNAs), a type of RNA that is widely expressed in human cells, have essential roles in the development and progression of cancer. CircRNAs contain microRNA (miRNA) binding sites and can function as miRNA sponges to regulate gene expression by removing the inhibitory effect of an miRNA on its target gene. METHODS: We used the bioinformatics software TargetScan and miRanda to predict circRNA-miRNA and miRNAi-Mrna interactions. Rate of inhibiting of proliferation was measured using a WST-8 cell proliferation assay. Clone formation ability was assessed with a clone formation inhibition test. Cell invasion and migration capacity was evaluated by performing a Transwell assay. Relative gene expression was assessed using quantitative real-time polymerase chain reaction and relative protein expression levels were determined with western blotting. circRNA and miRNA interaction was confirmed by dual-luciferase reporter and RNA-pull down assays. RESULTS: In the present study, the miRNA hsa-miR-21-5p was a target of circRNA-ACAP2, and T lymphoma invasion and metastasis protein 1 (Tiam1) was identified as a target gene of hsa-miR-21-5p. CircRNA-ACAP2 and Tiam1 were shown to be highly expressed in colon cancer tissue and colon cancer SW480 cells, but miR-21-5p was expressed at a low level. SW480 cell proliferation was suppressed when the expression of circRNA-ACAP2 and Tiam1 was decreased and the expression of miR-21-5p was increased in vivo and in vitro. SW480 cell migration and invasion were also inhibited under the same circumstance. The circRNA-ACAP2 interaction regulated the expression of miR-21-5p, and miR-21-5p regulated the expression of Tiam1. Down-regulation of circRNA-ACAP2 promoted miR-21-5p expression, which further suppressed the transcription and translation of Tiam1. CONCLUSION: The present study shows that the circRNA-ACAP2/hsa-miR-21-5p/Tiam1 regulatory feedback circuit could affect the proliferation, migration, and invasion of colon cancer SW480 cells. This was probably due to the fact that circRNA-ACAP2 could act as a miRNA sponge to regulate Tiam1 expression by removing the inhibitory effect of miR-21-5p on Tiam1 expression. The results from this study have revealed new insights into the pathogenicity of colon cancer and may provide novel therapeutic targets for the treatment of colon cancer.

MiR-145 affected the circular RNA expression in prostate cancer LNCaP cells.

Recent evidence has demonstrated that circular RNAs (circRNAs) played crucial roles in fine-tuning the levels of gene expression by sequestering the corresponding microRNA (miRNAs). Their interaction with disease-associated miRNAs indicates that circRNAs are important for the development of disease, and miR-145 has been previously shown to have antitumor effect in prostate cancer. In the current study, we successfully established the miR-145-overexpressed prostate cancer LNCaP cells (LNCaP-miR-145) using lentiviral vectors. LNCaP cells expressing the empty vector (LNCaP-NC) were used as the negative control. The circRNA expression in LNCaP-miR-145 cells was detected by microarray analysis, and the miRNA targets of circRNAs were predicted using the bioinformatics software TargetScan and miRanda. Quantitative reverse transcription polymerase chain reaction was used to detect the expression levels of circRNAs in the prostate cancer tissue, nonmalignant tissue, LNCaP-miR-145 cells, and LNCaP-NC cells. The interaction of miRNA and circRNA was further confirmed by the dual-luciferase reporter assay. A total of 267 and 149 circRNAs were significantly up- and downregulated in LNCaP-miR-145 cells, respectively. hsa_circRNA_101981, hsa_circRNA_101996 and hsa_circRNA_09142 were the 3 circRNAs that interacted with hsa-miR-145-5p; hsa_circRNA_008068 and hsa_circRNA_406557 were the 2 circRNAs that interacted with hsa-miR-145-3p. Most of the circRNAs corresponded to the protein-coding exons. The expression levels of hsa_circRNA_101981, hsa_circRNA_00806, and hsa_circRNA_406557 were upregulated in the LNCaP-miR-145 cells, but downregulated in the prostate cancer tissue. In contrast, the expression levels of hsa_circRNA_101996 and hsa_circRNA_091420 were downregulated in the LNCaP-miR-145 cells, but upregulated in the prostate cancer tissue. Moreover, miR-145-5P might regulate the expression of hsa_circRNA_101981, hsa_circRNA_101996, and hsa_circRNA_09142, while miR-145-3P might regulate the expression of hsa_circRNA_008068 and hsa_circRNA_406557. Overexpression of miR-145 promoted the expression of hsa_circRNA_101981, hsa_circRNA_008068, and hsa_circRNA_406557 but suppressed the expressions of hsa_circRNA_101996 and hsa_circRNA_091420 in LNCaP cells. The results from the current study should give us a clue to clarify the tumor suppressive effect of miR-145.

