MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

miR-17-92 cluster-BTG2 axis regulates B-cell receptor signaling in mantle cell lymphoma.

B-cell receptor (BCR) signaling is critically activated and stable for mantle cell lymphoma (MCL), but the underlying mechanism of the activated BCR signaling pathway is not clear. The pathogenic basis of miR-17-92 cluster remains unclear although the oncogenic microRNA (miRNA) miR-17-92 cluster is highly expressed in patients with MCL. We revealed that miR-17-92 cluster overexpression is partly dependent on SOX11 expression and chromatin acetylation of MIR17HG enhancer regions. Moreover, miR-17-92 cluster regulates not only cell proliferation but BCR signaling activation in MCL cell lines. To comprehensively identify miR-17-92 cluster target genes, we performed pulldown-seq, where target RNA of miRNA was captured using the biotinylated miRNA mimics and magnetic bead-coated streptavidin, and quantified using next-generation sequencing. The pulldown-seq identified novel miRNA target genes, including tumor suppressors such as BTG2 (miR-19b), CDKN2A (miR-17), SYNE1 (miR-20a), TET2 (miR-18, miR-19b, and miR-92a), TNFRSF10A (miR-92a), and TRAF3 (miR-17). Notably, the gene expression profile data of patients with MCL revealed that BTG2 expression was negatively associated with that of BCR signature genes, and low BTG2 expression was associated with poor overall survival. Moreover, BTG2 silencing in MCL cell lines significantly induced BCR signaling overactivation and cell proliferation. Our results suggest an oncogenic role of miR-17-92 cluster-activating BCR signaling throughout BTG2 deregulation in MCL. Furthermore, this may contribute to the prediction of the therapeutic efficacy and improved outcomes of MCL.

miR-92b-3p protects retinal tissues against DNA damage and apoptosis by targeting BTG2 in experimental myopia.

BACKGROUND: Myopia is one of the eye diseases that can damage the vision of young people. This study aimed to explore the protective role of miR-92b-3p against DNA damage and apoptosis in retinal tissues of negative lens-induced myopic (LIM) guinea pigs by targeting BTG2. METHODS: Biometric measurements of ocular parameters, flash electroretinogram (FERG), and retinal thickness (RT) were performed after miR-92b-3p intravitreal injection in LIM guinea pigs. The apoptotic rate was detected by Annexin V-FITC/PI double staining, and the change in mitochondrial membrane potential was measured by JC-1 staining. Retinal apoptosis and expression of p53, BTG2, and CDK2 were explored by TdT-mediated dUTP-biotin nick labeling (TUNEL) and immunofluorescence staining assays, respectively. BTG2 and its upstream and downstream molecules at gene and protein levels in retinal tissues were measured by real-time quantitative PCR (qPCR) and Western blotting. RESULTS: Compared with normal controls (NC), the ocular axial length of LIM guinea pig significantly increased, whereas refraction decreased. Meanwhile, dMax-a and -b wave amplitudes of ERG declined, retinal thickness was decreased, the number of apoptotic cells and apoptotic rate in LIM eyes was exaggerated, and the mitochondrial membrane potential significantly decreased. In addition, results of qPCR and Western blot assays showed that the expression levels of p53, BTG2, CDK2, and BAX in LIM guinea pigs were higher than the levels of the NC group, whereas the BCL-2 expression level was decreased. By contrast, the miR-92b-3p intravitreal injection in LIM guinea pigs could significantly inhibit axial elongation, alleviate DNA damage and apoptosis, and thus protect guinea pigs against myopia. CONCLUSION: In conclusion, p53 and BTG2 were activated in the retinal tissue of myopic guinea pigs, and the activated BTG2 could elevate the expression of CDK2 and BAX, and attenuate the expression of BCL-2, which in turn promote apoptosis and eventually lead to retinal thinning and impaired visual function in myopic guinea pigs. The miR-92b-3p intravitreal injection can attenuate the elongation of ocular length and retinal thickness, and inhibit the CDK2, BAX, and p53 expression by targeting BTG2, thereby ameliorating DNA damage and apoptosis in LIM guinea pigs and protecting ocular tissues.

Btg1 and Btg2 regulate neonatal cardiomyocyte cell cycle arrest.

