The study on circRNA profiling uncovers the regulatory function of the hsa_circ_0059665/miR-602 pathway in breast cancer.

Abnormal expression of circRNAs has been observed in different types of carcinomas, and they play significant roles in the biology of these cancers. Nevertheless, the clinical relevance and functional mechanisms of the majority of circRNAs implicated in breast cancer progression remain unclear. The primary objective of our investigation is to uncover new circRNAs in breast cancer and elucidate the underlying mechanisms by which they exert their effects. The circRNA expression profile data for breast cancer and RNA-sequencing data were acquired from distinct public databases. Differentially expressed circRNAs and mRNA were identified through fold change filtering. The establishment of the competing endogenous RNAs (ceRNAs) network relied on the interplay between circular RNAs, miRNAs, and mRNAs. The hub genes were identified from the protein-protein interaction (PPI) regulatory network using the CytoHubba plugin in Cytoscape. Moreover, the expression levels and prognostic value of these hub genes in the PPI network were assessed using the GEPIA and Kaplan-Meier plotter databases. Fluorescence in situ hybridization (FISH) was used to identified the expression and intracellular localization of hsa_circ_0059665 by using the tissue microarray. Transwell analysis and CCK-8 analysis were performed to assess the invasion, migration, and proliferation abilities of breast cancer cells. Additionally, we investigated the interactions between hsa_circ_0059665 and miR-602 through various methods, including FISH, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assay. Rescue experiments were conducted to determine the potential regulatory role of hsa_circ_0059665 in breast cancer progression. A total of 252 differentially expressed circRNAs were identified. Among them, 246 circRNAs were up-regulated, while 6 circRNAs were down-regulated. Based on prediction and screening of circRNA-miRNA and miRNA-mRNA binding sites, we constructed a network consisting of circRNA-miRNA-mRNA interactions. In addition, we constructed a Protein-Protein Interaction (PPI) network and identified six hub genes. Moreover, the expression levels of these six hub genes in breast cancer tissues were found to be significantly lower. Furthermore, the survival analysis results revealed a significant correlation between low expression levels of KIT, FGF2, NTRK2, CAV1, LEP and poorer prognosis in breast cancer patients. The FISH experiment results indicated that hsa_circ_0059665 exhibits significant downregulation in breast cancer, and its decreased expression is linked to poor prognosis in breast cancer patients. Functional in vitro experiments revealed that overexpression of hsa_circ_0059665 can inhibit proliferation, migration and invasion abilities of breast cancer cells. Further molecular mechanism studies showed that hsa_circ_0059665 exerts its anticancer gene role by acting as a molecular sponge for miR-602. In our study, we constructed and analyzed a circRNA-related ceRNA regulatory network and found that hsa_circ_0059665 can act as a sponge for miR-602 and inhibit the proliferation, invasion and migration of breast cancer cells.

Wu Z ，Wu M ，Jiang X ，Shang F ，Li S ，Mi Y ，Geng C ，Tian Y ，Li Z ，Zhao Z ... -  《Scientific Reports》

The investigation of circRNA profiling reveals the regulatory role of the hsa_circ_0000375/miR-7706 pathway in breast cancer.

Circular RNAs (circRNAs) take an effect on tumorigenesis and progression. However, circRNAs have not been systematically identified in breast cancer (BC) as crucial regulators in multitudinous biological processes. This study is conducted to explore novel circRNAs in BC and the corresponding mechanisms of their action. The circRNA expression profile and RNA-sequencing data about BC were respectively downloaded from public database. Differentially expressed circRNAs, miRNAs, and mRNAs were identified by fold change filtering. The competing endogenous RNAs (ceRNAs) network was established based on the relationship between circular RNAs, miRNAs and mRNAs. GO and KEGG enrichment analysis of the overlapped genes were carried out to predict the potential functions and mechanisms of circRNAs in BC. The CytoHubba plugin in Cytoscape was applied to identify the hub genes from the PPI regulatory network. Kaplan-Meier plotter was used to perform survival analysis of these hub genes further. Real-time PCR was performed to test the expression of circRNA in BC tissues. Cell function studies including transwell analysis and CCK-8 analysis were used to investigate circRNAs' biological functions. A total of seven circRNAs exhibiting differential expression were identified in this study. After the intersection between the predicted target miRNA and the down-regulated differential miRNAs (DEmiRNAs), circRNA-miRNA interactions involving 3 circRNAs and 4 miRNAs were identified. Venn diagram was utilized to intersect the predicted target genes of the 4 miRNAs and the down-regulated differential genes in BC, and 149 overlapped genes were screened out ulteriorly. Additionally, we built a protein-protein interaction (PPI) network and selected six hub genes. Moreover, the survival data of BC patients suggested that low expression of ADIPOQ, LPL and LEP were significantly correlated with poor prognosis. Results from real-time PCR indicated that hsa_circ_0000375 was significantly down-regulated in breast cancer tissues. Functional in vitro experiments showed that over-expression of hsa_circ_0000375 can restrain proliferation, migration and invasion abilities of breast cancer cells. Further verification indicated that hsa_circ_0000375 exerted its anti-oncogene effect via sponge of miR-7706. This study constructed and analyzed a circRNA-associated ceRNA regulatory network and uncovered that hsa_circ_0000375 exerted its anti-oncogene effect via sponge of miR-7706.

Wang H ，Liu F ，Xue J ，Liu Y ，Gao W ，Yang S ，Mi Y ，Zhang X ，Gao S ，Geng C ... -  《-》

Identification of epithelial-mesenchymal transition-related circRNA-miRNA-mRNA ceRNA regulatory network in breast cancer.

