Silencing SNHG1 Suppresses Viability, Proliferation and Invasion of Gallbladder Carcinoma Cells via Targeting miR-194-5p.

OBJECTIVE: Long non-coding RNA small nuclear host gene 1 (LncRNA SNHG1) was implicated in several malignancies, but its role and interaction with microRNAs (miRs) in gallbladder cancer (GBC) were awaited to be addressed. METHODS: Clinical GBC tissues were collected. After transfection, GBC cell viability, proliferation and invasion were determined by MTT assay, colony formation assay and Transwell assay, respectively. Target gene and potential binding sites were predicted, followed by confirmation with dual-luciferase reporter assay. Relative expressions of SNHG1, miR-194-5p, epithelial-mesenchymal transition (EMT)-related markers, LIF interleukin 6 family cytokine (LIF), stathmin1 (STMN1), platelet derived growth factor subunit A (PDGFA), insulin like growth factor 1 receptor (IGF1R) and signal transducer and activator of transcription 1 (STAT1) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot as needed. Correlation between SNHG1 and miR-194-5p in GBC was analyzed. RESULTS: High expression of SNHG1 was observed in both GBC tissues and cells, and it was associated with tumor sizes. ShSNHG1 inhibited SNHG1 expression and retarded the viability, proliferation and invasion of GBC cells, accompanied with downregulated N-Cadherin and Vimentin yet upregulated E-Cadherin. MiR-194-5p could competitively bind with SNHG1 and was low-expressed in GBC, revealing the negative correlation between SNHG1 and miR-194-5p. Downregulated miR-194-5p reversed the effects of SNHG1 silencing on viability, proliferation, invasion and EMT-related marker expressions in GBC cells. Additionally, LIF and PDGFA were the target genes of miR-194-5p. CONCLUSIONS: SNHG1 silencing suppressed the viability, proliferation and invasion of GBC cells via targeting miR-194-5p, revealing a new role of SNHG1 in GBC.

Mesenchymal stem cell extracellular vesicles-derived microRNA-194-5p delays the development of intervertebral disc degeneration by targeting TRAF6.

OBJECTIVE: Mesenchymal stem cells-derived extracellular vesicles (MSCs-EVs) can improve intervertebral disc degeneration (IDD). Considering that, their concrete mechanisms from microRNA-194-5p/tumor receptor-associated factor 6 (miR-194-5p/TRAF6) axis in IDD ask for disclosure in a scientific way. METHODS: Nucleus pulposus (NP) cells and MSCs were obtained. EVs were isolated from the obtained MSCs and identified. miR-194-5p expression in MSC-EVs was altered by sequence transfection. Subsequently, MSCs-EVs were co-cultured with NP cells intervened by tumor necrosis factor α (TNF-α). NP cell proliferation and apoptosis, along with their osteogenic differentiation ability were evaluated. miR-194-5p and TRAF6 expression and their interaction were determined. RESULTS: In TNF-α-intervened NP cells, miR-194-5p was down-regulated and TRAF6 was up-regulated. Restoring miR-194-5p effectively enhanced proliferation and osteogenic differentiation, and reduced apoptosis of TNF-α-intervened NP cells. miR-194-5p-enriched MSCs-EVs protected TNF-α-intervened NP cells. miR-194-5p targeted TRAF6, TRAF6 overexpression exerted negatively for the growth of TNF-α-intervened NP cells, and could reduce the protective effects of miR-194-5p on TNF-α-intervened NP cells. CONCLUSION: It is elucidated that miR-194-5p derived from MSCs-EVs protects TNF-α-intervened NP cells through restricting TRAF6, replenishing a potential target for IDD treatment.

Circ-IGF1R plays a significant role in psoriasis via regulation of a miR-194-5p/CDK1 axis.

