EphA3 targeted by miR-3666 contributes to melanoma malignancy via activating ERK1/2 and p38 MAPK pathways.

Melanoma is a rare, fatal type of skin tumor. Although EPH receptor A3 (EphA3) is deregulated in melanoma, its detailed role remained uncharacterized. Using real time quantitative PCR analysis and western blotting, EphA3 was identified to be upregulated in melanoma tissues and cells, while miR-3666 showed an opposite expression trend. Cell counting kit-8, scratch wound, and in vivo assays proved that EphA3 silence inhibited the melanoma cell proliferation and migration and retarded tumor growth in vivo. Furthermore, western blotting results displayed that EphA3 silence resulted in a low expression of p38-MAPK and p-ERK1/2. Mechanically, miR-3666 was proved to target EphA3 3'UTR by the luciferase reporter assay. Furthermore, miR-3666 mimic compromised the driven melanoma cell proliferation and migration by EphA3 overexpression. In addition, induction of ERK1/2 and p38 MAPK pathways offset the positive effect of EphA3 overexpression on melanoma cells. In conclusion, miR-3666 downregulated EphA3 expression and retarded melanoma malignancy via inactivating ERK1/2 and p38 MAPK pathways. Hence, miR-3666/EphA3 axis may represent a druggable target against melanoma progression.

Long noncoding RNA KCNQ1OT1 contributes to tumor growth and activates Wnt/ß­catenin signaling in osteosarcoma by targeting the miR­3666/KLF7 axis.

Osteosarcoma (OS) is a type of primary malignant cancer occurring in the bone and poses a threat to the lives of children and young adults. Long non­coding RNAs (lncRNAs) have been certified to play important roles in various human malignant tumors, including OS. lncRNA KCNQ1OT1 has been investigated in certain types of cancer; however, its role and molecular mechanisms in OS remain to be determined. In the present study, a high KCNQ1OT1 expression was detected in human OS tissues and cell lines. Moreover, patients with OS with a high expression of KCNQ1OT1 presented a worse prognosis. Loss­of­function assays demonstrated that KCNQ1OT1 silencing suppressed cell proliferative, migratory and invasive abilities in OS. Importantly, the knockdown of KCNQ1OT1 suppressed the Wnt/ß­catenin signaling pathway in OS. In vivo assays displayed the inhibitory role of the silencing of KCNQ1OT1 in OS tumor growth. As regards the underlying mechanisms, KCNQ1OT1 could sponge miR­3666, and its expression was negatively associated with that of miR­3666 in OS tissues. Thereafter, Kruppel­like factor 7 (KLF7), upregulated in OS tissues and cells, was discerned as a target gene of miR­3666. Furthermore, KLF7 expression negatively correlated with miR­3666 expression, whereas it positively correlated with KCNQ1OT1 expression. A rescue assay delineated that the overexpression of KLF7 counteracted the KCNQ1OT1 knockdown­induced suppression of OS cell proliferation, migration, invasion and Wnt/ß­catenin signaling. Collectively, the present study demonstrates that KCNQ1OT1 facilitates OS progression and activates Wnt/ß­catenin signaling by targeting the miR­3666/KLF7 axis.

MiR-3666 serves as a tumor suppressor in ovarian carcinoma by down-regulating AK4 via targeting STAT3.

As a result of metastasis and high recurrence, ovarian carcinoma (OC) is one of the most frequent gynecological carcinomas affecting women up to now. In spite of advances in OC treatments, the molecular mechanisms underlying OC progression are still needed to be deeply understood. MicroRNAs (miRNAs) with aberrant expressions are widely known to regulate target genes so as to mediate diverse biological activities of tumor cells. In the present study, we inspected the expression profile and latent mechanism of miR-3666 in OC. First of all, our research revealed the down-regulated miR-3666 in OC cells. Furthermore, miR-3666 up-regulation could repress cell proliferation and migration as well as induce cell apoptosis in OC. In addition, we unmasked that miR-3666 targeted STAT3 (signal transducer and activator of transcription 3) and further down-regulated STAT3 expression. Moreover, adenylate kinase 4 (AK4) was transcriptionally enhanced by STAT3, and then miR-3666 restrained AK4 expression by mediating STAT3. In the end, rescue experiments depicted that miR-3666 suppressed the development of OC via STAT3-mediated AK4. We uncovered that miR-3666 inhibited the tumorigenesis and even development of OC via suppressing STAT3/AK4 axis, offering a novel biomarker and therapeutic target for OC.

miR-3666 inhibits development of hepatic steatosis by negatively regulating PPARγ.