Rodent cardiomyocytes undergo mitotic arrest in the first postnatal week. Here, we investigate the role of transcriptional co-regulator Btg2 (B-cell translocation gene 2) and functionally-similar homolog Btg1 in postnatal cardiomyocyte cell cycling and maturation. Btg1 and Btg2 (Btg1/2) are expressed in neonatal C57BL/6 mouse left ventricles coincident with cardiomyocyte cell cycle arrest. Btg1/2 constitutive double knockout (DKO) mouse hearts exhibit increased pHH3+ mitotic cardiomyocytes compared to Wildtype at postnatal day (P)7, but not at P30. Similarly, neonatal AAV9-mediated Btg1/2 double knockdown (DKD) mouse hearts exhibit increased EdU+ mitotic cardiomyocytes compared to Scramble AAV9-shRNA controls at P7, but not at P14. In neonatal rat ventricular myocyte (NRVM) cultures, siRNA-mediated Btg1/2 single and double knockdown cohorts showed increased EdU+ cardiomyocytes compared to Scramble siRNA controls, without increase in binucleation or nuclear DNA content. RNAseq analyses of Btg1/2-depleted NRVMs support a role for Btg1/2 in inhibiting cell proliferation, and in modulating reactive oxygen species response pathways, implicated in neonatal cardiomyocyte cell cycle arrest. Together, these data identify Btg1 and Btg2 as novel contributing factors in mammalian cardiomyocyte cell cycle arrest after birth.

LncRNA CARMN inhibits cervical cancer cell growth via the miR-92a-3p/BTG2/Wnt/ß-catenin axis.

Long noncoding RNA (lncRNA) cardiac mesoderm enhancer-associated noncoding RNA (CARMN) is a newly discovered tumor-suppressor lncRNA in cancers. However, its role in cervical cancer (CC) remains elusive. This study was conducted to analyze the molecular mechanism of CARMN in CC cell growth and provide a novel theoretical basis for CC treatment. RT-qPCR and clinical analysis revealed that CARMN and B-cell translocation gene 2 (BTG2) were downregulated, whereas miR-92a-3p was upregulated in CC tissues and cells and their expressions were correlated with clinicopathological characteristics and prognosis. MTT assay, flow cytometry, and Transwell assays revealed that CARMN overexpression reduced proliferation, migration, and invasion and increased apoptosis rate in CC cells. Mechanically, CARMN repressed miR-92a-3p to promote BTG2 transcription. Functional rescue assays revealed that miR-92a-3p overexpression or BTG2 downregulation reversed the inhibitory role of CARMN overexpression in CC cell growth. Western blot analysis elicited that Wnt3a and ß-catenin were elevated in CC cells and CARMN blocked the Wnt/ß-catenin signaling pathway via the miR-92a-3p/BTG2 axis. Overall, our findings demonstrated that CARMN repressed miR-92a-3p to upregulate BTG2 transcription and then blocked the Wnt/ß-catenin signaling pathway, thereby suppressing CC cell growth.

BTG2 acts as an inducer of muscle stem cell senescence.

BACKGROUND: Muscle aging is associated with muscle stem cell (MuSC) senescence, a process of whose DNA damage accumulation is considered as one of the leading causes. BTG2 had been identified as a mediator of genotoxic and cellular stress signaling pathways, however, its role in senescence of stem cells, including MuSC, remains unknown. METHOD: We first compared MuSCs isolated from young and old mice to evaluate our in vitro model of natural senescence. CCK8 and EdU assays were utilized to assess the proliferation capacity of the MuSCs. Cellular senescence was further assessed at biochemical levels by SA-ß-Gal and γHA2.X staining, and at molecular levels by quantifying the expression of senescence-associated genes. Next, by performing genetic analysis, we identified Btg2 as a potential regulator of MuSC senescence, which was experimentally validated by Btg2 overexpression and knockdown in primary MuSCs. Lastly, we extended our research to humans by analyzing the potential links between BTG2 and muscle function decline in aging. RESULTS: BTG2 is highly expressed in MuSCs from elder mice showing senescent phenotypes. Overexpression and knockdown of Btg2 stimulates and prevents MuSCs senescence, respectively. In humans, high level of BTG2 is associated with low muscle mass in aging, and is a risk factor of aging-related diseases, such as diabetic retinopathy and HDL cholesterol. CONCLUSION: Our work demonstrates BTG2 as a regulator of MuSC senescence and may serve as an intervention target for muscle aging.