Psoriasis is a skin disorder that is classed as an autoimmune disease. It is characterized by excessive proliferation, abnormal migration and differentiation of keratinocytes, as well as inflammatory cell infiltration. Circular RNAs (circRNAs/circ) have been reported to play an important role in several aspects of psoriasis. Thus in the present study, the role of circ-insulin-like growth factor 1 receptor (circ-IGF1R) in the development of psoriasis was assessed, and the involvement of microRNA (miR)-194-5p was also investigated as its expression was downregulated in psoriasis. StarBase analysis and dual luciferase reporter assays confirmed the interaction between circ-IGF1R with miR-194-5p. The increased expression of circ-IGF1R and decreased expression of miR-194-5p were further confirmed by reverse transcription-quantitative polymerase chain reaction in interleukin (IL-22)-stimulated HaCaT cells. The increased proliferation, migration and invasion, as well as decreased apoptosis, caspase 3 activity and cleaved-caspase 3/caspase 3 ratio were observed in IL-22-stimulated HaCaT cells. Conversely, transfection of circ-IGF1R-small interfering (si)RNA resulted in significantly increased expression of miR-194-5p with or without stimulation of IL-22 in HaCaT cells, and also overcame the effects of the miR-194-5p inhibitor. Additionally, transfection of circ-IGF1R-siRNA inhibited cell proliferation, migration and invasion, which were reversed by transfection of a miR-194-5p inhibitor. Similarly, circ-IGF1R-siRNA promoted apoptosis, caspase 3 activity and the cleaved-caspase 3/caspase 3 ratio, which were reversed by miR-194-5p inhibitor. These results showed that circ-IGF1R could affect the proliferation, apoptosis, migration and invasion of IL-22-stimulated HaCaT cells by regulating the expression of miR-194-5p. Based on TargetScan prediction and dual luciferase reporter assays, it was shown that cyclin-dependent kinase (CDK)1 was targeted by miR-194-5p. Additionally, the expression of CDK1 was upregulated following stimulation with IL-22 in HaCaT cells at the mRNA and protein levels. Transfection of miR-194-5p mimic or miR-194-5p inhibitor negatively regulated CDK1 expression in the IL-22 induced HaCaT cells. In conclusion, circ-IGF1R-siRNA could inhibit the cell proliferation, migration and invasion, and induce apoptosis by regulating the miR-194-5p/CDK1 axis. circ-IGF1R may thus serve as a potential treatment target for psoriasis.

MicroNAR-194-5p hinders the activation of NLRP3 inflammasomes and alleviates neuroinflammation during intracerebral hemorrhage by blocking the interaction between TRAF6 and NLRP3.

The possible role of miR-194-5p in brain and neurodegenerative diseases has been reported, but its role in intracerebral hemorrhage (ICH) has not been studied. This study estimated the mechanism of miR-194-5p in ICH. ICH rat model was established by injecting collagenase type VII. miR-194-5p expression in brain tissue of ICH rats was overexpressed by injection of miR-194-5p agomir. Then neurological function score and brain water content were measured. The morphological changes of brain tissue and neuronal apoptosis were evaluated by histological staining. Levels of NLRP3 inflammasomes, IL-1ß and IL-18 were measured. The target relation between miR-194-5p and TRAF6 was verified and the binding of TRAF6 to NLRP3 was explored. miR-194-5p was decreased in ICH rats. After overexpression of miR-194-5p, the neuropathological injury in ICH rats was significantly reduced, and NLRP3-mediated inflammatory injury was inhibited. miR-194-5p targeted TRAF6. TRAF6 interacted with NLRP3 to promote the activation of NLRP3 inflammasomes. Overexpression of miR-194-5p reduced the interaction between TRAF6 and NLRP3, thereby alleviating the neuroinflammation. Collectively, overexpression of miR-194-5p reduced the TRAF6/NLRP3 interaction, thus inhibiting the activation of NLRP3 inflammasomes and reducing neuroinflammation during ICH. This study may shed new light on ICH treatment.

miR-194-5p serves a suppressive role in human keloid fibroblasts via targeting NR2F2.

