Cyclin G2 upregulation impairs migration, invasion, and network formation through RNF123/Dvl2/JNK signaling in the trophoblast cell line HTR8/SVneo, a possible role in preeclampsia.

Disruption of extravillous trophoblast (EVT) migration and invasion is considered to be responsible for pathological placentation in preeclampsia (PE). Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression. However, its biological function and underlying molecular mechanism in PE are poorly understood. In this study, clinical data demonstrated that CCNG2 was significantly upregulated in PE placenta and associated with invasive EVT dysfunction. Additionally, Ccng2 knockout led to an attenuation of PE-like symptoms in the PE mouse model produced via treatment with NG-nitro-L-arginine methyl ester (L-NAME). In vitro, CCNG2 inhibited the migration, invasion, and endothelial-like network formation of human trophoblast cell line HTR8/SVneo. Mechanically, CCNG2 suppressed JNK-dependent Wnt/PCP signaling and its downstream indicators including epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) via promoting the polyubiquitination degradation of dishevelled 2 (Dvl2) protein in HTR8/SVneo cells. We also discovered that the E3 ligase Ring finger protein 123 (RNF123), as a novel CCNG2 target among HTR8/SVneo cells, interacted with Dvl2 and participated in CCNG2-induced polyubiquitination degradation of Dvl2. Moreover, we verified that the treatment of HTR8/SVneo cells with RNF123-specific siRNA improved polyubiquitination-induced degradation of Dvl2 and the activity of Wnt/PCP-JNK signaling mediated by CCNG2. Taken together, our results reveal that the CCNG2/RNF123/Dvl2/JNK axis may be involved in the pathogenesis and progression of PE through trophoblastic cell function modulation, thus probably providing us with new therapeutic strategies for PE treatment.

miR-17-5p drives G2/M-phase accumulation by directly targeting CCNG2 and is related to recurrence of head and neck squamous cell carcinoma.

BACKGROUND: The human miR-17-92 polycistron is the first reported and most well-studied onco-miRNA with a cluster of seven miRNAs. miR-17-5p, a member of the miR-17-92 family, plays an important role in tumor cell proliferation, apoptosis, migration and invasion. However, few studies have shown the role of miR-17-5p in the cell cycle of head and neck squamous cell carcinoma (HNSCC). METHODS: RT-qPCR was used to detect miR-17-5p expression levels in 64 HNSCC tissues and 5 cell lines. The relationship between the expression of miR-17-5p in the tissues and the clinical characteristics of the patients was analyzed. HNSCC cells were transfected with an miR-17-5p mimic or inhibitor to evaluate cell cycle distribution by flow cytometry. Cell cycle distribution of cells transfected with target gene was evaluated using flow cytometry. Dual-luciferase reporter assay was used to detect the regulatory effect of miR-17-5p on target gene expression. RESULTS: In the present study, we found that miR-17-5p expression in HNSCC tissues and cell lines was remarkably increased, and miR-17-5p is related to recurrence in HNSCC patients. Silencing miR-17-5p blocked HNSCC cells in G2/M phase, whereas its overexpression propelled cell cycle progression. More importantly, we verified that miR-17-5p negatively regulated CCNG2 mRNA and protein expression by directly targeting its 3'UTR. CONCLUSION: These findings suggest that miR-17-5p might act as a tumor promoter and prognostic factor for recurrence in HNSCC patients.

Lack of miR-1246 in small extracellular vesicle blunts tumorigenesis of laryngeal carcinoma cells by regulating Cyclin G2.

Small extracellular vesicle (sEV) has precise impacts on tumor microenvironment and play vital functions in intercellular interaction. However, the functional role of sEV miRNA on laryngeal squamous cell carcinoma (LSCC) is largely unresolved. Here, the expression of miR-1246 in LSCC tissues and plasma sEV was examined. The internalization ability of sEV was determined by uptake assay. Then, the source and purity of sEV were checked through RNase and/or pharmacological inhibitors application. The invasion, migration, proliferation, and cell cycle assays were used to determine the altered abilities of miR-1246 in sEV in LSCC. Finally, target gene of miR-1246, Cyclin G2 (CCNG2), was stained immunohistochemically. In addition, the relationship between CCNG2 and clinicopathological features of patients was analyzed. We found that miR-1246 was higher in LSCC tissues and plasma sEV. MiR-1246 was enriched in sEV rather than soluble form. SEV could be internalized into adjacent cells. Lack of miR-1246 in sEV abrogated the tumorigenesis of LSCC. Furthermore, CCNG2 knockdown arrested the cell cycle and correlated to clinicopathological features and prognosis of LSCC patients. Taken together, we found that the function of sEV miR-1246 by regulating CCNG2 is responsible for LSCC advancement with emphasis on the main source of miR-1246 mainly root in sEV rather than in soluble form.

