MiR-302b Suppresses Tumor Metastasis by Targeting Frizzled 6 in OSCC.

Oral squamous cell carcinoma (OSCC) accounts for approximately 90% of malignant epithelial tumors of the oral and maxillofacial region. OSCC has high rate of metastasis and poor prognosis. Tobacco and/or alcohol consumption and human papillomavirus infection are relatively exact susceptibility factors for OSCC, but the specific process of oral mucosal carcinogenesis and progression is very complicated. microRNA-302b (miR-302b) could regulate various characteristics of many tumor cells, such as proliferation and apoptosis, but its role and mechanism in OSCC have not been reported. This research aims to study the effect of miR-302b on the invasion and migration ability of OSCC and the mechanism by which it functions as well as to identify new prognostic indicators and therapeutic targets for OSCC patients. Functional studies showed that the miR-302b level was negatively correlated with the invasion and migration ability of OSCC. The studies also showed that the overexpression of miR-302b could attenuate the invasion and migration ability of OSCC cells and reduce lymphangiogenesis and the lung metastasis rate of OSCC cells in a mouse model. Mechanistic studies were performed by quantitative polymerase chain reactions, luciferase assays, and RNA pull-down experiments. The results verified that frizzled class receptor 6 (FZD6) is a target gene of miR-302b in OSCC that could promote cell invasion and migration. Clinical studies demonstrate that the protein expression level of FZD6 was higher in OSCC and metastatic lymph nodes than in normal oral mucosa epithelium. Taken together, these data showed that miR-302b could inhibit the invasion and migration ability of OSCC cells by targeting and downregulating FZD6, thereby inhibiting OSCC metastasis. As a new target gene of miR-302b, FZD6 has the potential to become a prognostic and therapeutic target for OSCC patients.

MicroRNA-302b mitigates renal fibrosis via inhibiting TGF-ß/Smad pathway activation

Renal fibrosis is one of the most significant pathological changes after ureteral obstruction. Transforming growth factor-β (TGF-β) signaling pathway plays essential roles in kidney fibrosis regulation. The aims of the present study were to investigate effects of microRNA-302b (miR-302b) on renal fibrosis, and interaction between miR-302b and TGF-β signaling pathway in murine unilateral ureteral obstruction (UUO) model. Microarray dataset GSE42716 was downloaded by retrieving Gene Expression Omnibus database. In accordance with bioinformatics analysis results, miR-302b was significantly down-regulated in UUO mouse kidney tissue and TGF-β1-treated HK-2 cells. Masson's trichrome staining showed that miR-302b mimics decreased renal fibrosis induced by UUO. The increased mRNA expression of collagen I and α-smooth muscle actin (α-SMA) and decreased expression of E-cadherin were reversed by miR-302b mimics. In addition, miR-302b up-regulation also inhibited TGF-β1-induced epithelial mesenchymal transition (EMT) of HK-2 cells by restoring E-cadherin expression and decreasing α-SMA expression. miR-302b mimics suppressed both luciferase activity and protein expression of TGF-βR2. However, miR-302b inhibitor increased TGF-βR2 luciferase activity and protein expression. Meanwhile, miR-302b mimics inhibited TGF-βR2 mRNA expression and decreased Smad2 and Smad3 phosphorylation in vivo and in vitro. Furthermore, over-expression of TGF-βR2 restored the miR-302b-induced decrease of collagen I and α-SMA expression. In conclusion, this study demonstrated that miR-302b attenuated renal fibrosis by targeting TGF-βR2 to suppress TGF-β/Smad signaling activation. Our findings showed that elevating renal miR-302b levels may be a novel therapeutic strategy for preventing renal fibrosis.

RACK1 promotes miR-302b/c/d-3p expression and inhibits CCNO expression to induce cell apoptosis in cervical squamous cell carcinoma.

BACKGROUND: Cervical squamous cell carcinoma (CSCC) is one of the main causes of cancer-related deaths in women worldwide. The present study was conducted with the main objective of determining the potential role of receptor for activated protein kinase C1 (RACK1) in CSCC through regulation of microRNA (miR)-302b/c/d-3p and Cyclin O (CCNO). METHODS: The expression of RACK1, miR-302b/c/d-3p and CCNO in CSCC tissues and cells was measured by RT-qPCR and Western blot analysis. The interaction among RACK1, miR-302b/c/d-3p, and CCNO was determined by dual luciferase reporter assay. Subsequently, effects of RACK1, miR-302b/c/d-3p and CCNO on CSCC cell cycle entry, proliferation and apoptosis were investigated with the use of flow cytometry, EdU, and TUNEL assays. Furthermore, mouse xenograft model of CSCC cells was established to verify the function of RACK1 in vivo. RESULTS: RACK1 and miR-302b/c/d-3p were down-regulated and CCNO was overexpressed in CSCC. CCNO was identified as the target of miR-302b/c/d-3p. Importantly, overexpressed miR-302b-3p, miR-302c-3p or miR-302d-3p or RACK1 enhanced the apoptosis and suppressed the proliferation of CSCC cells in vitro, while inhibiting tumor growth in vivo by targeting CCNO. CONCLUSIONS: On all accounts, overexpressed RACK1 could dampen the progression of CSCC through miR-302b/c/d-3p-mediated CCNO inhibition.

