Circular RNA MYLK promotes tumour growth and metastasis via modulating miR‐513a‐5p/VEGFC signalling in renal cell carcinoma

Growing evidence indicates that circular RNAs (circRNAs) are promising biomarkers, as they play significant roles in the development of various cancers. The circular RNA MYLK (circMYLK) has been reported to be involved in the development of malignant tumours, including liver, prostate and bladder cancers. Nevertheless, the biological function of circMYLK in renal cell carcinoma (RCC) remains unclear. In this study, we observed that circMYLK is notably up‐regulated in RCC. Increased circMYLK expression led to a larger tumour size, distant metastasis and poor prognosis of RCC patients. Moreover, circMYLK silencing repressed RCC growth and metastasis in vitro and in vivo. Mechanistically, circMYLK can capture miR‐513a‐5p to facilitate VEGFC expression and further promote the tumorigenesis of RCC cells. In summary, our findings demonstrate that circMYLK has an oncogenic role in RCC growth and metastasis by modulating miR‐513a‐5p/VEGFC signalling. Thus, circMYLK has potential as a diagnostic biomarker and therapeutic target in the treatment of RCC.     

Overexpression of Circular RNA ciRS‐7 Abrogates the Tumor Suppressive Effect of miR‐7 on Gastric Cancer via PTEN/PI3K/AKT Signaling Pathway

Gastric cancer (GC) has one of the highest mortality rates of malignancies globally. Currently, ciRS‐7, a novel circular RNA, has emerged as a potential sponge for miR‐7. However, few studies on ciRS‐7 in GC have been performed. In this study, we investigated the clinical significance and function of ciRS‐7 in GC. First, the expression levels of ciRS‐7 in 102 primary GC tissues and the matched para‐carcinoma tissues were evaluated and the clinical relevance was confirmed in an independent validation cohort (n = 154). Second, the effects of ciRS‐7 on miR‐7, PTEN, and PI3K were evaluated. Finally, the function of ciRS‐7 in GC was analyzed with cell lines and nude mice. The expression of ciRS‐7 was significantly upregulated in GC tissues compared with the matched para‐carcinoma tissues (P = 0.0023), and the upregulation of ciRS‐7 was linked to poor survival in the testing (P = 0.0143) and validation cohort (P = 0.0061). Multivariate survival analysis revealed that ciRS‐7 was probably an independent risk factor of overall survival (P < 0.05). Furthermore, overexpression of ciRS‐7 blocked the miR‐7‐induced tumor suppression in MGC‐803 and HGC‐27 cells and led to a more aggressive oncogenic phenotype, via antagonizing miR‐7‐mediated PTEN/PI3K/AKT pathway. ciRS‐7 may act as a prospective prognostic biological marker and a promising therapeutic target for GC. J. Cell. Biochem. 119: 440–446, 2018. © 2017 Wiley Periodicals, Inc.     

Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer

Mounting evidence has illustrated the vital roles of long non‐coding RNAs (lncRNAs in gastric cancer (GC). Nevertheless, the majority of their roles and mechanisms in GC are still largely unknown. In this study, we investigate the roles of lncRNA SLC25A5‐AS1 on tumourigenesis and explore its potential mechanisms in GC. The results showed that the expressions of SLC25A5‐AS1 in GC were significantly lower than that of adjacent normal tissues, which were significantly associated with tumour size, TNM stage and lymph node metastasis. Moreover, SLC25A5‐AS1 could inhibit GC cell proliferation, induce G1/G1 cell cycle arrest and cell apoptosis in vitro, as well as GC growth in vivo. Dual‐luciferase reporter assay confirmed the direct interaction between SLC25A5‐AS1 and miR‐19a‐3p, rescue experiment showed that co‐transfection miR‐19a‐3p mimics and pcDNA‐SLC25A5‐AS1 could partially restore the ability of GC cell proliferation and the inhibition of cell apoptosis. The mechanism analyses further found that SLC25A5‐AS1 might act as a competing endogenous RNAs (ceRNA), which was involved in the derepression of PTEN expression, a target gene of miR‐19a‐3p, and regulate malignant phenotype via PI3K/AKT signalling pathway in GC. Taken together, this study indicated that SLC25A5‐AS1 was down‐regulated in GC and functioned as a suppressor in the progression of GC. Moreover, it could act as a ceRNA to regulate cellular behaviours via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway. Thus, SLC25A5‐AS1 might be served as a potential target for cancer therapeutics in GC.     

