Exosomal derived miR-1246 from hydroquinone-transformed cells drives S phase accumulation arrest by targeting cyclin G2 in TK6 cells.

BACKGROUND: Hydroquinone (HQ), a major metabolite of benzene and known hematotoxic carcinogen. MicroRNA 1246 (miR-1246), an oncogene, regulates target genes in carcinogenesis including leukemia. This study investigates the impact of exosomal derived miR-1246 from HQ-transformed (HQ19) cells on cell-to-cell communication in recipient TK6 cells. METHODS: RNA sequencing was used to identify differentially expressed exosomal miRNAs in HQ19 cells and its phosphate buffered solution control cells (PBS19), which were then confirmed using qRT-PCR. The impact of exosomal miR-1246 derived from HQ-transformed cells on cell cycle distribution was investigated in recipient TK6 cells. RESULTS: RNA sequencing analysis revealed that 34 exosomal miRNAs were upregulated and 158 miRNAs were downregulated in HQ19 cells compared with PBS19 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses predicted that their targets are enriched in cancer development-related pathways, such as MAPK signaling, microRNAs in cancer, apoptosis, PI3K-Akt signaling, cell cycle, Ras signaling, and Chronic myeloid leukemia. Eleven miRNAs were confirmed to have differential expression through qRT-PCR, with 6 upregulated (miR-140-3p, miR-551b-3p, miR-7-5p, miR-1290, miR-92a-3p, and miR-1246) and 5 downregulated (miR-183-5p, miR-26a-5p, miR-30c-5p, miR-205-5p, and miR-99b-3p). Among these, miR-1246 exhibited the highest expression level. HQ exposure resulted in a concentration-dependent increase in miR-1246 levels and decrease Cyclin G2 (CCNG2) levels in TK6 cells. Similarly, exosomes from HQ19 exhibited similar effects as HQ exposure. Dual luciferase reporter gene assays indicated that miR-1246 could band to CCNG2. After HQ exposure, exosomal miR-1246 induced cell cycle arrest at the S phase, elevating the expression of genes like pRb, E2F1, and Cyclin D1 associated with S phase checkpoint. However, silencing miR-1246 caused G2/M-phase arrest. CONCLUSION: HQ-transformed cells' exosomal miR-1246 targets CCNG2, regulating TK6 cell cycle arrest, highlighting its potential as a biomarker for HQ-induced malignant transformation.

Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma.

BACKGROUND: IFN-γ is a key mediator of tumor immunity that can induce macrophage polarization to suppress tumor growth. Cyclin G2 functions as a tumor suppressor in various cancer cells; however, its role in macrophages remains unclear. This study aimed to investigate the role and underlying mechanisms of cyclin G2 in macrophages in vitro and in vivo. METHODS: Mouse tumor models were used to determine the effect of cyclin G2 in macrophages on tumor growth in vivo following IFN-γ treatment. Immunohistochemistry staining, immunofluorescence staining and flow cytometry were used to evaluate the number of cytotoxic T lymphocytes (CTLs) and blood vessels in the mouse tumors. Moreover, the biological roles of cyclin G2 in macrophages with regard to CTL chemotaxis, cytotoxic function, and vascular endothelial cell tube formation were assessed using in vitro functional experiments. Immunoprecipitation (IP), real-time PCR, and enzyme-linked immunosorbent assays (ELISAs) were conducted to investigate the underlying mechanisms by which cyclin G2 regulates CTLs and vascular endothelial cells. RESULTS: We found that cyclin G2 expression was upregulated in macrophages after IFN-γ treatment. Upregulated cyclin G2 inhibited lung and colon cancer growth by increasing the secretion of its downstream effector CXCL9, which promoted CTL chemotaxis and suppressed vascular endothelial cell tube formation. Moreover, cyclin G2 increased CXCL9 mRNA levels by promoting STAT1 nuclear translocation. In addition, cyclin G2 promoted the activation of the STAT1 signaling pathway, which was dependent on PP2Ac. CONCLUSIONS: Cyclin G2 is upregulated by IFN-γ in macrophages, promotes the secretion of CXCL9 to increase CTL chemotaxis and inhibit angiogenesis to suppress tumor growth. Our findings suggest that targeting cyclin G2 could benefit future immunotherapy.