Bioinformatic analysis and prediction of the function and regulatory network of long non-coding RNAs in hepatocellular carcinoma.

Computational analysis and bioinformatics have significantly advanced the ability of researchers to process and analyze biological data. Molecular data from human and model organisms may facilitate drug target validation and identification of biomarkers with increased predictive accuracy. The aim of the present study was to investigate the function of long non-coding RNAs (lncRNAs) in hepatocellular carcinoma (HCC) using online databases, and to predict their regulatory mechanism. HCC-associated lncRNAs, their downstream transcription factors and microRNAs (miRNAs/miRs), as well as the HCC-associated target genes, were identified using online databases. HCC-associated lncRNAs, including HOX antisense intergenic RNA (HOTAIR) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) were selected based on established databases of lncRNAs. The interaction between the HCC-associated lncRNAs and miRNAs (hsa-miR-1, hsa-miR-20a-5p) was predicted using starBase2.0. Signal transducer and activator of transcription 1, hepatocyte nuclear factor 4α (HNF4A), octamer-binding transcription factor 4, Nanog homeobox (NANOG), caudal type homeobox 2 (CDX2), DEAD-box helicase 5, brahma-related gene 1, MYC-associated factor X and MYC proto-oncogene, bHLH transcription factor have been identified as the transcription factors for HOTAIR and MALAT1 using ChIPBase. Additionally, CDX2, HNF4A, NANOG, ETS transcription factor, Jun proto-oncogene and forkhead box protein A1 were identified as the transcription factors for hsa-miR-1 and hsa-miR-20a-5p. CDX2, HNF4A and NANOG were the transcriptional factors in common between the lncRNAs and miRNAs. Cyclin D1, E2F transcription factor 1, epithelial growth factor receptor, MYC, MET proto-oncogene, receptor tyrosine kinase and vascular endothelial growth factor A were identified as target genes for the HCC progression, two of which were also the target genes of hsa-miR-1 and hsa-miR-20a-5p using the miRwalk and OncoDN. HCC databases. Additionally, these target genes may be involved in biological functions, including the regulation of cell growth, cell cycle progression and mitosis, and in disease progression, as demonstrated using DAVID clustering analysis. The present study aimed to predict a regulatory network of lncRNAs in HCC progression using bioinformatics analysis.

Gene-gene interaction network analysis of hepatocellular carcinoma using bioinformatic software.

Information processing tools and bioinformatics software have markedly advanced the ability of researchers to process and analyze biological data. Data from the genomes of humans and model organisms aid researchers to identify topics to study, which in turn improves predictive accuracy, facilitates the identification of relevant genes and simplifies the validation of laboratory data. The objective of the present study was to investigate the regulatory network constituted by long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and mRNA in hepatocellular carcinoma (HCC). Microarray data from HCC datasets were downloaded from The Cancer Genome Atlas database, and the Limma package in R was used to identify the differentially expressed genes (DEGs) between HCC and normal samples. Gene ontology enrichment analysis of DEGs was conducted using the Database for Annotation, Visualization, and Integrated Discovery. TargetScan, microcosm, miRanda, miRDB and PicTar were used to predict target genes. lncRNAs associated with HCC were probed using the lncRNASNP database, and a lncRNA-miRNA-mRNA regulatory network was visualized using Cytoscape. The present study identified 114 differentially expressed miRNAs and 2,239 differentially expressed mRNAs; of these, 725 were downregulated genes that were primarily involved in complement and coagulation cascades, fatty acid metabolism and butanoate metabolism, among others. The remaining 1,514 were upregulated genes principally involved in DNA replication, oocyte meiosis and homologous recombination, among others. Through the integrated analysis of associations between different types of RNAs and target gene prediction, the present study identified 203 miRNA-mRNA pairs, including 28 miRNAs and 170 mRNAs, and identified 348 lncRNA-miRNA pairs, containing 28 miRNAs. Therefore, owing to the association between lncRNAs-miRNAs-mRNAs, the present study screened out 2,721 regulatory associations. The data in the present study provide a comprehensive bioinformatic analysis of genes, functions and pathways that may be involved in the pathogenesis of HCC.

