LncRNA BDNF‐AS inhibits proliferation,migration, invasion and EMT in oesophageal cancer cells by targeting miR‐214

This study was aimed at exploring the effect of lncRNA BDNF‐AS on cell proliferation, migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of oesophageal cancer (EC) cells. The expression of BDNF‐AS and miR‐214 in tissue samples and cells was measured by qRT‐PCR. The targeted relationship between BDNF‐AS and miR‐214 was analysed by dual‐luciferase reporter assay. After cell transfection, the cell proliferation activity was assessed by MTS method, while the migrating and invading abilities were evaluated by transwell assay. LncRNA BDNF‐AS was remarkably down‐regulated, while miR‐214 was up‐regulated in EC tissues and cells in comparison with normal tissues and cells. Overexpression of BDNF‐AS significantly inhibited the abilities of cell proliferation, migration and invasion as well as the EMT processes of EC cells. The bioinformatics analysis and luciferase assay indicated that BDNF‐AS could be directly bound by miR‐214. Furthermore, overexpression of miR‐214 and BDNF‐AS exerted suppressive influence on EC cell multiplication, migration, invasion and EMT processes. LncRNA BDNF‐AS restrained cell proliferation, migration, invasion and EMT processes in EC cells by targeting miR‐214.     

Up‐regulation of ZFAS1 indicates dismal prognosis for cholangiocarcinoma and promotes proliferation and metastasis by modulating USF1 via miR‐296‐5p

LncRNAs has been demonstrated to modulate neoplastic development by modulating downstream miRNAs and functional genes. In this study, we aimed to detect the interaction among lncRNA ZFAS1 miR‐296‐5p and USF1. We explored the proliferation, migration and invasion of cholangiocarcinoma. The differentially expressed ZFAS1 was discovered in both tissues and cell lines by qRT‐PCR. The targeting relationship between miR‐296‐5p and ZFAS1 or USF1 was validated by dual‐luciferase assay. The impact of ZFAS1 on CCA cell proliferation was observed by CCK‐8 assay. The protein expression of USF1 was determined by Western blot. The effects of ZFAS1, miR‐296‐5p and USF1 on tumour growth were further confirmed using xenograft model. LncRNA ZFAS1 expression was relatively up‐regulated in tumour tissues and cells while miR‐296‐5p was significantly down‐regulated. Knockdown of ZFAS1 significantly suppressed tumour proliferation, migration, invasion and USF1 expression. Overexpressed miR‐296‐5p suppressed cell proliferation and metastasis. Knockdown of USF1 inhibited cell proliferation and metastasis and xenograft tumour growth. In conclusion, ZFAS1 might promote cholangiocarcinoma proliferation and metastasis by modulating USF1 via miR‐296‐5p.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.     

Hypoxia‐induced miR‐3677‐3p promotes the proliferation,migration and invasion of hepatocellular carcinoma cells by suppressing SIRT5

Hepatocellular carcinoma (HCC), with life‐threatening malignant behaviours, often develops distant metastases and is the fourth most common primary cancer in the world, having taken millions of lives in Asian countries such as China. The novel miR‐3677‐3p is involved in a high‐expression‐related poor prognosis in HCC tissues and cell lines, indicating oncogenesis functions in vitro and in vivo. Initially, we confirmed the inhibition of proliferation, migration and invasion in miR‐3677‐3p knock‐down MHCC‐97H and SMMC‐7721 cell lines, which are well known for their high degree of invasiveness. Then, we reversed the functional experiments in the low‐miR‐3677‐3p‐expression Hep3B cell line via overexpressing miR‐3677‐3p. In nude mice xenograft and lung metastasis assays, we found suppressor behaviours, smaller nodules and low density of organ spread, after injection of cells transfected with shRNA‐miR‐3677‐3p. A combination of databases (Starbase, TargetScan and MiRgator) illustrated miR‐3677‐3p targets, and it was shown to suppress the expression of SIRT5 in a dual‐luciferase reporter system. To clarify the conclusions of previous ambiguous research, we up‐regulated SIRT5 in Hep3B cells, and rescue tests were established for confirmation that miR‐3677‐3p suppresses SIRT5 to enhance the migration and invasion of HCC. Interestingly, we discovered hypoxia‐induced miR‐3677‐3p up‐regulation benefited HCC malignancy and invasiveness. In conclusion, the overexpression of miR‐3677‐3p mediated SIRT5 inhibition, which could increase proliferation, migration and invasion of HCC in hypoxic microenvironments.     