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.

Cyclin G2 reverses immunosuppressive tumor microenvironment and potentiates PD-1 blockade in glioma.

BACKGROUND: Expression of aberrant cyclin G2 is a key factor contributing to cancer biological processes, including glioma. However, the potential underlying mechanisms of cyclin G2 in the glioma tumor immune microenvironment remain unclear. METHODS: Co-immunoprecipitation (co-IP), in situ proximity ligation assay (PLA), and in vitro kinase assay were conducted to reveal the underlying mechanism by which cyclin G2 regulates Y10 phosphorylation of LDHA. Further, the biological roles of cyclin G2 in cell proliferation, migration, invasion capacity, apoptosis, glycolysis, and immunomodulation were assessed through in vitro and in vivo functional experiments. Expressions of cyclin G2 and Foxp3 in glioma specimens was determined by immunohistochemistry. RESULTS: In this study, we found that cyclin G2 impeded the interaction between LDHA and FGFR1, thereby decreasing Y10 phosphorylation of LDHA through FGFR1 catalysis. Cyclin G2 inhibited proliferation, migration, invasion capacity, and glycolysis and promoted apoptosis glioma cells via suppressing Y10 phosphorylation of LDHA. Moreover, we further verified that cyclin G2 reversed the immunosuppressive to antitumor immune microenvironment through inhibiting lactate production by glioma cells. Besides, cyclin G2 potentiated PD-1 blockade and exerted strong antitumor immunity in the glioma-bearing mice model. CONCLUSIONS: Cyclin G2 acts as a potent tumor suppressor in glioma and enhances responses to immunotherapy. Our findings may be helpful in selecting glioma patients for immunotherapy trials in the future.

Control of preadipocyte proliferation, apoptosis and early adipogenesis by the forkhead transcription factor FoxO6.

AIMS: Previous studies have shown that the forkhead transcription factor FoxO6 involved in memory consolidation and hepatic glucose homeostasis. Here we asked whether chicken FoxO6 may regulate preadipocyte proliferation, apoptosis and early adipogenesis. MAIN METHODS: Overexpression and knockdown of FoxO6 were performed and evaluated through cell proliferation methods, Oil-Red-O staining, and specific marker expression. Chromatin immunoprecipitation (ChIP) assay was performed to confirm cyclin G2 (CCNG2) as a direct target gene of FoxO6. KEY FINDINGS: FoxO6 is ubiquitously expressed in different chicken tissues and highly expressed in liver, abdominal fat, and preadipocytes in cultured cell. FoxO6 overexpression decreased preadipocyte proliferation by causing G1-phase cell-cycle arrest, whereas inhibition of FoxO6 showed the opposite effects. Overexpression or knockdown of FoxO6 significantly altered the mRNA and protein levels of cell-cycle related markers, such as CCNG2, cyclin dependent kinase inhibitor 1B (CDKN1B), cyclin dependent kinase inhibitor 1A (CDKN1A) and cyclin D2 (CCND2). During preadipocyte proliferation, FoxO6 targets and induces expression of CCNG2, as confirmed by ChIP assay and qPCR. In addition, FoxO6 induces preadipocyte apoptosis through increasing the protein expression levels of cleaved caspase-3 and cleaved caspase-8. Moreover, FoxO6 at the early stage of adipogenesis suppressed mRNA and protein levels of the key early regulators of adipogenesis, such as PPARγ and C/EBPα. SIGNIFICANCE: The results demonstrate that FoxO6 controls preadipocyte proliferation, apoptosis and early adipogenesis, and point to new approaches for further studies related to obesity.

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.

Cyclin G2 Is Involved in the Proliferation of Placental Trophoblast Cells and Their Interactions with Endothelial Cells.