AIMS: PPARγ is a crucial transcription factor involved in development of hepatic steatosis, an early stage of NAFLD. PPARγ is tightly regulated through various positive and negative regulators including miRNAs. In this study, we report for the first time miR-3666 as a negative regulator of PPARγ and its involvement in development of hepatic steatosis. METHODS: Binding of miR-3666 to regulate PPARγ was checked by luciferase assay and was confirmed by mutating PPARγ 3'UTR. Regulation of PPARγ was determined by overexpression of miR-3666 in HepG2 cells. Hepatic steatotic state in HepG2 cells was developed by exposure to excess palmitic acid and expression of PPARγ, miR-3666 and some PPARγ target and non-target genes was checked. Involvement of mir-3666 by regulating PPARγ in hepatic steatosis was also examined in liver of HFD fed mice. RESULTS: On overexpression of miR-3666, PPARγ expression decreased significantly in a dose-dependent manner in HepG2 cells. Binding of miR-3666 to PPARγ was confirmed as the luciferase activity using pMIR-REPORT with PPARγ 3'UTR decreased in PA treated HepG2 cells overexpressing miR-3666 and remained unchanged when PPARγ 3'UTR was mutated. In PA treated HepG2 cells during development of hepatic steatosis PPARγ was significantly up-regulated concomitant with down-regulation of miR-3666. Overexpression of miR-3666 in these cells decreased the extent of hepatic steatosis. Significant up-regulation of PPARγ and down-regulation of miR-3666 was also observed in liver of HFD fed mice indicating that miR-3666 regulates PPARγ in vivo. CONCLUSIONS: miR-3666 negatively regulates PPARγ by binding to its 3'UTR during development of hepatic steatosis.

Downregulation of circular RNA circPVT1 restricts cell growth of hepatocellular carcinoma through downregulation of Sirtuin 7 via microRNA-3666.

Circular RNAs (circRNAs) have been identified recently as pivotal regulators in the development and progression of cancers, generally by acting as competing endogenous RNAs of microRNAs (miRNAs) to regulate gene expression. The dysregulation of circRNAs in hepatocellular carcinoma (HCC) has attracted much attention, but the precise role of circRNAs in HCC remains largely unknown. In this study, we aimed to investigate the potential role of circular RNA PVT1 (circPVT1), a newly identified cancer-related circRNA, in HCC. Herein, we found that circPVT1 expression was significantly upregulated in HCC tissues and cell lines. Knockdown of circPVT1 significantly reduced the growth and colony formation, and increased cell apoptosis, of HCC cells. Our results further identified circPVT1 as a sponge for miR-3666. Knockdown of circPVT1 significantly increased miR-3666 expression in HCC cells. Moreover, miR-3666 expression was significantly downregulated in HCC tissues and was inversely correlated with circPVT1 expression. In addition, the overexpression of miR-3666 inhibited the growth of HCC cells by targeting Sirtuin 7 (SIRT7). Notably, miR-3666 inhibition or SIRT7 overexpression partially reversed the circPVT1 knockdown-mediated inhibitory effect on HCC cell growth. Overall, these results demonstrate that downregulation of circPVT1 represses HCC cell growth by upregulating miR-3666 to inhibit SIRT7, suggesting circPVT1 as a potential therapeutic target for HCC. Our study highlights the involvement of circPVT1/miR-3666/SIRT7 in regulating HCC cell growth.