Overexpression of geranylgeranyl diphosphate synthase contributes to tumour metastasis and correlates with poor prognosis of lung adenocarcinoma

This study aimed to evaluate the biological role of geranylgeranyl diphosphate synthase (GGPPS) in the progression of lung adenocarcinoma. GGPPS expression was detected in lung adenocarcinoma tissues by qRT‐PCR, tissue microarray (TMA) and western blotting. The relationships between GGPPS expression and the clinicopathological characteristics and prognosis of lung adenocarcinoma patients were assessed. GGPPS was down‐regulated in SPCA‐1, PC9 and A549 cells using siRNA and up‐regulated in A549 cells using an adenoviral vector. The biological roles of GGPPS in cell proliferation, apoptosis, migration and invasion were determined by MTT and colony formation assays, flow cytometry, and transwell and wound‐healing assays, respectively. In addition, the regulatory roles of GGPPS on the expression of several epithelial‐mesenchymal transition (EMT) markers were determined. Furthermore, the Rac1/Cdc42 prenylation was detected after knockdown of GGPPS in SPCA‐1 and PC9 cells. GGPPS expression was significantly increased in lung adenocarcinoma tissues compared to that in adjacent normal tissues. Overexpression of GGPPS was correlated with large tumours, high TNM stage, lymph node metastasis and poor prognosis in patients. Knockdown of GGPPS inhibited the migration and invasion of lung adenocarcinoma cells, but did not affect cell proliferation and apoptosis. Meanwhile, GGPPS inhibition significantly increased the expression of E‐cadherin and reduced the expression of N‐cadherin and vimentin in lung adenocarcinoma cells. In addition, the Rac1/Cdc42 geranylgeranylation was reduced by GGPPS knockdown. Overexpression of GGPPS correlates with poor prognosis of lung adenocarcinoma and contributes to metastasis through regulating EMT.     

HEF1 promotes epithelial mesenchymal transition and bone invasion in prostate cancer under the regulation of microRNA‐145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bones, and it's crucial to understand the mechanism of tumor progression to metastasis in order to develop therapies that may reduce the morbidity and mortality of PCa patients. Although we had identified that microRNA(miR)‐145 could repress bone metastasis of PCa via regulating epithelial–mesenchymal transition (EMT) in previous study, it is still unknown how miR‐145 regulated EMT. In the present study, we constructed a luciferase reporter system and identified HEF1 as a direct target of miR‐145. More importantly, HEF1 was shown to promote migration, invasion and EMT of PC‐3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. And HEF1 was also shown to partially mediate miR‐145 suppression of EMT and invasion. Furthermore, inhibition of HEF1 repressed bone invasion of PC‐3 cells in vivo. Expression of HEF1 was negatively correlated with miR‐145 in primary PCa and bone metastatic specimens, but HEF1 was higher in samples which were more likely to commit to bone metastasis or those with higher free prostate‐specific antigen (fPSA) levels and Gleason scores. Taken together, these findings indicate that HEF1 promotes EMT and bone invasion in prostate cancer by directly targeted by miR‐145, and miR‐145 suppresses EMT and invasion, at least in part, through repressing HEF1. J. Cell. Biochem. 114: 1606–1615, 2013. © 2013 Wiley Periodicals, Inc.     