BACKGROUND Remodeling of maternal spiral arteries after embryo implantation relies on well-regulated trophoblast functions. Although cyclin G2 (CCNG2) is thought to be involved in placental development and function, its role in trophoblasts and the mechanisms underlying placental development and function remain unclear. The present study investigated the potential role of CCNG2 in trophoblast cell proliferation and their interactions with endothelial cells. MATERIAL AND METHODS CCNG2 levels were modified by stable infection of HTR8/SVneo cells with lentiviruses overexpressing and silencing CCNG2. Cell proliferation was measured using CCK-8 assays. Network formation assays were performed using trophoblasts alone and co-cultured trophoblasts and endothelial cells to measure angiogenesis of trophoblasts and trophoblast-endothelial interactions. Levels of angiogenic factors (VEGF and sFlt-1) in the supernatant were measured by ELISA, and the expression of cell cycle regulatory (cyclin D1) and invasive (MMP2, MMP3, MMP9) markers implicated in artery remodeling were measured by western blotting. RESULTS Ectopic expression of CCNG2 blocked the proliferation of HTR8/SVneo cells, as well as their abilities to form networks and integrate into human umbilical vein endothelial cells, whereas CCNG2 inhibition had the opposite effects. CCNG2 upregulation significantly reduced the expression of VEGF, cyclin D1, MMP2, MMP3, and MMP9, but enhanced the expression of sFlt-1. In contrast, CCNG2 downregulation had the opposite effects. CONCLUSIONS CCNG2 plays a critical role in trophoblast proliferation and trophoblast-endothelial cell interactions by significant affecting cell cycle, angiogenic, and invasive markers. CCNG2 may thus be a novel marker for the treatment of placental disorders.

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.

Cyclin G2 regulates canonical Wnt signalling via interaction with Dapper1 to attenuate tubulointerstitial fibrosis in diabetic nephropathy.

Cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. Cyclin G2 in the occurrence and development of diabetic nephropathy (DN), one of the most severe diabetic complications, has not been fully identified. In this study, we investigated the function and regulatory mechanism of cyclin G2 in DN. In vivo studies revealed that a deficiency of cyclin G2 significantly increased albuminuria and promoted tubulointerstitial fibrosis in established DN. Cyclin G2 regulated the expression of fibrosis-related proteins via the canonical Wnt signalling pathway in renal tubular epithelial cells. Moreover, the binding of cyclin G2 to Dapper1 (Dpr1/DACT1), a protein involved in Wnt signalling, decreased the phosphorylation of Dpr1 at Ser762 by casein kinase 1 (CK1) and suppressed the Wnt signalling pathway. These findings reveal that cyclin G2 can protect against renal injury and fibrosis associated with DN and, thus, is a new target for the prevention and treatment of diabetic complications.

MiR-1290 targets CCNG2 to promote the metastasis of oral squamous cell carcinoma.

OBJECTIVE: MicroRNAs (miRNAs) have been demonstrated to be involved in the pathogenesis of various human cancers, including oral squamous cell carcinoma (OSCC). Here, we designed this study to explore the potential effect of miR-1290 on tumorigenesis of OSCC. PATIENTS AND METHODS: The expressions of miR-1290 and cyclin G2 (CCNG2) in OSCC were observed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Dual-Luciferase Reporter Assay was performed to confirm the relationship between miR-1290 and CCNG2. The functions of miR-1290 and CCNG2 were analyzed using transwell assay. The Western blot analysis was used to detect epithelial-mesenchymal transition (EMT). RESULTS: Upregulation of miR-1290 and downregulation of CCNG2 were identified in OSCC. And upregulation of miR-1290 was associated with clinicopathological characteristics and poor prognosis in OSCC patients. Moreover, the downregulation of miR-1290 inhibited cell metastasis and EMT in OSCC cells. Furthermore, CCNG2 was a direct target of miR-1290. Its expression was inversely regulated by miR-1290 in OSCC cells. At the same time, the suppressive effect of CCNG2 was observed in OSCC. Furthermore, overexpression of CCNG2 weakened the promoted effect of miR-1290 on cell metastasis in OSCC. CONCLUSIONS: MiR-1290 promoted cell metastasis and EMT, inhibiting CCNG2 expression in OSCC.

miR-590-3p Targets Cyclin G2 and FOXO3 to Promote Ovarian Cancer Cell Proliferation, Invasion, and Spheroid Formation.