Analyzing the LncRNA, miRNA, and mRNA Regulatory Network in Prostate Cancer with Bioinformatics Software.

Information processing tools and bioinformatics software have significantly advanced researchers' ability to process and analyze biological data. Molecular data from human and model organism genomes help researchers identify topics for study, which, in turn, improves predictive accuracy, facilitates the identification of relevant genes, and simplifies the validation of laboratory data. The objective of this study was to explore the regulatory network constituted by long noncoding RNA (lncRNA), miRNA, and mRNA in prostate cancer (PCa). Microarray data of PCa were downloaded from The Cancer Genome Atlas database and DESeq package in R language were used to identify the differentially expressed genes (DEGs) between PCa and normal samples. Gene ontology enrichment analysis of DEGs was conducted using the Database for Annotation, Visualization, and Integrated Discovery. TargetScan, microcosm, miRanda, miRDB, and PicTar were used to predict target genes. LncRNA associated with PCa was exploited in the lncRNASNP database, and the LncRNA-miRNA-mRNA regulatory network was visualized using Cytoscape. Our study identified 57 differentially expressed miRNAs and 1252 differentially expressed mRNAs; of these, 691 were downregulated genes primarily involved in focal adhesion, vascular smooth muscle contraction, calcium signaling pathway, and so on. The remaining 561 were upregulated genes principally involved in systemic lupus erythematosus, progesterone-mediated oocyte maturation, oocyte meiosis, and so on. Through the integrated analysis of correlation and target gene prediction, our studies identified 1214 miRNA:mRNA pairs, including 52 miRNAs and 395 mRNAs, and screened out 455 lncRNA-miRNA pairs containing 52 miRNAs. Therefore, owing to the interrelationship of lncRNAs and miRNAs with mRNAs, our study screened out 19,075 regulatory relationships. Our data provide a comprehensive bioinformatics analysis of genes, functions, and pathways that may be involved in the pathogenesis of PCa.

Overexpression of Long Non-Coding RNA MEG3 Inhibits Proliferation of Hepatocellular Carcinoma Huh7 Cells via Negative Modulation of miRNA-664.

Evidence is accumulating that long non-coding RNAs (lncRNAs) are involved in human tumorigenesis and dysregulated in many cancers, including hepatocellular carcinoma (HCC). Because lncRNAs can regulate essential pathways that contribute to tumor initiation and progression with their tissue specificity, lncRNAs are valuable biomarkers and therapeutic targets. Maternally expressed gene 3 (MEG3) is a lncRNA overexpressed in HCC cells that inhibits HCC progression, however, the mechanism remains largely unknown. Recently, a novel regulatory mechanism has been proposed in which RNAs can cross-talk with each other via competing for shared microRNAs (miRNAs). The proposed competitive endogenous RNAs could mediate the bioavailability of miRNAs on their targets, thus imposing another level of post-transcriptional regulation. In the current study, we demonstrated that MEG3 is down-regulated in HCC tissues. MEG3 over-expression imposes another level of post-transcriptional regulation, whereas MEG3 overexpression increase the expression of the miR-664 target gene, ADH4, through competitive "sponging" miR-664. In addition, NF-κB may affect transcription of MEG3 by directly binding to the promoter region. Our data revealed that NF-κB may affect the transcript of MEG3. MEG3 overexpression inhibited the proliferation of HCC cells, at least in part by affecting miR-664mediated regulation of ADH4. Together, these results suggest that MEG3 is a suppressor of tumor which acts in part through "sponging" miR-664. J. Cell. Biochem. 118: 3713-3721, 2017. © 2017 Wiley Periodicals, Inc.