Exosomal MicroRNA MiR-1246 Promotes Cell Proliferation, Invasion and Drug Resistance by Targeting CCNG2 in Breast Cancer.

BACKGROUND/AIMS: Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246) as a serum biomarker for breast cancer and understand the underlying mechanism in breast cancer progression. METHODS: The expression levels of endogenous and exosomal miRNAs were examined by real time PCR, and the expression level of the target protein was detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study their uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-1246 was estimated by invasion assay and cell viability assay. RESULTS: In this study, we demonstrate that exosomes carrying microRNA can be transferred among different cell lines through direct uptake. miR-1246 is highly expressed in metastatic breast cancer MDA-MB-231 cells compared to non-metastatic breast cancer cells or non-malignant breast cells. Moreover, miR-1246 can suppress the expression level of its target gene, Cyclin-G2 (CCNG2), indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could enhance the viability, migration and chemotherapy resistance of non-malignant HMLE cells. CONCLUSIONS: Together, our results support an important role of exosomes and exosomal miRNAs in regulating breast tumor progression, which highlights their potential for applications in miRNA-based therapeutics.

Methylation associated miR-1246 contributes to poor prognosis in gliomas treated with temozolomide.

OBJECTIVE: Glioblastoma (GBM) is the most aggressive type of glioma. In this study, we aimed to investigate the biological functions and the possible mechanisms of miR-1246 in glioma. METHODS: A miRNA-seq array was conducted in both the tumor tissues and the glioma cell lines treated with 5-Aza to determine the methylation statues of miRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to verify the miR-1246 expressions. We used overall survival (OS) and the progress-free survival (PFS) to investigate the clinical significance of miR-1246 in the prognosis of glioma patients. Additionally, bioinformatic analysis was used for discovering the potential targets of miR-1246. Cell viability, wound-healing assay and protein expression tests were conducted after the transfection or knockdown of miR-1246 and CCNG2, respectively. RESULTS: We found the reduced expression of miR-1246 in IDH1MUT tumor tissues and the increased expression in the glioma cell lines treated with 5-Aza. Therefore, miR-1246 was selected as a candidate for further analysis. Kaplan-Meier analysis showed that the glioma patients with the high level of miR-1246 had the worst survival rate compared to the low level counterparts. Overexpression of miR-1246 promoted cell proliferation, migration and invasion in glioma cells. Moreover, the results showed that the downregulation of miR-1246 decreased chemoresistance by targeting CCNG2. In addition, Gene ontology (GO) analysis revealed that miR-1246 was associated with the regulations of transcription, cell cycle, cell proliferation, cell adhesion and apoptosis. CONCLUSION: These results indicated that the miR-1246/CCNG2 axis might be a potential target for improving the drug resistance in glioma.

CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells.

The cyclin family protein CCNG2 has an important inhibitory role in cancer initiation and progression, but the exact mechanism is still unknown. In this study, we examined the relationship between CCNG2 and the malignancy of astrocytomas and whether the AKT pathway, which is upregulated in astrocytomas, may inhibit CCNG2 expression. CCNG2 expression was found to be negatively associated with the pathological grade and proliferative activity of astrocytomas, as the highest expression was found in control brain tissue (N = 31), whereas the lowest expression was in high-grade glioma tissue (N = 31). Additionally, CCNG2 overexpression in glioma cell lines, T98G and U251 inhibited proliferation and arrested cells in the G0/G1 phase. Moreover, CCNG2 overexpression could increase glioma cells apoptosis. In contrast, AKT activity increased in glioma cells that had low CCNG2 expression. Expression of CCNG2 was higher in cells treated with the AKT kinase inhibitor MK-2206 indicating that the presence of phosphorylated AKT may inhibit the expression of CCNG2. Inhibition of AKT also led to decreased colony formation in T98G and U251 cells and knocked down of CCNG2 reversed the result. Finally, overexpression of CCNG2 in glioma cells reduced tumor volume in a murine model. To conclude, low expression of CCNG2 correlated with the severity astrocytoma and CCNG2 overexpression could induce apoptosis and inhibit proliferation. Inhibition of AKT activity increased the expression of CCNG2. The present study highlights the regulatory consequences of CCNG2 expression and AKT activity in astrocytoma tumorigenesis and the potential use of CCNG2 in anticancer treatment.

miR-1246 Targets CCNG2 to Enhance Cancer Stemness and Chemoresistance in Oral Carcinomas.