MicroRNA-3666 inhibits lung cancer cell proliferation, migration, and invasiveness by targeting BPTF.

A previous study by our group indicted that overexpression of bromodomain PHD-finger transcription factor (BPTF) occurs in lung adenocarcinoma, and is closely associated with advanced clinical stage, higher numbers of metastatic lymph nodes, the occurrence of distant metastasis, low histological grade, and poor prognosis. Down-regulation of BPTF inhibited lung adenocarcinoma cell proliferation and promoted lung adenocarcinoma cell apoptosis. The purpose of this study is to identify valuable microRNAs (miRNAs) that target BPTF to modulate lung adenocarcinoma cell proliferation. In our results, we found that miR-3666 was notably reduced in lung adenocarcinoma tissues and cell lines. Using an miR-3666 mimic, we discovered that cell proliferation, migration, and invasiveness were suppressed by miR-3666 overexpression, but these were all enhanced when the expression of miR-3666 was reduced. Moreover, bioinformatics analysis using the TargetScan database and miRanda software suggested a putative target site in BPTF 3'-UTR. Furthermore, using a luciferase reporter assay, we verified that miR-3666 directly targets the 3'-UTR of BPTF. Using Western blot we discovered that overexpression of miR-3666 negatively regulates the protein expression of BPTF. Finally, we identified that the PI3K-AKT and epilthelial-mesenchymal transition (EMT) signaling pathways were inhibited by miR-3666 overexpression in lung cancer cells. In conclusion, our data indicate that miR-3666 could play an essential role in cell proliferation, migration, and invasiveness by targeting BPTF and partly inhibiting the PI3K-AKT and EMT signaling pathways in human lung cancers.

Pancreatic ß-cell function is inhibited by miR-3666 in type 2 diabetes mellitus by targeting adiponectin

Type 2 diabetes mellitus (T2D) is a common endocrine and metabolic disorder, and poses threats to human health worldwide. Recently, microRNAs (miRNAs) have been suggested to play important roles in the pathophysiology of T2D. In this study, we explored the role of miR-3666 in T2D. miR-3666 was significantly down-regulated in the serum of T2D patients when compared to that of healthy volunteers, and miR-3666 expression level was negatively correlated with blood glucose levels of T2D patients. Overexpression of miR-3666 inhibited cell proliferation, reduced insulin secretion, and promoted cell apoptosis of pancreatic β-cell line (INS-1 cells). On the other hand, knockdown of miR-3666 had the opposite effects in INS-1 cells. The bio-informatics analysis using TargetScan revealed that adiponectin (ADIPOQ) was a downstream target of miR-3666, and the interaction between miR-3666 and ADIPOQ was validated by luciferase reporter assay. In addition, miR-3666 negatively regulated the mRNA and protein expression of ADIPOQ. Overexpression of ADIPOQ promoted insulin secretion after glucose stimulation, promoted cell proliferation, inhibited cell apoptosis, and partially abolished the effects of miR-3666 overexpression on insulin secretion, cell proliferation, and cell apoptosis of INS-1 cells. In conclusion, our results revealed that miR-3666 inhibited pancreatic cell proliferation, reduced insulin sensitivity, and promoted apoptosis by targeting ADIPOQ.

miR­3666 suppresses cellular proliferation and invasion in colorectal cancer by targeting SATB2.

MicroRNA­3666 (miR­3666) acts as a tumor suppressor in cervical cancer, non­small cell lung cancer and thyroid carcinoma; however, the function of miR­3666 in colorectal cancer (CRC) remains largely unknown. In the present study, was demonstrated that miR­3666 was significantly downregulated in CRC tissues compared with in adjacent normal tissues by reverse transcription­quantitative polymerase chain reaction. Additionally, miR­3666 may serve as a prognostic biomarker for patients with CRC. Via functional experiments, the present study reported that miR­3666 overexpression significantly inhibited the proliferation, migration and invasion of CRC cells as determined by Cell Counting Kit­8 and Transwell assays, and vice versa. In addition, miR­3666 was reported to directly target special AT­rich sequence binding protein 2 (SATB2) in CRC cells; overexpression of miR­3666 significantly suppressed the expression of SATB2 in CRC cells as determined by western blotting. Furthermore, an inverse correlation was observed between the expression levels of miR­3666 and SATB2 in CRC tissues. Restoration of SATB1 expression significantly reversed the effects of miR­3666 mimic on CRC cells. In summary, the results of the present study indicated that miR­3666 may serve as a tumor suppressor in CRC by targeting SATB2.