Downregulation of miR-302b is associated with poor prognosis and tumor progression of breast cancer.

BACKGROUND: MicroRNAs (miRNAs) are well known to play crucial role in various types of cancers, including breast cancer (BC). METHODS: The present study aimed to investigate the expression, clinical value, and functional role of miR-302b in BC. The expression level of miR-302b was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The clinical value of miR-302b in BC prognosis was calculated via Kaplan-Meier survival analysis and Cox regression analysis. Cell experiments were applied to investigate the functional role of miR-302b in BC. RESULTS: miR-302b was significantly downregulated in BC tissues and cell lines compared to the corresponding controls (all P < 0.01). Notably, the expression of miR-302b was significantly associated with lymph node metastasis and TNM stage (all P < 0.05). Patients with lower miR-302b expression had shorter survival time than those with higher miR-302b expression (log-rank P = 0.002). Furthermore, miR-302b expression and TNM stage were proven to be independent prognostic factors for BC. Overexpression of miR-302b inhibited BC cell proliferation, migration, and invasion in BT549 and MCF-7 cell lines, while silence of miR-302b exhibited an opposite effects on BC cells (all P < 0.05). RUNX2 was determined to be the target gene of miR-302b. CONCLUSIONS: The present study suggests that miR-302b functions as a tumor suppressor in BC and inhibits the tumor progression of BC via targeting RUNX2. Downregulation of miR-302b might be a significant prognostic factor for poor survival in BC patients.

Fentanyl inhibits proliferation and invasion via enhancing miR-302b expression in esophageal squamous cell carcinoma.

Fentanyl is one of the most commonly used intravenous anesthetic agents during cancer resection surgery, but the effect of fentanyl on esophageal squamous cell carcinoma (ESCC) remains unclear. The aim of the present study was to investigate the involvement of microRNA 302b (miR-302b) in the anti-proliferation and anti-invasion effects of fentanyl in ESCC. In the present study, the effects of fentanyl on cell proliferation, apoptosis and invasion were detected using MTT assays, flow cytometry and Transwell assays in ESCC Eca109 and TE1 cell lines. Subsequently, expression of miR-302b was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RT-qPCR and western blot analysis were performed in order to evaluate the expression of ErbB4, a target of miR-302b. Furthermore, anti-miR were used to inhibit miR-302b in fentanyl-treated ESCC cells in order to evaluate the role of miR-302b in the effect of fentanyl on malignant behaviors. Fentanyl inhibited the proliferation of Eca109 and TE1 cells in a dose- and time-dependent manner. Following exposure to fentanyl for 48 h, Eca109 and TE1 cells exhibited increased apoptosis and decreased invasion. Furthermore, fentanyl upregulated miR-302b expression, but downregulated ErbB4 expression. Finally, loss of miR-302b using the anti-miR technique reversed the effect of fentanyl on cell proliferation, apoptosis and invasion in the two ESCC cell lines. Taken together, the results of the present study indicated that fentanyl inhibits the proliferation and invasion of ESCC cells through upregulation of miR-302b.

MicroRNA-302b negatively regulates IL-1ß production in response to MSU crystals by targeting IRAK4 and EphA2.

BACKGROUND: Interleukin-1ß (IL-1ß) is a pivotal proinflammatory cytokine that is strongly associated with the inflammation of gout. However, the underlying mechanism through which the production of IL-1ß is regulated has not been fully elucidated. Our previous work identified that miR-302b had an important immune regulatory role in bacterial lung infections. This study was conducted to evaluate the function of miR-302b on monosodium urate (MSU) crystal-induced inflammation and its mechanism. METHODS: The expression pattern and the immune-regulatory role of miR-302b were evaluated both in vitro and in vivo. The functional targets of miR-302b were predicted by bioinformatics, and then validated by genetic approaches. In addition, the clinical feature of miR-302b was analyzed using serum samples of patients with gouty arthritis. RESULTS: The extremely high expression of miR-302b was observed in both macrophages and mouse air membranes treated with MSU. Intriguingly, overexpression of miR-302b regulated NF-κB and caspase-1 signaling, leading to significantly attenuate MSU-induced IL-1ß. By genetic analysis, miR-302b exhibited inhibitory function on IRAK4 and EphA2 by binding to their 3'-UTR regions. Corporately silencing IRAK4 and EphA2 largely impaired MSU-induced IL-1ß protein production. Moreover, it was also found that miR-302b and EphA2 suppressed the migration of macrophages. Finally, it was observed that high expression of miR-302b was a general feature in patients with gouty arthritis. CONCLUSIONS: These results suggest that miR-302b can regulate IL-1ß production in MSU-induced inflammation by targeting NF-κB and caspase-1 signaling, and may be a potential therapeutic target for gouty arthritis.