Linoleic acid derivatives target miR-361-3p/BTG2 to confer anticancer effects in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a deadly hematologic malignancy. In this study, miR-361-3p and BTG2 gene expression in AML blood and healthy specimens were analyzed using quantitative real-time reverse transcription polymerase chain reaction. A significant negative correlation between miR-361-3p and BTG2 was observed. The cell viability and apoptosis were measured by CCK-8 assay, EdU incorporation assay and flow cytometry. A dual-luciferase reporter gene assay was performed to confirm the binding sequence between miR-361-3p and BTG2 messenger RNA 3'-untranslated region. 9s-Hydroxyoctadecadienoic acid (9s-HODE), a major active derivative of linoleic acid, reduced the viability and induced cell apoptosis of HL-60 cells. Furthermore, the miR-361-3p mimics and siBTG2 reversed the above effects of 9s-HODE. 9s-HODE exerted an anti-AML effect through, at least partly, regulating the miR-361-3p/BTG2 axis.

miR-103-3p Regulates the Proliferation and Differentiation of C2C12 Myoblasts by Targeting BTG2.

Skeletal muscle, a vital and intricate organ, plays a pivotal role in maintaining overall body metabolism, facilitating movement, and supporting normal daily activities. An accumulating body of evidence suggests that microRNA (miRNA) holds a crucial role in orchestrating skeletal muscle growth. Therefore, the primary aim of this study was to investigate the influence of miR-103-3p on myogenesis. In our study, the overexpression of miR-103-3p was found to stimulate proliferation while suppressing differentiation in C2C12 myoblasts. Conversely, the inhibition of miR-103-3p expression yielded contrasting effects. Through bioinformatics analysis, potential binding sites of miR-103-3p with the 3'UTR region of BTG anti-proliferative factor 2 (BTG2) were predicted. Subsequently, dual luciferase assays conclusively demonstrated BTG2 as the direct target gene of miR-103-3p. Further investigation into the role of BTG2 in C2C12 myoblasts unveiled that its overexpression impeded proliferation and encouraged differentiation in these cells. Notably, co-transfection experiments showcased that the overexpression of BTG2 could counteract the effects induced by miR-103-3p. In summary, our findings elucidate that miR-103-3p promotes proliferation while inhibiting differentiation in C2C12 myoblasts by targeting BTG2.

Heat shock transcription factor HSF2 modulates the autophagy response through the BTG2-SOD2 axis.

The heat shock transcription factor HSF1 regulates the inducible Hsp gene transcription, whereas HSF2 is involved in the constitutive transcription. HSFs can work for the non-heat shock genes transcription in a case-specific manner to facilitate normal cellular functions. Here, we demonstrate that HSF2 acts as an upstream regulator of heat shock-induced autophagy response in a rat histiocytoma. The heat-induced HSF2 transactivates the B-cell translocation gene-2 (BTG2) transcription, and the latter acts as a transcriptional coactivator for superoxide dismutase (SOD2). The altered HSF2 promoter occupancy on the BTG2 promoter enhances BTG2 transcription. Since SOD2 regulation is linked to mitochondrial redox sensing, HSF2 appears to act as a redox sensor in deciding the cell fate. The HSF2 shRNA or NFE2L2/BTG2 siRNA treatments have interfered with the autophagy response. We demonstrate that HSF2 is an upstream activator of autophagy response, and the HSF2-BTG2-SOD2 axis acts as a switch between the non-selective (micro/macro) and selective (chaperone-mediated) autophagy.

Lowly expressed LNC01136 fails to aid HIF-1α to induce BTG2 expression resulting in increased proliferation of retinal microvascular endothelial cells.