Inhibition of miR‐103a‐3p suppresses lipopolysaccharide‐induced sepsis and liver injury by regulating FBXW7 expression

Inflammation, apoptosis, and oxidative stress are involved in septic liver dysfunction. Herein, the role of miR‐103a‐3p/FBXW7 axis in lipopolysaccharides (LPS)‐induced septic liver injury was investigated in mice. Hematoxylin‐eosin staining was used to evaluate LPS‐induced liver injury. Quantitative real‐time polymerase chain reaction was performed to determine the expression of microRNA (miR) and messenger RNA, and western blot analysis was conducted to examine the protein levels. Dual‐luciferase reporter assay was used to confirm the binding between miR‐103a‐3p and FBXW7. Both annexin V‐fluoresceine isothiocyanate/propidium iodide staining and caspase‐3 activity were employed to determine cell apoptosis. First, miR‐103a‐3p was upregulated in the septic serum of mice and patients with sepsis, and miR‐103a‐3p was elevated in the septic liver of LPS‐induced mice. Then, interfering miR‐103a‐3p significantly decreased apoptosis by suppressing Bax expression and upregulating Bcl‐2 levels in LPS‐induced AML12 and LO2 cells, and septic liver of mice. Furthermore, inhibition of miR‐103a‐3p repressed LPS‐induced inflammation by downregulating the expression of tumor necrosis factor, interleukin 1β, and interleukin 6 in vitro and in vivo. Meanwhile, interfering miR‐103a‐3p obviously attenuated LPS‐induced overactivation of oxidation via promoting expression of antioxidative enzymes, including catalase, superoxide dismutase, and glutathione in vitro and in vivo. Moreover, FBXW7 was a target of miR‐103a‐3p, and overexpression of FBXW7 significantly ameliorated LPS‐induced septic liver injury in mice. Finally, knockdown of FBXW7 markedly reversed anti‐miR‐103a‐3p‐mediated suppression of septic liver injury in mice. In conclusion, interfering miR‐103a‐3p or overexpression of FBXW7 improved LPS‐induced septic liver injury by suppressing apoptosis, inflammation, and oxidative reaction.     

Long non‐coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR‐204‐5p and miR‐125a‐3p

A growing number of long non‐coding RNAs (lncRNAs) have been found to be involved in diverse biological processes such as cell cycle regulation, embryonic development, and cell differentiation. However, limited knowledge is available concerning the underlying mechanisms of lncRNA functions. In this study, we found down‐regulation of TCONS_00041960 during adipogenic and osteogenic differentiation of glucocorticoid‐treated bone marrow mesenchymal stem cells (BMSCs). Furthermore, up‐regulation of TCONS_00041960 promoted expression of osteogenic genes Runx2, osterix, and osteocalcin, and anti‐adipogenic gene glucocorticoid‐induced leucine zipper (GILZ). Conversely, expression of adipocyte‐specific markers was decreased in the presence of over‐expressed TCONS_00041960. Mechanistically, we determined that TCONS_00041960 as a competing endogenous RNA interacted with miR‐204‐5p and miR‐125a‐3p to regulate Runx2 and GILZ, respectively. Overall, we identified a new TCONS_00041960‐miR‐204‐5p/miR‐125a‐3p‐Runx2/GILZ axis involved in regulation of adipogenic and osteogenic differentiation of glucocorticoid‐treated BMSCs.     