The protective role of all-transretinoic acid (ATRA) against colorectal cancer development is achieved via increasing miR-3666 expression and decreasing E2F7 expression.

OBJECTIVE: Colorectal cancer (CRC) is one of the most common malignancies with high morbidity and mortality rates worldwide. This study aimed to investigate whether miR-3666 was involved in inhibitory effects of all-transretinoic acid (ATRA) on the development of colorectal cancer (CRC). MATERIAL AND METHODS: Surgical specimens of CRC tissues and adjacent non-tumor mucosa were collected for determining miR-3666 expression. Human CRC HCT116 cells were treated with different doses of ATRA (10, 20, 40, and 60 µM, respectively) and/or transfected with miR-3666 mimic, miR-3666 inhibitor, E2F7 siRNAs or their controls, respectively. After different treatments, cell viability, apoptosis, migration and invasion were detected. The regulatory relationship between miR-3666 and E2F7 was investigated. Furthermore, the association between MAPK/ERK pathway and ATRA or miR-3666/E2F7 was explored. RESULTS: The miR-3666 was lowly expressed in CRC tissues, while E2F7 was highly expressed. ATRA decreased HCT116 cell viability, migration, and invasion, and induced apoptosis, indicating that ATRA inhibited the malignant behaviors of HCT116 cells. Moreover, ATRA increased miR-3666 expression, and effects of ATRA on the malignant behaviors of HCT116 cells were achieved by positive regulating miR-3666 expression. Furthermore, E2F7 was a target gene of miR-3666, and knockdown of E2F7 reversed the combined effects of ATRA and miR-3666 inhibitor on the malignant behaviors of HCT116 cells. Besides, ATRA inhibited the activation of MAPK/ERK signaling pathway, which was reversed by inhibition of miR-3666. CONCLUSIONS: Our results reveal that ATRA protects against CRC development possible via increasing miR-3666 expression and decreasing E2F7 expression. MiR-3666/E2F7 may play a key role in regulating the inhibitory effects of ATRA on HCT116 cells via suppressing the activation of MAPK/ERK signaling pathway.

Pituitary tumor-transforming gene 1 enhances metastases of cervical cancer cells through miR-3666-regulated ZEB1.

Early cancer metastases often occur in cervical cancer (CC) patients, resulting in poor prognosis and poor therapeutic outcome after resection of primary cancer. Hence, there is a compelling requirement for elucidating the molecular mechanisms underlying the CC cell invasiveness. Recently, the role of microRNAs (miRNAs) and pituitary tumor-transforming gene 1 (Pttg1) in the carcinogenesis of CC has been reported. Nevertheless, the relationship between miRNAs and Pttg1 remains ill-defined. Here, we showed that the levels of miR-3666 were significantly decreased and the levels of zinc finger E-box binding homeobox 1 (ZEB1) and Pttg1 were significantly increased in the CC specimens from patients, compared to the paired non-tumor tissue. Moreover, the levels of miR-3666 and ZEB1 inversely correlated. Bioinformatics analyses showed that miR-3666 targeted the 3'-untranslated region (3'-UTR) of ZEB1 messenger RNA (mRNA) to inhibit its translation, which was confirmed by luciferase reporter assay. Moreover, Pttg1 overexpression inhibited miR-3666 and subsequently increased ZEB1 and cell invasion, while Pttg1 depletion increased miR-3666 and subsequently decreased ZEB1 and cell invasion. Together, our data suggest that Pttg1 may increase CC cell metastasis, possibly through miR-3666-regulated ZEB1 levels.