BACKGROUND: Retinal neovascularization (RN), a major cause of blindness occurring in multiple types of ophthalmic diseases, is closely associated with hypoxic conditions. However, the underlying pathological mechanisms of RN have not been fully elucidated. BTG2 is anti-proliferative factor. The up-stream of BTG2 gene within 3000 bp expresses a long non-coding RNA, LNC01136. METHODS: we initially compared the expression of BTG2 and LNC01136 in human retinal microvascular endothelial cells (hRMECs) with other eye-associated cells, including Muller cells, ARPE19 cells and RGC-5, in response to a hypoxia mimetic agent (CoCl2). FISH and PCR tests were performed to determine the enrichment of LNC01136 in different cellular components. LNC01136 were overexpressed or knockdown to determine the effect on BTG2 expression. Finally, ChIP, RIP and Co-IP assays were performed to determine the interaction among BTG2, HIF-1α, LNC01136 and CNOT7. RESULTS: After the treatment with CoCl2, expression levels of BTG2 and LNC01136 were strongly induced in Muller cells, ARPE19 cells and RGC-5, but weakly in hRMECs. LNC01136 is prominently located in cell nucleus and aids HIF-1α to enhance transcription of BTG2, which consequently inhibits cell growth. The anti-proliferative effect of BTG2 is probably associated to the interaction with CNOT7 and the regulation of multiple cell cycle-related proteins. CONCLUSIONS: This study revealed that LNC01136 is a cell growth suppressor by recruiting HIF-1α to induce BTG2 expression. However the low expression of LNC01136 in hRMECs compared to other eye-associated cells promoted hRMECs' proliferation, which is probably a cause of RN under hypoxia.

DSCR9/miR-21-5p axis inhibits pancreatic cancer proliferation and resistance to gemcitabine via BTG2 signaling.

The outcome of pancreatic adenocarcinoma (PAAD) patients is poor, given resistance to gemcitabine. Long noncoding RNA (lncRNA) has been implicated in the carcinogenesis of pancreatic cancer; however, its function and mechanism in PAAD resistance to gemcitabine (GEM) are yet unknown. Herein, we demonstrate that lncRNA DSCR9 is significantly reduced in PAAD in vitro and in vivo. CCK-8, BrdU and flow cytometry assays show that overexpression of DSCR9 markedly suppresses pancreatic cancer cell proliferation and invasion, and promotes apoptosis under gemcitabine treatment. BTG2 acts as a tumor suppressor by reducing the proliferation and invasion of pancreatic cancer cells and increasing gemcitabine-induced apoptosis. Immunofluorescence (IF) staining combined with fluorescence in situ hybridization (FISH) of pancreatic cancer tissues shows that DSCR9 and BTG2 are both increased in pancreatic cancer tissues. Luciferase assay shows that miR-21-5p simultaneously binds to DSCR9 and 3'UTR of BTG2; DSCR9 relieves miR-21-5p-induced inhibition of BTG2 by competing with BTG2 for miR-21-5p binding. Overexpression of miR-21-5p enhances the invasiveness of pancreatic cancer cells by promoting cancer cell proliferation and invasion and attenuating gemcitabine-induced apoptosis. Overexpression of miR-21-5p attenuates the effect of DSCR9 overexpression on BTG2 expression and invasiveness of pancreatic cancer cells. Finally, miR-21-5p expression is increased, while BTG2 expression is decreased in pancreatic cancer tissues. miR-21-5p is negatively correlated with DSCR9 and BTG2. In conclusion, the DSCR9/miR-21-5p/BTG2 axis modulates pancreatic cancer proliferation, invasion, and gemcitabine resistance.

miR-365 secreted from M2 Macrophage-derived extracellular vesicles promotes pancreatic ductal adenocarcinoma progression through the BTG2/FAK/AKT axis.

Clinical and experimental evidence indicates that tumour-associated macrophages support cancer progression. Moreover, macrophage-derived extracellular vesicles (EVs) are involved in pathogenesis of multiple cancers, yet the functions of molecular determinants in which have not been fully understood. Herein, we aim to understand whether macrophage modulates pancreatic ductal adenocarcinoma (PDAC) progression in an EV-dependent manner and the underlying mechanisms. microRNA (miR)-365 was experimentally determined to be enriched in the EVs from M2 macrophages (M2-EVs), which could be transferred into PDAC cells. Using a co-culture system, M2-EVs could enhance the proliferating, migrating and invading potentials of PDAC cells, while inhibition of miR-365 in M2-EVs could repress these malignant functions. B-cell translocation gene 2 (BTG2) was identified to be a direct target of miR-365, while the focal adhesion kinase (F/ATP)-dependent tyrosine kinase (AKT) pathway was activated by miR-365. We further demonstrated that overexpression of BTG2 could delay the progression of PDAC in vitro, whereas by impairing BTG2-mediated anti-tumour effect, M2-EV-miR-365 promoted PDAC progression. For validation, a nude mouse model of tumorigenesis was established, in which we found that targeting M2-EV-miR-365 contributed to suppression of tumour growth. Collectively, M2-EVs carry miR-365 to suppress BTG2 expression, which activated FAK/AKT pathway, thus promoting PDAC development.