Circular RNAs (circRNAs) have attracted lots of attention in tumorigenesis and progression. However, circRNAs as crucial regulators in epithelial-mesenchymal transition have not been systematically identified in breast cancer. The purpose of our research was to investigate the circRNA network associated with epithelial-mesenchymal transition in breast cancer. Expression profiling data of circRNAs were identified by circRNA microarray in transfected ZEB1 and control breast cancer cells. The differentially expressed circRNAs, miRNAs, and mRNAs were determined via fold change filtering. The competing endogenous RNAs (ceRNAs) network was established on the foundation of the relationship between circular RNAs, miRNAs and mRNAs. The CytoHubba was used to determine the hub genes from the protein-protein interaction (PPI) regulatory network. The GEPIA database was used to observe the expression of the hub genes mRNA between breast cancer tissues and normal tissues. The HPA database was applied to investigate the expression of six hub genes at the protein level. Morever, we further used Kaplan-Meier plotter to perform survival analysis of these hub genes. The top three up-regulated differential expressed circRNAs were identified by circRNA microarray. Following the Real-time PCR validation of the three circRNAs, two circRNAs (hsa_circRNA_002082 and hsa_circRNA_400031) were selected for further analysis. After the predicted target miRNA, ten circRNA-miRNA interactions including two circRNAs and ten miRNAs were determined. Furthermore, the Venn diagram was used to intersect the predicted target genes and the differentially expressed genes, and screened 174 overlapped genes. Subsequently, we constructed a PPI network, and selecting six hub genes, containing KIF4A, CENPF, OIP5, ZWINT, DEPDC1, BUB1B. The mRNA expression levels of the six hub genes were obviously up-regulated in breast cancer. The protein expression levels of KIF4A, CENPF, OIP5, and DEPDC1 were significantly increased in breast cancer tissues. Moreover, the survival analysis results revealed that high expression of the six hub genes were obviously correlated with poor prognosis of breast cancer patients. Our study constructed and analyzed a circRNA-associated ceRNA regulatory network and discovered that hsa_circRNA_002082 and hsa_circRNA_400031 may mechanism as ceRNAs to serve key roles in breast cancer epithelial-mesenchymal transition.

Sang M ，Wu M ，Meng L ，Zheng Y ，Gu L ，Liu F ，Sang M ... -  《-》

Construct a circRNA/miRNA/mRNA regulatory network to explore potential pathogenesis and therapy options of clear cell renal cell carcinoma.

Bai S ，Wu Y ，Yan Y ，Shao S ，Zhang J ，Liu J ，Hui B ，Liu R ，Ma H ，Zhang X ，Ren J ... -  《Scientific Reports》

Identification of potentially functional circular RNAs hsa_circ_0070934 and hsa_circ_0004315 as prognostic factors of hepatocellular carcinoma by integrated bioinformatics analysis.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide, which has a high mortality rate and poor treatment outcomes with yet unknown molecular basis. It seems that gene expression plays a pivotal role in the pathogenesis of the disease. Circular RNAs (circRNAs) can interact with microRNAs (miRNAs) to regulate gene expression in various malignancies by acting as competitive endogenous RNAs (ceRNAs). However, the potential pathogenesis roles of the ceRNA network among circRNA/miRNA/mRNA in HCC are unclear. In this study, first, the HCC circRNA expression data were obtained from three Gene Expression Omnibus microarray datasets (GSE164803, GSE94508, GSE97332), and the differentially expressed circRNAs (DECs) were identified using R limma package. Also, the liver hepatocellular carcinoma (LIHC) miRNA and mRNA sequence data were retrieved from TCGA and differentially expressed miRNAs (DEMIs) and mRNAs (DEGs) were determined using the R DESeq2 package. Second, CSCD website was used to uncover the binding sites of miRNAs on DECs. The DECs' potential target miRNAs were revealed by conducting an intersection between predicted miRNAs from CSCD and downregulated DEMIs. Third, candidate genes were uncovered by intersecting targeted genes predicted by miRWalk and targetscan online tools with upregulated DEGs. The ceRNA network was then built using the Cytoscape software. The functional enrichment and the overall survival time of these potential targeted genes were analyzed, and a PPI network was constructed in the STRING database. Network visualization was performed by Cytoscape, and ten hub genes were detected using the CytoHubba plugin tool. Four DECs (hsa_circ_0000520, hsa_circ_0008616, hsa_circ_0070934, hsa_circ_0004315) were obtained and six miRNAs (hsa-miR-542-5p, hsa-miR-326, hsa-miR-511-5p, hsa-miR-195-5p, hsa-miR-214-3p, and hsa-miR-424-5p) which are regulated by the above DECs were identified. Then 543 overlapped genes regulated by six miRNAs mentioned above were predicted. Functional enrichment analysis showed that these genes are mostly associated with regulatory pathways in cancer. Ten hub genes (TTK, AURKB, KIF20A, KIF23, CEP55, CDC6, DTL, NCAPG, CENPF, PLK4) have been screened from the PPI network of the 204 survival-related genes. KIF20A, NCAPG, TTK, PLK4, and CDC6 were selected for the highest significance p-values. At the end, a circRNA-miRNA-mRNA regulatory axis was established for five final selected hub genes. This study implies the potential pathogenesis of the obtained network and proposes that the two DECs (has_circ_0070934 and has_circ_0004315) may be important prognostic markers for HCC.

Morovat P ，Morovat S ，Ashrafi AM ，Teimourian S ... -  《Scientific Reports》