Hsa_circ_0030586 promotes epithelial-mesenchymal transition in prostate cancer via PI3K-AKT signaling.

Circular RNAs (CircRNAs) gain importance as regulatory molecules in prostate cancer (PCa), but molecular mechanism of most circRNAs in pathogenesis of PCa remains to be studied. This study aimed to explore the role of hsa_circ_0030586 in PCa. Gene Expression Omnibus database (GSE77661) was used to screen out candidate circRNAs. Quantitative real-time PCR was used to verify the relative expressions of circRNAs, miRNAs, and genes in PCa cells. A CCK-8 assay was used to evaluate the cells' proliferation. Transwell and wound healing assay were used to determine the cells' migration and invasion. Western blotting and immunohistochemistry were used to detect the protein expression of PI3K/AKT signaling proteins and epithelial-mesenchymal transition (EMT) markers. Furthermore, a nude mice tumorigenesis experiment in vivo was conducted to determine the function of hsa_circ_0030586 on PCa. Our results showed that hsa_circ_0030586 is significantly upregulated in PCa cells (p < 0.05). Its circular structure was confirmed via agarose gel electrophoresis and Sanger sequencing. Interfering with hsa_circ_0030586 in PC3 cells inhibited cell proliferation, migration, and invasion and led to the significant upregulation of E-cadherin and the significant downregulation of p-AKT/AKT, IKKα, PIK3CB, and Twist (all p < 0.05). Conversely, the hsa_circ_003058 interference fragment combined with the transfection of a miR-145-3p inhibitor could reverse the above effects. In vivo tumorigenesis of the xenograft model confirmed that interfering with hsa_circ_0030586 suppressed tumor cell proliferation and inhibited PI3K-AKT signaling and EMT in PC3 cells. Hsa_circ_0030586 is significantly upregulated in PCa cells and may promote EMT via PI3K-AKT signaling.

CORO6 Promotes Cell Growth and Invasion of Clear Cell Renal Cell Carcinoma via Activation of WNT Signaling.

Renal cell carcinoma (RCC) constitutes the most lethal type of genitourinary cancer. Understanding of RCC tumor biology helps to identify novel targets and develop directed treatments for patients with this type of cancer. Analysis from both The Cancer Genome Atlas Kidney Renal Clear Cell Carcinoma dataset and our RCC samples demonstrated that the expression level of CORO6 was significantly higher in RCC patients than in normal kidney tissues, and its level was highly associated with tumor stage and grade. Importantly, CORO6 expression level was an independent predictor of tumor metastasis and overall survival in RCC patients. Our cell line data also confirmed that CORO6 knockdown could suppress RCC cell growth as well as cell migration and invasion. The depletion of CORO6 led to cell cycle arrest at the G0/G1 phase and caused cell apoptosis. Further, mechanistic dissection showed that CORO6 mediated RCC cell growth, and cell invasion relied on WNT signaling. Moreover, the in vivo data suggested that CORO6 knockdown indeed suppressed RCC tumor growth. Overall, our study defines the oncogenic role of CORO6 in RCC progression and provides a rationale for developing CORO6-targeted therapies for improved treatment of RCC patients.

Effects of NRP1 on angiogenesis and vascular maturity in endothelial cells are dependent on the expression of SEMA4D.

Angiogenesis and vascular maturation play important roles in tumorigenesis and tumor development. The expression of neuropilin 1 (NRP1) is closely associated with angiogenesis in tumors; however, the molecular mechanisms of action in angiogenesis and tumor maturation, as well as the potential clinical value of NRP1 remain unclear. The importance of NRP1 expression in tumor progression was determined using The Cancer Genome Atlas (TCGA) database analysis. Gain­ and loss­of­function experiments of NRP1 were performed in vascular endothelial cells (ECs) to investigate the functions in angiogenesis. CCK­8, flow cytometry, Transwell experiments and a series of in vitro experiments were used to detect cell functions. A combination of angiogenesis antibody arrays and RNA­Seq analyses were performed to reveal the proangiogenic mechanisms of action. The function of semaphorin 4D (SEMA4D) was also investigated separately. NRP1 mRNA levels were significantly increased in primary tumors compared with normal tissues based on TCGA data (P<0.01) and were associated with tumor development in patients. Gain­ and loss­of­function experiments highlighted the function of NRP1 in promoting EC proliferation, motility and capillary­like tube formation and in reducing apoptosis. NRP1 overexpression led to significantly decreased EC markers (PECAM­1, angiogenin, PIGF and MMP­9) expression levels and reduced the vascular maturity. MAPK7, TPM1, RRBP1, PTPRK, HSP90A, PRKD2, PFKFB3, RGS4 and SPARC were revealed to play important roles in this process. SEMA4D was revealed to be a key protein associated with NRP1 in ECs. These data indicated that NRP1­promoted angiogenesis may be induced at the cost of reducing maturity of the ECs. NRP1 may also be a therapeutic target for antiangiogenic strategies and a candidate prognostic marker for tumors.