MiR‐671‐5p plays a promising role in restraining osteosarcoma cell characteristics through targeting TUFT1

The aim of our study was to explore the roles of miR‐671‐5p in mediating biological processes of osteosarcoma (OS) cells and clinical implications. On the basis of the OS samples acquired from the GEO database, the expression difference and overall survival analyses of miR‐671‐5p and TUFT1 were determined. The expression of MiR‐671‐5p was verified using OS cell lines. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, wound‐healing, and Transwell assays were respectively carried out to probe whether miR‐671‐5p regulated OS cell vitality, migration, and invasion. The expression of miR‐671‐5p was downregulated in OS tissues and cell lines. High expression of MiR‐671‐5p blocked OS cell growth, migration, and invasion. TUFT1 was predicted and validated as the target of miR‐671‐5p in OS cells using in silico analysis and luciferase reporter assays. Forced expression of TUFT1 reversed the suppressive influence of miR‐671‐5p on cell viability, migration, and invasion of OS cells. Moreover, the low expression of miR‐671‐5p and the high expression of TUFT1 led to poor prognosis. Taken together, targeting miR‐671‐5p/TUFT1 may be a promising strategy for treating OS.     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.     

Retracted: microRNA-539 functions as a tumor suppressor in papillary thyroid carcinoma via the transforming growth factor β1/Smads signaling pathway by targeting secretory leukocyte protease inhibitor

Papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancy, with growing incidence every year. microRNAs (miRs) are known to regulate the physiological and pathological processes of cancers, such as proliferation, migration, invasion, survival, and epithelial-mesenchymal transition (EMT). Herein, this study aimed to investigate the effect of miR-539 on cell proliferation, apoptosis, and EMT by targeting secretory leukocyte protease inhibitor (SLPI) via the transforming growth factor β1 (TGF-β1)/Smads signaling pathway in PTC. First, PTC-related differentially expressed genes and regulatory miR were screened using bioinformatics analysis, dual luciferase reporter gene assay, and ribonucleoprotein immunoprecipitation, which identified the SLPI gene and the regulatory miR-539 for this study. We identified SLPI as a highly expressed gene in PTC tissues, and SLPI was targeted and negatively regulated by miR-539. Then, we introduced a series of miR-539 mimics, miR-539 inhibitors, and small interfering RNA against SLPI plasmids into CGTHW-3 cells to examine the effects of miR-539 and SLPI on the expression of TGF-β1/Smads signaling pathway-, EMT-, and apoptosis-related factors, as well as cell proliferation, migration, invasion, and apoptosis. The obtained results indicated that CGTHW-3 cells treated with silenced SLPI or overexpressed miR-539 suppressed the cell proliferation, migration, invasion abilities, and resistance to apoptosis of PTC cells, corresponding to increased expression of Bcl-2-associated X protein, TGF-β1, Sekelsky mothers against dpp 4, and epithelial cadherin, and decreased B cell lymphoma 2, Vimentin, and N-cadherin. Altogether, we concluded that overexpressed miR-539 could inhibit the PTC cell proliferation and promote apoptosis and EMT by targeting SPLI via activation of the TGF-β1/Smads signaling pathway.     

LncRNA MEG3 inhibits the progression of prostate cancer by modulating miR‐9‐5p/QKI‐5 axis

This study was designed to detecting the influences of lncRNA MEG3 in prostate cancer. Aberrant lncRNAs expression profiles of prostate cancer were screened by microarray analysis. The qRT‐PCR and Western blot were employed to investigating the expression levels of lncRNA MEG3, miR‐9‐5p and QKI‐5. The luciferase reporter assay was utilized to testifying the interactions relationship among these molecules. Applying CCK‐8 assay, wound healing assay, transwell assay and flow cytometry in turn, the cell proliferation, migration and invasion abilities as well as apoptosis were measured respectively. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer tissues and cells and could inhibit the expression of miR‐9‐5p, whereas miR‐9‐5p down‐regulated QKI‐5 expression. Overexpressed MEG3 and QKI‐5 could decrease the abilities of proliferation, migration and invasion in prostate cancer cells effectively and increased the apoptosis rate. On the contrary, miR‐9‐5p mimics presented an opposite tendency in prostate cancer cells. Furthermore, MEG3 inhibited tumour growth and up‐regulated expression of QKI‐5 in vivo. LncRNA MEG3 was a down‐regulated lncRNA in prostate cancer and impacted the abilities of cell proliferation, migration and invasion, and cell apoptosis rate, this regulation relied on regulating miR‐9‐5p and its targeting gene QKI‐5.     