Ovarian cancer is the leading cause of death from gynecological cancers. MicroRNAs (miRNAs) are small, non-coding RNAs that interact with the 3' untranslated region (3' UTR) of target genes to repress their expression. We have previously reported that miR-590-3p promoted ovarian cancer growth and metastasis, in part by targeting Forkhead box A (FOXA2). In this study, we further investigated the mechanisms by which miR-590-3p promotes ovarian cancer development. Using luciferase reporter assays, real-time PCR, and Western blot analyses, we demonstrated that miR-590-3p targets cyclin G2 (CCNG2) and Forkhead box class O3 (FOXO3) at their 3' UTRs. Silencing of CCNG2 or FOXO3 mimicked, while the overexpression of CCNG2 or FOXO3 reversed, the stimulatory effect of miR-590-3p on cell proliferation and invasion. In hanging drop cultures, the overexpression of mir-590 or the transient transfection of miR-590-3p mimics induced the formation of compact spheroids. Transfection of the CCNG2 or FOXO3 plasmid into the mir-590 cells resulted in the partial disruption of the compact spheroid formation. Since we have shown that CCNG2 suppressed ß-catenin signaling, we investigated if miR-590-3p regulated ß-catenin activity. In the TOPFlash luciferase reporter assays, mir-590 increased ß-catenin/TCF transcriptional activity and the nuclear accumulation of ß-catenin. Silencing of ß-catenin attenuated the effect of mir-590 on the compact spheroid formation. Taken together, these results suggest that miR-590-3p promotes ovarian cancer development, in part by directly targeting CCNG2 and FOXO3.

A novel DNA-binding motif in prostate tumor overexpressed-1 (PTOV1) required for the expression of ALDH1A1 and CCNG2 in cancer cells.

PTOV1 is a transcription and translation regulator and a promoter of cancer progression. Its overexpression in prostate cancer induces transcription of drug resistance and self-renewal genes, and docetaxel resistance. Here we studied PTOV1 ability to directly activate the transcription of ALDH1A1 and CCNG2 by binding to specific promoter sequences. Chromatin immunoprecipitation and electrophoretic mobility shift assays identified a DNA-binding motif inside the PTOV-A domain with similarities to known AT-hooks that specifically interacts with ALDH1A1 and CCNG2 promoters. Mutation of this AT-hook-like sequence significantly decreased the expression of ALDH1A1 and CCNG2 promoted by PTOV1. Immunohistochemistry revealed the association of PTOV1 with mitotic chromosomes in high grade prostate, colon, bladder, and breast carcinomas. Overexpression of PTOV1, ALDH1A1, and CCNG2 significantly correlated with poor prognosis in prostate carcinomas and with shorter relapse-free survival in colon carcinoma. The previously described interaction with translation complexes and its direct binding to ALDH1A1 and CCNG2 promoters found here reveal the PTOV1 capacity to modulate the expression of critical genes at multiple levels in aggressive cancers. Remarkably, the AT-hook motifs in PTOV1 open possibilities for selective targeting its nuclear and/or cytoplasmic activities.

Cyclin G2 Inhibits the Warburg Effect and Tumour Progression by Suppressing LDHA Phosphorylation in Glioma.

Cyclin G2 has been identified as a tumour suppressor in several cancers. However, its regulatory roles and underlying mechanisms in tumours are still unknown. In this study, we demonstrated that cyclin G2 was expressed at low levels in glioma, which was as a poor prognostic factor for this disease. We also found that, cyclin G2 could suppress cell proliferation, initiate cell apoptosis and reduce aerobic glycolysis, suggesting that cyclin G2 plays a tumour suppressive role in glioma. Mechanistically, cyclin G2 could negatively regulate tyrosine-10 phosphorylation of a critical glycolytic enzyme, lactate dehydrogenase A, through direct interaction. Taken together, these results indicate that cyclin G2 acts as a tumour suppressor in glioma by repressing glycolysis and tumour progression through its interaction with LDHA.