LINC01089 inhibits the progression of cervical cancer via inhibiting miR-27a-3p and increasing BTG2.

BACKGROUND: Increasing evidence confirms that long non-coding RNA (lncRNA) has a vital impact on the procession of cervical cancer (CC). The present study aimed to investigate the clinical significance of LINC01089 in CC, as well as explore its biological functions and potential molecular mechanisms. METHODS: A quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to investigate the expression of LINC01089 and miR-27a-3p in CC cells and tissues. Analysis of the correlation between the expression level of LINC01089 and the clinical pathological parameters of CC was then conducted. The human CC cell lines HeLa and SiHa were utilized for transfection to establish a gain-of-function model and loss-of-function models. Western blotting and a qRT-PCR were performed to detect B-cell translocation gene-2 (BTG2) expression in CC cells. Cell counting kit (CCK)-8 and 5-bromo-2-deoxyuridine (BrdU) assays were performed to detect the proliferation of CC cells. The transwell method was employed to evaluate the migration and invasion of CC cells. The interactions between LINC01089 and miR-27a-3p were verified by bioinformatics, a dual luciferase reporter gene experiment and a RNA immunoprecipitation experiment, respectively. RESULTS: The expression of LINC01089 in CC was markedly down-regulated. The low expression of LINC01089 in CC was closely associated with a larger tumor size and positive lymph node metastasis. Moreover, overexpression of LINC01089 impeded the proliferation and metastasis of CC cells, whereas knockdown of LINC01089 had the opposite biological functions. In terms of mechanism, LINC01089 could sponge miR-27a-3p and indirectly up-regulate BTG2 expression. CONCLUSIONS: LINC01089, as a tumor suppressor, impedes the development of CC by targeting miR-27a-3p to up-regulate BTG2 expression.

Urinary Exosomal miRNAs as biomarkers of bladder Cancer and experimental verification of mechanism of miR-93-5p in bladder Cancer.

BACKGROUND: Bladder cancer (BC) is one of the most common malignancies globally. Early diagnosis of it can significantly improve patients' survival and quality of life. Urinary exosomes (UEs)-derived miRNAs might be a promising biomarker for BC detection. METHOD: A total of 12 patients with BC and 4 non-cancerous participants (as healthy control) were recruited from a single center between March 2018 and December 2019 as the discovery set. Midstream urine samples from each participants were collected and high-throughput sequencing and differentially expression analysis were conducted. Combined with miRNA expression profile of BC tissue from The Cancer Genome Atlas (TCGA), miRNAs biomarkers for BC were determined. Candidate miRNAs as biomarkers were selected followed by verification with a quantitative reverse-transcription polymerase chain reaction assay in an independent validation cohort consisting of 53 BC patients and 51 healthy controls. The receiver-operating characteristic (ROC) curve was established to evaluate the diagnostic performance of UE-derived miRNAs. The possible mechanism of miRNAs were revealed by bioinformatic analysis and explored in vitro experiments. RESULTS: We identified that miR-93-5p, miR-516a-5p were simultaneously significantly increased both in UEs from BC compared with healthy control and BC tissue compared with normal tissue, which were verified by RT-qPCR in the validation cohort. Subsequently, the performance to discover BC of the miR-93-5p, miR-516a-5p was further verified with an area under ROC curve (AUC) of 0.838 and 0.790, respectively, which was significantly higher than that of urine cytology (AUC = 0.630). Moreover, miR-93-5p was significantly increased in muscle-invasive BC compared with non-muscle-invasive BC with an AUC of 0.769. Bioinformatic analysis revealed that B-cell translocation gene 2(BTG2) gene may be the hub target gene of miR-93-5p. In vitro experiments verified that miR-93-5p suppressed BTG2 expression and promoted BC cells proliferation, invasion and migration. CONCLUSION: Urine derived exosomes have a distinct miRNA profile in BC patients, and urinary exosomal miRNAs could be used as a promising non-invasive tool to detect BC. In vitro experiments suggested that miR-93-5p overexpression may contribute to BC progression via suppressing BTG2 expression.

TINCR inhibits the proliferation and invasion of laryngeal squamous cell carcinoma by regulating miR-210/BTG2.