Snail-activated long non-coding RNA PCA3 up-regulates PRKD3 expression by miR-1261 sponging, thereby promotes invasion and migration of prostate cancer cells.

Rapidly accumulated evidence has shown that long non-coding RNA (lncRNAs) disregulation is involved in human tumorigenesis in many cancers, including prostate cancer (PCa). LncRNAs can regulate essential pathways that contribute to tumor initiation and progression with tissue specificity, which suggests that lncRNAs could be valuable biomarkers and therapeutic targets. Prostate cancer antigen 3 (PCA3), also known as differential display code 3 (DD3), is one such lncRNA that maps to chromosome 9q21-22. PCA3 expression is highly specific to PCa. In the present study, the level of PCA3 expression in prostate cancer cells was reduced by small interfering RNA (siRNA). Subsequently, the ability of LNCaP cell proliferation, invasion, and migration of PCa was compromised both in vivo and in vitro with the occurrence of cell autophagy. Recently, a novel regulatory mechanism has been proposed in which RNAs cross talk via competing with the shared microRNAs (miRNAs). In addition, lncRNAs can directly interact with RNA-binding proteins and then bind to the gene promoter region to further regulate gene expression. The proposed competitive endogenous RNAs mediate the bioavailability of miRNAs on their targets, thus imposing another level of post-transcriptional regulation. Here, we demonstrated that binding of Snail to the promoter region of PCA3 could activate the expression of PCA3. Down-regulation of PCA3 by silencing could increase the expression of the miRNA-1261, which then targeted at the PRKD3 gene (protein kinase D3) through competitive sponging. In summary, these results suggest that the transcription factor, Snail, activated the expression of lncRNA PCA3, which could inhibit the translation of PRKD3 protein via competitive miR-1261 sponging, and thus high expression of PRKD3 further promoted invasion and migration of prostate cancer.

Reciprocal regulation of PCGEM1 and miR-145 promote proliferation of LNCaP prostate cancer cells.

Prostate cancer gene expression marker 1 (PCGEM1) is a long non-coding RNA (lncRNA) overexpressed in prostate cancer (PCa) cells that promotes PCa initiation and progression, and protects against chemotherapy-induced apoptosis. The microRNA miR-145 functions as a tumor suppressor in PCa. We speculate that reciprocal regulation of PCGEM1 and miR-145 promote proliferation of LNCaP prostate cancer cells. To test this hypothesis, the interaction between PCGEM1 and miR-145 was examined using a luciferase reporter assay. Expression levels were selectively altered in LNCaP cells and noncancerous RWPE-1 prostate cells by transfection of miR-145 or small interfering RNA sequences against (siRNA) PCGEM1. Relative expression levels were detected by RT-PCR, tumor cell growth and early apoptosis by the MTT assay and flow cytometry, respectively, and tumor cell migration and invasion properties by transwell assays. The effect of siRNA PCGEM1 and miR-145 transfection on prostate cancer growth in vivo was examined in the (nu/nu) mouse model. PCGEM1 and miR-145 exhibited reciprocal regulation; downregulation of PCGEM1 expression in LNCaP cells increased expression of miR-145, while overexpression of miR-145 decreased PCGEM1 expression. Transfection of the miR-145 expression vector and siRNA PCGEM1 inhibited tumor cell proliferation, migration, and invasion, and induced early apoptosis both in vitro. In contrast, there was no effect on RWPE-1 cells. We demonstrate a reciprocal negative control relationship between PCGEM1 and miR-145 that regulates both LNCaP cell proliferation and nu/nu PCa tumor growth. The results also identify PCGEM1 and associated regulators as possible targets for PCa therapy.