Cyclin G2 suppresses Wnt/ß-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1.

BACKGROUND: Gastric cancer is one of the most common malignant tumors. Cyclin G2 has been shown to be associated with the development of multiple types of tumors, but its underlying mechanisms in gastric tumors is not well-understood. The aim of this study is to investigate the role and the underlying mechanisms of cyclin G2 on Wnt/ß-catenin signaling in gastric cancer. METHODS: Real-time PCR, immunohistochemistry and in silico assay were used to determine the expression of cyclin G2 in gastric cancer. TCGA datasets were used to evaluate the association between cyclin G2 expression and the prognostic landscape of gastric cancers. The effects of ectopic and endogenous cyclin G2 on the proliferation and migration of gastric cancer cells were assessed using the MTS assay, colony formation assay, cell cycle assay, wound healing assay and transwell assay. Moreover, a xenograft model and a metastasis model of nude mice was used to determine the influence of cyclin G2 on gastric tumor growth and migration in vivo. The effects of cyclin G2 expression on Wnt/ß-catenin signaling were explored using a TOPFlash luciferase reporter assay, and the molecular mechanisms involved were investigated using immunoblots assay, yeast two-hybrid screening, immunoprecipitation and Duolink in situ PLA. Ccng2-/- mice were generated to further confirm the inhibitory effect of cyclin G2 on Wnt/ß-catenin signaling in vivo. Furthermore, GSK-3ß inhibitors were utilized to explore the role of Wnt/ß-catenin signaling in the suppression effect of cyclin G2 on gastric cancer cell proliferation and migration. RESULTS: We found that cyclin G2 levels were decreased in gastric cancer tissues and were associated with tumor size, migration and poor differentiation status. Moreover, overexpression of cyclin G2 attenuated tumor growth and metastasis both in vitro and in vivo. Dpr1 was identified as a cyclin G2-interacting protein which was required for the cyclin G2-mediated inhibition of ß-catenin expression. Mechanically, cyclin G2 impacted the activity of CKI to phosphorylate Dpr1, which has been proved to be a protein that acts as a suppressor of Wnt/ß-catenin signaling when unphosphorylated. Furthermore, GSK-3ß inhibitors abolished the cyclin G2-induced suppression of cell proliferation and migration. CONCLUSIONS: This study demonstrates that cyclin G2 suppresses Wnt/ß-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1.

Cyclin G2 Suppresses Glomerulosclerosis by Regulating Canonical Wnt Signalling.

Recent data has shown that cyclin G2 (CCNG2) is an atypical cyclin that inhibits cell cycle progression and is often dysregulated in human cancers. The involvement of cyclin G2 in the occurrence and development of diabetic nephropathy (DN) has not been determined. In the present study, we conducted cyclin G2 knockout studies to determine whether this protein regulates glomerulosclerosis in DN mice. We found that cyclin G2 regulated the expression of renal glomerulosclerosis-related proteins via the canonical Wnt signalling pathway in glomerular mesangial cells. A cyclin G2 deficiency resulted in more severe renal injury in DN mice. These findings provided new insight into the pathogenesis of DN, revealing that cyclin G2 has a protective role in glomerulosclerosis and is a potential new target for the prevention and treatment of DN.

Transcription Factor FOXO3a Is a Negative Regulator of Cytotoxicity of Fusarium mycotoxin in GES-1 Cells.