Keloids are a skin fibrotic disease that cause a number of problems for reconstructive surgeons. MicroRNAs (miRs) are crucial for the development of keloids. The present study aimed to investigate the function of the miR­194­5p/nuclear receptor subfamily 2 group F member 2 (NR2F2) interactome in human keloid fibroblasts. Microarray analysis was performed to identify key genes that may participate in keloid progression. The expression levels of miR­194­5p and NR2F2 mRNA in normal human skin fibroblasts (HSFs) and human keloid fibroblasts (KEL­FIBs) were measured via reverse transcription­quantitative PCR. Furthermore, cell proliferation, apoptosis, migration and invasion were assessed in KEL­FIB cells. Following NR2F2 knockdown and miR­194­5p inhibition, NR2F2 expression was measured via western blotting. The microarray analysis identified NR2F2 as a key gene related to keloids. The regulatory association between miR­194­5p and NR2F2 was identified using TargetScan Human (version 7.2) and verified by performing a dual­luciferase reporter assay. miR­194­5p expression was decreased in KEL­FIB cells compared with HSF cells, and miR­194­5p overexpression inhibited the aggressive phenotypes of KEL­FIB cells compared with the negative control group. Meanwhile, NR2F2 expression was negatively correlated with miR­194­5p expression. NR2F2 knockdown and miR­194­5p overexpression displayed similar effects on KEL­FIB cells. Moreover, NR2F2 knockdown effectively reversed miR­194­5p inhibitor­mediated effects in keloid fibroblasts. The present study indicated that the novel miR/194­5p/NR2F2 interactome might be involved in the progression of keloid aggression and may serve as a potential therapeutic target for human keloid in the future.

LncRNA CASC2 targets CAV1 by competitively binding with microRNA-194-5p to inhibit neonatal lung injury.

Long non-coding RNAs (lncRNAs) are vital regulators of different biological processes during bronchopulmonary dysplasia (BPD). This study was conducted to probe the biological roles of lncRNA CASC2 in the pathogenesis of BPD and neonatal lung injury. Firstly, a hyperoxia-induced mouse model with BPD was established. LncRNAs with differential expression in lung tissues of normal and BPD mice were analyzed by microarray. An adenovirus vector overexpressing CASC2 was constructed and its functions on BPD symptoms in model mice were analyzed. Gain- and loss-of function studies of CASC2 were performed in a bronchial epithelial cell line BEAS-2B to determine its role in cell apoptosis and proliferation under normoxic and hyperoxic conditions. The downstream mechanical molecules of lncRNA CASC2 were predicted on bioinformatics systems and confirmed by luciferase assays. The functional interactions among lncRNA CASC2, miR-194-5p, and CAV1 in BPD were determined by rescue experiments. Consequently, lncRNA CASC2 was found to be poorly expressed in BPD mice. Besides, overexpressed CASC2 was found to relieve the symptoms of BPD in neonatal mice and suppress apoptosis as well as promote proliferation in hyperoxia-induced BEAS-2B cells. Importantly, CASC2 was found to regulate CAV1 expression by competitively binding to miR-194-5p and downregulate the activity of the TGF-ß1 signaling pathway, thereby suppressing lung injury. Either miR-194-5p upregulation or CAV1 downregulation blocked the roles of CASC2. To sum up, this study evidenced that CASC2 alleviates hyperoxia-induced lung injury in mouse and cell models with the involvement of a miR-194-5p-CAV1 crosstalk and the TGF-ß1 inactivation.

Long non­coding RNA Unigene56159 promotes glioblastoma multiforme cell proliferation and invasion through negatively regulating microRNA­194­5p.

Long non­coding RNAs (lncRNA) serve a vital role in tumor progression. The present study identified a fundamental role for a novel lincRNA, Unigene56159, in the progression of glioblastoma (GBM). Unigene56159 gene expression was found to be significantly upregulated in tissue samples from patients with GBM as well as in GBM cell lines by reverse transcription­quantitative PCR, while microRNA (miR)­194­5p expression levels were decreased. This higher expression level of Unigene56159 was positively correlated with poor overall survival in patients with GBM. However, the mechanism by which this occurs remains to be elucidated. lncRNAs may act as endogenous miRNA sponges for binding to miRNAs or participating in the competitive endogenous RNAs (ceRNA) regulatory network. Small interfering RNA (siRNA) was used to silence the expression of Unigene56159 and inhibit the proliferation and invasion of GBM cell lines by MTT and Transwell assay. Unigene56159 was found to directly interact with miR­194­5p, and rescue assay was performed to further confirm that Unigene56159 contributed to glioma progression by regulating miR­194­5p. Thus, Unigene56159 may function as a competing endogenous RNA by sequestering miR­194­5p in GBM cells. These findings suggested that Unigene56159 may serve an oncogenic role in GBM and may promote disease progression through interacting with miR­194­5p. This could be a potential therapeutic target for the treatment of GBM.