Long non‐coding RNA deleted in lymphocytic leukaemia 1 promotes hepatocellular carcinoma progression by sponging miR‐133a to regulate IGF‐1R expression

Long non‐coding RNA (lncRNA) deleted in lymphocytic leukaemia 1 (DLEU1) was reported to be involved in the occurrence and development of multiple cancers. However, the exact expression, biological function and underlying mechanism of DLEU1 in hepatocellular carcinoma (HCC) remain unclear. In this study, real‐time quantitative polymerase chain reaction (qRT‐PCR) in HCC tissues and cell lines revealed that DLEU1 expression was up‐regulated, and the increased DLEU1 was closely associated with advanced tumour‐node‐metastasis stage, vascular metastasis and poor overall survival. Function experiments showed that knockdown of DLEU1 significantly inhibited HCC cell proliferation, colony formation, migration and invasion, and suppressed epithelial to mesenchymal transition (EMT) process via increasing the expression of E‐cadherin and decreasing the expression of N‐cadherin and Vimentin. Luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay demonstrated that DLEU1 could sponge miR‐133a. Moreover, miR‐133a inhibition significantly reversed the suppression effects of DLEU1 knockdown on HCC cells. Besides, we found that silenced DLEU1 significantly decreased insulin‐like growth factor 1 receptor (IGF‐1R) expression (a target of miR‐133a) and its downstream signal PI3K/AKT pathway in HCC cells, while miR‐133a inhibitor partially reversed this trend. Furthermore, DLEU1 knockdown impaired tumour growth in vivo by regulating miR‐133a/IGF‐1R axis. Collectively, these findings indicate that DLEU1 promoted HCC progression by sponging miR‐133a to regulate IGF‐1R expression. Deleted in lymphocytic leukaemia 1/miR‐133a/IGF‐1R axis may be a novel target for treatment of HCC.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

miR‐20a‐5p regulates pulmonary surfactant gene expression in alveolar type II cells

MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR‐20a‐5p had such regulatory functions on alveolar type II (AT‐II) cells. To accomplish this, miR‐20a‐5p–overexpressed and miR‐20a‐5p–inhibited adenoviral vectors were constructed and transfected into cultured AT‐II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR‐20a‐5p levels were verified by real‐time PCR. The CCK‐8 assay was used to compare the proliferation ability of AT‐II cells that had over‐ or underexpressed miR‐20a‐5p. The expression of surfactant‐associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real‐time PCR and Western blotting. In AT‐II cells, transfection resulted in over‐ or under‐regulation of miR‐20a‐5p. While overexpression of miR‐20a‐5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR‐20a‐5p regulation of PTEN. As a result, when miR‐20a‐5p was rendered overexpressed, PTEN was down‐regulated. By contrast, when miR‐20a‐5p was underexpressed, PTEN was up‐regulated. Neither overexpression nor underexpression of miR‐20a‐5p altered the cell proliferation. miR‐20a‐5p plays no role in proliferation of foetal AT‐II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.     

Clinical utility of plasma miR‐371a‐3p in germ cell tumors

Germ cell tumours predominantly of the testis ((T)GCTs) are remarkably chemotherapy sensitive. However, a small proportion of patients fail to be cured with cisplatin‐based combination chemotherapy. miR‐371a‐3p is a new liquid biopsy biomarker for (T)GCTs. The aim of this study was to evaluate clinical utility of plasma miR‐371a‐3p level in patients starting systemic chemotherapy. Patients were included before the first cycle (N = 180) and second cycle (N = 101) of systemic first line chemotherapy, treated between July 2010 and May 2017. Plasma miR‐371a‐3p levels were measured with the ampTSmiR test and compared to disease characteristics and outcome. Pretreatment plasma miR‐371a‐3p levels were increased in 51.7% of cases and associated with number of metastatic sites, presence of lung, retroperitoneal, and mediastinal lymph node metastases, S – stage, IGCCCG risk group, and response to therapy. Patients with a negative pretreatment plasma level had better progression‐free survival (PFS) and overall survival (OS) compared to patients being positive for miR‐371a‐3p (hazard ratio [HR] = 0.26, 95% confidence interval [CI] 0.09‐0.71, P = 0.02 for PFS and HR = 0.21, 95% CI 0.07‐0.67, P = 0.03 for OS, respectively). Patients negative for miR‐371a‐3p in both samples had a superior PFS (HR = 0.10, 95% CI 0.01‐21.49, P = 0.02) and OS (HR = 0.08, 95% CI 0.01‐27.81, P = 0.008) compared to patients with miR‐371a‐3p positive in both samples (multivariate analyses were non‐significant). In total 68% of the patients were S0. This study demonstrates clinical value of plasma miR‐371a‐3p level in chemotherapy naïve (T)GCT patients starting first line of chemotherapy to predict prognosis.