MiRNAs have been recognized as crucial components in carcinogenesis, but whether miR-1246 affects the cancer stemness and drug resistance in oral squamous cell carcinoma (OSCC) has not been fully understood and its downstream targets still need to be unraveled. In the present work, we employed miRNAs RT-PCR analysis to evaluate the expression of miR-1246 in tumor tissues and oral cancer stem cells (OCSC). Stemness phenotypes, including self-renewal, migration, invasion, colony formation capacities, and in vivo oncogenicity of oral cancer cells following transfected with miR-1246 inhibitors or mimics were examined. Our results suggested that the expression level of miR-1246 was significantly upregulated in the tumor tissues and OCSC. Kaplan-Meier survival analysis of OSCC patients with high levels of miR-1246 had the worst survival rate compared to their low-expression counterparts. Inhibition of miR-1246 in OCSC significantly reduced the stemness hallmarks, while overexpression of miR-1246 enhanced these characteristics. Moreover, we showed that downregulation of miR-1246 decreased chemoresistance. In addition, we verified that miR-1246-inhibited CCNG2 contributed to the cancer stemness of OSCC. These results demonstrated the significance of miR-1246 in the regulation of OSCC stemness. Targeting miR-1246-CCNG2 axis may be beneficial to suppress cancer relapse and metastasis in OSCC patients.

Cyclin G2 promotes cell cycle arrest in breast cancer cells responding to fulvestrant and metformin and correlates with patient survival.

Definition of cell cycle control proteins that modify tumor cell resistance to estrogen (E2) signaling antagonists could inform clinical choice for estrogen receptor positive (ER+) breast cancer (BC) therapy. Cyclin G2 (CycG2) is upregulated during cell cycle arrest responses to cellular stresses and growth inhibitory signals and its gene, CCNG2, is directly repressed by E2-bound ER complexes. Our previous studies showed that blockade of HER2, PI3K and mTOR signaling upregulates CycG2 expression in HER2+ BC cells, and that CycG2 overexpression induces cell cycle arrest. Moreover, insulin and insulin-like growth factor-1 (IGF-1) receptor signaling strongly represses CycG2. Here we show that blockade of ER-signaling in MCF7 and T47D BC cell lines enhances the expression and nuclear localization of CycG2. Knockdown of CycG2 attenuated the cell cycle arrest response of E2-depleted and fulvestrant treated MCF7 cells. These muted responses were accompanied by sustained inhibitory phosphorylation of retinoblastoma (RB) protein, expression of cyclin D1, phospho-activation of ERK1/2 and MEK1/2 and expression of cRaf. Our work indicates that CycG2 can form complexes with CDK10, a CDK linked to modulation of RAF/MEK/MAPK signaling and tamoxifen resistance. We determined that metformin upregulates CycG2 and potentiates fulvestrant-induced CycG2 expression and cell cycle arrest. CycG2 knockdown blunts the enhanced anti-proliferative effect of metformin on fulvestrant treated cells. Meta-analysis of BC tumor microarrays indicates that CCNG2 expression is low in aggressive, poor-prognosis BC and that high CCNG2 expression correlates with longer periods of patient survival. Together these findings indicate that CycG2 contributes to signaling networks that limit BC.

Modulation of insulin degrading enzyme activity and liver cell proliferation.

Diabetes mellitus type 2 (T2DM), insulin therapy, and hyperinsulinemia are independent risk factors of liver cancer. Recently, the use of a novel inhibitor of insulin degrading enzyme (IDE) was proposed as a new therapeutic strategy in T2DM. However, IDE inhibition might stimulate liver cell proliferation via increased intracellular insulin concentration. The aim of this study was to characterize effects of inhibition of IDE activity in HepG2 hepatoma cells and to analyze liver specific expression of IDE in subjects with T2DM. HepG2 cells were treated with 10 nM insulin for 24 h with or without inhibition of IDE activity using IDE RNAi, and cell transcriptome and proliferation rate were analyzed. Human liver samples (n = 22) were used for the gene expression profiling by microarrays. In HepG2 cells, IDE knockdown changed expression of genes involved in cell cycle and apoptosis pathways. Proliferation rate was lower in IDE knockdown cells than in controls. Microarray analysis revealed the decrease of hepatic IDE expression in subjects with T2DM accompanied by the downregulation of the p53-dependent genes FAS and CCNG2, but not by the upregulation of proliferation markers MKI67, MCM2 and PCNA. Similar results were found in the liver microarray dataset from GEO Profiles database. In conclusion, IDE expression is decreased in liver of subjects with T2DM which is accompanied by the dysregulation of p53 pathway. Prolonged use of IDE inhibitors for T2DM treatment should be carefully tested in animal studies regarding its potential effect on hepatic tumorigenesis.