MiR‐552 promotes the proliferation,migration and EMT of hepatocellular carcinoma cells by inhibiting AJAP1 expression

Our goal was to explore the function of miR‐552 and its potential target AJAP1 in hepatocellular carcinoma (HCC) oncogenesis and progression. In this study, bioinformatics analysis was performed to detect abnormally expressed miRNAs. The relationship between miR‐552 and AJAP1 was validated using luciferase reporter assays. RT‐qPCR and Western blot assays were applied to explore the expression level of miR‐552, AJAP1 and epithelial‐mesenchymal transition (EMT) markers. HCC cell proliferation was examined using CCK8 assays, while migration and invasion were investigated using Transwell assays. Nude mouse tumourigenesis models were established to facilitate observation of HCC progression in vivo. Finally, prognostic analysis was performed to discover how the prognosis of HCC patients correlated with miR‐552 and AJAP1 expression. MiR‐552 overexpression in HCC cells promoted HCC cell migration, invasion and EMT by targeting/suppressing AJAP1. Poorer prognosis appeared in HCC patients with higher miR‐552 expression or lower AJAP1 levels. Our findings suggested that miR‐552 promotes HCC oncogenesis and progression by inhibiting AJAP1 expression.     

Downregulation of lncRNA HCP5 has inhibitory effects on gastric cancer cells by regulating DDX21 expression

LncRNA HCP5 has been confirmed to play crucial roles in many types of cancers. However, the role of lncRNA HCP5 in regulating the occurrence and development of gastric cancer (GC) remains unknown. In the current study, we aimed to investigate the precise effects of lncRNA HCP5 on cell proliferation, migration and invasion and molecular mechanisms in gastric cancer. Using RT-qPCR analysis, we found that lncRNA HCP5 was differentially expressed in GC cell lines. CCK-8, wound healing and transwell assay indicated that the proliferation, migration and invasion of gastric cancer cells were inhibited by downregulation of lncRNA HCP5 and lncRNA HCP5 overexpression exhibited the opposite effects in gastric cancer cells. Mechanistically, RNA binding protein immunoprecipitation and dual luciferase reporter assay confirmed the interaction between lncRNA HCP5 and DDX21. The effects of lncRNA HCP5 overexpression the proliferation, migration and invasion of GC cells were partly rescued by DDX21 silencing. Taken together, downregulation of lncRNA HCP5 exerted inhibitory effects on GC cell proliferation, migration and invasion through modulation of DDX21 expression, demonstrating the function of lncRNA HCP5 and DDX21 in GC progression.     

Shikonin suppresses progression and epithelial–mesenchymal transition in hepatocellular carcinoma (HCC) cells by modulating miR‐106b/SMAD7/TGF‐β signaling pathway