Molecular mechanism and key factors responsible for cytotoxicity against mycotoxin deoxynivalenol (DON) from Fusarium pathogens are rarely elucidated. In this study, rapid increases of ROS were first observed in human gastric epithelial (GES-1) cells under DON exposure. Mitochondrial DNA damage, impaired respiratory chain, and decreased oxygen consumption rate (OCR) values, as well as G2/M cell cycle arrest and apoptosis, were also detected. Via combinatorial approaches of a large-scale microarray of differentially expressed genes, high content and RNAi analysis, a transcription factor of Forkhead box O3 (FOXO3a) was found with crucial functionalities, regulated some apoptotic genes associated with mitochondrial toxicity and cell death after activation by nuclear translocation. Namely, knockdown of FOXO3a decreased the cytotoxicity of DON to GES-1 cells. Moreover, knockdown of the FOXO ortholog DAF16 in Caenorhabditis elegans increased the resistance to DON-induced cytotoxicity. Simultaneously, the signaling pathway of ROS/JNK/FOXO3a of DON-induced cytotoxicity was newly proposed. In total, FOXO3a via ROS/JNK/FOXO3a plays a critical role to function as negative regulator associating with DON-induced cytotoxicity, with the potential extending to other substances.

Farnesoid X receptor inhibits proliferation of human colorectal cancer cells via the miR­135A1/CCNG2 signaling pathway.

Colorectal cancer (CRC) is among the most common malignancies of the digestive system. Dysregulation of miRNAs and the farnesoid X receptor (FXR) are involved in the progression of CRC. In the present study, the effects of FXR and miR­135A1 in CRC were evaluated. Reverse transcription quantitative­polymerase chain reaction (RT­qPCR) was used to examine the expression of miR­135A1 in patient CRC tissues and adjacent non­tumor tissues, as well as cell lines. The association between miR­135A1 and clinical characteristics of patients with CRC was also investigated. RT­qPCR and western blotting were used to evaluate the expression of miR­135A1 targets. Regulation of cyclin G2 (CCNG2) by miR­135A1was confirmed using luciferase assays. The biological effects of miR­135A1 were assessed in transfected and untransfected CRC cell lines using colony formation assays, cell­cycle analysis by flow cytometry, and CCK­8 assays. miR­135A1 was upregulated in CRC specimens and cell lines. miR­135A1 expression was strongly associated with poor cell differentiation, high expression of carbohydrate antigen (CA)125, CA199, carcinoembryonic antigen and survival rate of patients with CRC. Expression of CCNG2 was downregulated in CRC patients and cell lines, and was further demonstrated to be among the downstream targets of miR­135A1. The present study indicated that inhibition of miR­135A1 expression leads to cell cycle arrest and inhibition of proliferation of CRC cells via increasing CCNG2 expression. In the present study, activation of FXR by GW4064 increased CCNG2 expression via suppression of miR­135A1 expression, and the FXR/miR­135A1/CCNG2 axis was demonstrated to be involved in mediating cell proliferation. In conclusion, activation of FXR by GW4064 suppresses cell proliferation and causes cell cycle arrest in CRC, and the miR­135A1/CCNG2 pathway was suggested to be involved in this step.

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.

Morin inhibited lung cancer cells viability, growth, and migration by suppressing miR-135b and inducing its target CCNG2.

Lung cancer is one of the most severe threats with the highest mortality rate to humans in the world. Recently, morin has been reported to have anti-tumor properties observed in several types of cancers. However, its mechanism is still unclear. We assessed the influences of morin on cell viability, colony formation, and migration ability of A549 and employed microRNA array to identify the microRNAs affected by morin. We found that morin-treated A549 cells showed statistically decreased cell viability, colony formation, and migration rate when comparing with the dimethyl sulfoxide-treated cells. Microarray results showed that with the treatment of morin, the expression level of miR-135b significantly reduced compared the control group, suggesting that morin may exert its anti-cancer property by suppressing the expression of miR-135b. In addition, we found a potential binding site of miR-135b within 3' untranslated region of CCNG2-encoding cyclin homolog cyclin-G2. We evidenced that miR-135b directly targets CCNG2, which could be a potential biomarker of lung cancer prognosis. Morin exerts its anti-tumor function via downregulating the expression of miR-135b that directly targets and represses CCNG2.

Epidermal growth factor promotes cyclin G2 degradation via calpain-mediated proteolysis in gynaecological cancer cells.

Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.