Screening and identification of epithelial-to-mesenchymal transition-related circRNA and miRNA in prostate cancer.

Epithelial-to-mesenchymal transition (EMT) plays a vital role in the progression and metastasis of prostate cancer. However, the molecular mechanisms underlying prostate cancer metastasis are not fully demonstrated. In this study, EMT was induced by interferon-γ (IFN-γ) in PC-3M IE8 cells. High-throughput sequencing was used to screen the differentially expressed circular RNAs (circRNAs) and miRNAs in the cells with or without IFN-γ treatment. EMT-related circRNAs and miRNAs were further identified by quantitative real-time PCR (qPCR). In addition, the relationships among circRNAs, miRNAs, and mRNA were predicted. After cells were treated with IFN-γ, western blot analysis was conducted to detect the expression levels of EMT markers. E-cadherin expression levels were found to be downregulated, and Twist expression levels were found to be upregulated. Our results also found that IFN-γ promoted PC-3M IE8 cell migration and invasion, indicating that IFN-γ could induce EMT in PC-3M IE8 cells. Furthermore, high-throughput sequencing results revealed 827 upregulated and 1279 downregulated circRNAs and 39 upregulated and 2076 downregulated miRNAs in the IFN-γ group compared with the control group. KEGG analysis showed that both differentially expressed circRNAs and differentially expressed miRNAs were enriched in the MAPK signaling pathway related to EMT. Furthermore, the qPCR results revealed that the expression of hsa_circ_0001085, hsa_circ_0004916, hsa_circ_0001165, hsa-miR-196b-5p, and hsa-miR-187-3p in the IFN-γ group was consistent with the sequencing results. hsa_circ_0001165 and hsa_circ_0001085 were used to construct the network of circRNA-miRNA-mRNA. It was found that hsa_circ_0001165 may regulate TNF expression through hsa-miR-187-3p to induce EMT in prostate cancer cells. In addition, hsa_circ_0001085 may indirectly regulate the PI3K-Akt signaling and TGF-ß signaling pathways through hsa-miR-196b-5p and the MAPK signaling pathway through has-miR-451a, which played a regulatory role in prostate cancer cells in the EMT induction model. The results obtained in this study lay the foundation for future study.

Serum and tissue proteomic signatures of patients with hepatocellular carcinoma using 2­D gel electrophoresis.

Hepatocellular carcinoma (HCC) accounts for ~85% of primary liver cancer cases and is a leading cause of mortality worldwide. Effective early diagnosis is difficult for HCC; however, effective biomarkers may be beneficial for diagnosis. In the current study, serum samples, and HCC and adjacent tissue samples were obtained from patients with HCC for the detection of biomarkers using 2­D gel electrophoresis (2­DE) and matrix­assisted laser desorption/ionization­time of flight (TOF)/TOF mass spectrometry. The crude serum samples did not need to be prepared for removal of high abundance proteins. The mRNA expression levels of HCC­associated proteins were detected in tissues using reverse transcription­quantitative PCR. Statistical analysis and database matching were used to identify the differentially expressed proteins detected in the serum and tissue groups. Immunohistochemistry (IHC) was performed to detect the expression of significant proteins in HCC and adjacent tissues. The results revealed ~800 protein spots on a 2­DE gel that were detected in serum samples, and 1,200 spots were identified in the tissue samples. The protein and mRNA expression levels of oxysterol binding protein­like 11 (OSBPL11) in HCC serum and tissue samples were consistent. Pathway analysis demonstrated that members of the apolipoprotein family, particularly apolipoprotein E (APOE), and RAS family members were closely associated in HCC, either directly or via ferratin heavy polypeptide 1. IHC results demonstrated that the APOE protein serves an important role in liver cancer development. The lysis buffer used in the current study was effective for serum protein separation in 2­DE sample preparation. In addition, the present study revealed that downregulated OSBPL11 may be a potential indicator for HCC, and the apolipoprotein family, particularly APOE, and the RAS family may cooperatively serve an important role.