Upregulation of microRNA­194­5p inhibits hypopharyngeal carcinoma cell proliferation, migration and invasion by targeting SMURF1 via the mTOR signaling pathway.

Hypopharyngeal carcinoma (HPC) is an aggressive malignancy with the worst prognosis among all head and neck cancers. MicroRNAs (miRNAs) are involved in the development of many human cancers, and may function as oncogenes or tumor suppressors. The present study aimed to evaluate the effects of miRNA (miR)­194­5p on the proliferation and invasion of HPC cells and to identify the potential regulatory mechanism. First, miR­194­5p and Smad ubiquitin regulatory factor 1 (SMURF1) expression levels were examined in HPC tissues. Subsequently, to explore the effects of miR­194­5p on SMURF1, a dual­luciferase reporter gene assay was performed to verify the target relationship. To define the role of miR­194­5p in HPC progression, miR­194­5p upregulation and depletion were used to evaluate its effects on cell viability, invasion and migration. SMURF1 silencing and rapamycin [an inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway] treatment were also used to analyze the regulatory mechanism in HPC. Finally, tumor growth was assessed in xenografted tumors in nude mice. SMURF1 was demonstrated to be highly expressed, whereas miR­194­5p was poorly expressed in HPC tissues; SMURF1 was identified as a target gene of miR­194­5p. FaDu hypopharyngeal squamous cell carcinoma cells treated with miR­194­5p mimics exhibited decreased viability, invasion and migration. The results indicated that miR­194­5p may inactivate the mTOR signaling pathway by targeting SMURF1. In addition, the in vivo experiments further verified these regulatory effects. These data suggested that miR­194­5p­targeted SMURF1 inhibition may be involved in the disruption of HPC progression through the repression of the mTOR signaling pathway.

Tetraspanin 1 promotes epithelial-to-mesenchymal transition and metastasis of cholangiocarcinoma via PI3K/AKT signaling.

BACKGROUND: Numerous studies have demonstrated that tetraspanin 1 (TSPAN1), a transmembrane protein, functions as an oncoprotein in many cancer types. However, its role and underlying molecular mechanism in cholangiocarcinoma (CCA) progression remain unclear. METHODS: In the present study, the expression of TSPAN1 in human CCA and adjacent nontumor tissues was examined using real-time PCR, western blot and immunohistochemistry. The effect of TSPAN1 on proliferation and metastasis was evaluated by functional assays both in vitro and in vivo. A luciferase reporter assay was performed to investigate the interaction between microRNA-194-5p (miR-194-5p) and TSPAN1 3'-untranslated region. Co-immunoprecipitation (co-IP) was used to confirm the interaction between TSPAN1 protein and integrin α6ß1 and western blot was used to explore TSPAN1 mechanism. RESULTS: We found that TSPAN1 was frequently upregulated in CCA and high levels of TSPAN1 correlated with TNM stage, especially metastasis in CCA. TSPAN1 overexpression promoted CCA growth, metastasis, and induced epithelial-to-mesenchymal transition (EMT), while its silencing had the opposite effect both in vitro and in vivo. To explore the differential expression of TSPAN1, we screened miR-194-5p as the upstream regulator of TSPAN1. A combination of high-level TSPAN1 and low-level miR-194-5p predicted poor prognosis in patients with CCA. Furthermore, in accordance with the functional characteristics of the TSPAN superfamily, we proved that TSPAN1 interacted with integrin α6ß1 to amplify the phosphoinositide-3-kinase (PI3K)/AKT/glycogen synthase kinase (GSK)-3ß/Snail family transcriptional repressor (Snail)/phosphatase and tensin homolog (PTEN) feedback loop. CONCLUSION: The results indicate that TSPAN1 could be a potential therapeutic target for CCA.