Shikonin is a natural naphthoquinone component with antioxidant and anti‐tumor function and has been used for hepatocellular carcinoma (HCC) treatment. According to the previous study, many herbs can regulate cancer cell progression by targeting specific microRNA (miRNA) (Liu, 2016). However, the underlying pathological mechanism of shikonin in HCC therapy is still unclear. The detection of cell growth and death rate were performed by hemacytometry and trypan blue staining, respectively. The expression of miR‐106b and SMAD7 messenger RNA (mRNA) in HCC cells was evaluated by quantitative real‐time polymerase chain reaction. Cell proliferation, apoptosis, and migration ability were measured by cell counting kit‐8 (CCK‐8), flow cytometry, and transwell assay. The expression of proteins E‐cadherin, N‐cadherin, vimentin, SMAD7, TGF‐β1, p‐SMAD3, SMAD3, and GAPDH was examined by western blot. The interaction between SMAD7 and miR‐106b was assessed by luciferase reporter system. Shikonin inhibited Huh7 and HepG2 cell growth in a dose‐dependent manner while induced cell death in a time‐dependent manner. In addition, the expression of miR‐106b was reduced after shikonin treatment. Moreover, miR‐106b attenuated the suppressive effects of shikonin on HCC cell migration and epithelial–mesenchymal transition (EMT). SMAD7 was predicted as a target of miR‐106b and the prediction was confirmed by luciferase reporter system. Additionally, we observed that SMAD7 reversed the promotive effects of miR‐106b on HCC cell progression and EMT. The subsequent western blot assay revealed that shikonin could modulate SMAD7/TGF‐β signaling pathway by targeting miR‐106b. In conclusion, Shikonin suppresses cell progression and EMT and accelerates cell death of HCC cells via modulating miR‐106b/SMAD7/TGF‐β signaling pathway, suggesting shikonin could be an effective agent for HCC treatment.     

MiR-517a-3p accelerates lung cancer cell proliferation and invasion through inhibiting FOXJ3 expression

Aims

Aberrant expression of microRNAs (miRNAs) results in alterations of various biological processes (e.g., cell cycle, cell differentiation, and apoptosis) and cell transformation. Altered miRNAs expression was associated with lung carcinogenesis and tumor progression. This study aimed to investigate the function and underlying molecular events of miR-517a-3p on regulation of lung cancer cell proliferation and invasion.

Main methods

Transfected miR-517a-3p mimics or inhibitors into 95D and 95C cells respectively, the effects of miR-517a-3p on lung cancer cell proliferation, migration, and invasion were detected. Bioinformatics software forecasted potential target genes of miR-517a-3p and dual luciferase reporter gene system and western blot verified whether miR-517a-3p regulates FOXJ3 expression directly.

Key findings

MiR-517a-3p was differentially expressed in lung cancer 95D and 95C cell lines that have different metastatic potential. Manipulation of miR-517a-3p expression changed lung cancer cell proliferation, migration and invasion capacity. MiR-517a-3p directly regulated FOXJ3 expression by binding to FOXJ3 promoter.

Significance

This study demonstrated that miR-517a-3p promoted lung cancer cell proliferation and invasion by targeting of FOXJ3 expression.     

MircoRNA‐1275 promotes proliferation,invasion and migration of glioma cells via SERPINE1

This study was designed to explore the relationship between miR‐1275 and SERPINE1 and its effects on glioma cell proliferation, migration, invasion and apoptosis. Differentially expressed miRNAs and mRNAs in glioma tissues were screened out by bioinformatic analysis. Dual‐luciferase reporter gene assay was used to validate the targeted relationship between miR‐1275 and SERPINE1. qRT‐PCR was used to detect the expression of miR‐1275 and SERPINE1 in glioma tissues. The expressions of SERPINE1 and p53 pathway‐related proteins in glioma cells were detected by western blot. Glioma cell proliferation, apoptosis, migration and invasion were respectively detected by CCK‐8 assay, flow cytometry, wound healing assay and transwell assay. Tumour xenograft model was developed to study the influence of miR‐1275 and SERPINE1 on glioma growth in vivo. The results of microarray analysis, qRT‐PCR and western blot showed that miR‐1275 was low‐expressed while SERPINE1 was high‐expressed in glioma. Dual‐luciferase assay showed that miR‐1275 could bind to SERPINE1. Overexpression of miR‐1275 could promote the p53 pathway‐related proteins’ expression. Highly expressed miR‐1275 could repress the migration, proliferation and invasion of glioma cells while highly expressed SERPINE1 had inverse effects. Tumour xenograft showed that up‐regulated miR‐1275 or down‐regulated SERPINE1 could repress glioma growth in vivo. Up‐regulation of miR‐1275 activated p53 signalling pathway via regulating SERPINE1 and therefore suppressed glioma cell proliferation, invasion and migration, whereas promoted cell apoptosis.