BACKGROUND: Terminal differentiation-induced ncRNA (TINCR) plays an essential role in epidermal differentiation and is involved in the development of various cancers. METHODS: qPCR was used to detect the expression level of TINCR in tissues and cell lines of laryngeal squamous cell carcinoma (LSCC). The potential targets of TINCR were predicted by the bioinformation website. The expression of miR-210 and BTG2 genes were detected by qPCR, and the protein levels of BTG2 and Ki-67 were evaluated by western blot. CCK-8 assay, scratch test, and transwell chamber were used to evaluate the proliferation, invasion, and metastasis ability of LSCC cells. The relationships among TINCR, miR-210, and BTG2 were investigated by bioinformatics software and luciferase reporter assay. The in vivo function of TINCR was accessed on survival rate and tumor growth in nude mice. RESULTS: We used qRT-PCR to detect the expression of TINCR in laryngeal squamous cell carcinoma (LSCC) tissues and cells and found significantly lower levels in cancer tissues compared with adjacent tissues. Additionally, patients with high TINCR expression had a better prognosis. TINCR overexpression was observed to inhibit the proliferation and invasion of LSCC cells. TINCR was shown to exert its antiproliferation and invasion effects by adsorbing miR-210, which significantly promoted the proliferation and invasion of laryngeal squamous cells. Overexpression of miR-210 was determined to reverse the tumour-suppressive effects of TINCR. BTG2 (anti-proliferation factor 2) was identified as the target gene of miR-210, and BTG2 overexpression inhibited the proliferation and invasion of LSCC cells. BTG2 knockdown relieved the inhibitory effects of TINCR on the proliferation and invasion of LSCC. Finally, TINCR upregulation slowed xenograft tumour growth in nude mice and significantly increased survival compared with control mice. CONCLUSION: The results of this study suggest that TINCR inhibits the proliferation and invasion of LSCC by regulating the miR-210/BTG2 pathway, participates in cell cycle regulation, and may become a target for the treatment of LSCC.

Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression.

Hepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

SRXN1 stimulates hepatocellular carcinoma tumorigenesis and metastasis through modulating ROS/p65/BTG2 signalling.

Sulfiredoxin 1 (SRXN1) is a pivotal regulator of the antioxidant response in eukaryotic cells. However, the role of SRXN1 in hepatocellular carcinoma (HCC) is far from clear. The present study aims to elucidate whether SRXN1 participates in tumorigenesis and metastasis of HCC and to determine the molecular mechanisms. We found that SRXN1 expression was up-regulated in HCC tissue samples and correlated with poor prognosis in HCC patients. We also observed that SRXN1 knockdown by transient siRNA transfection inhibited HCC cell proliferation, migration and invasion. Overexpression of SRXN1 increased HCC cell migration and invasion. B-cell translocation gene 2 (BTG2) was identified as a downstream target of SRXN1. Mechanistic studies revealed that SRXN1-depleted reactive oxygen species (ROS) modulated migration and invasion of HCC cells. In addition, the ROS/p65/BTG2 signalling hub was found to regulate the epithelial-mesenchymal transition (EMT), which mediates the pro-metastasis role of SRXN1 in HCC cells. In vivo experiments showed SRXN1 promotes HCC tumour growth and metastasis in mouse subcutaneous xenograft and metastasis models. Collectively, our results revealed a novel pro-tumorigenic and pro-metastatic function of SRXN1 in HCC. These findings demonstrate a rationale to exploit SRXN1 as a therapeutic target effectively preventing metastasis of HCC.

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling.

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.

miR-487a performs oncogenic functions in osteosarcoma by targeting BTG2 mRNA.

Aberrant microRNA (miRNA) expression plays a critical role in osteosarcoma (OS) pathogenesis. In this study, we elucidated the involvement of miR-487a in OS and the underlying molecular mechanisms. We found that miR-487a was upregulated in OS clinical samples and cell lines. Knockdown of miR-487a suppressed OS cell growth and invasion and induced apoptosis; however, overexpression of miR-487a promoted OS cell growth and invasion. Accordingly, downregulation of miR-487a significantly suppressed tumor growth of OS xenografts in vivo. Furthermore, B-cell translocation gene 2 (BTG2) mRNA was found to be a novel target of miR-487a. Knockdown of BTG2 using small interfering RNA (siRNA) recapitulated the oncogenic effects of miR-487a, whereas BTG2 overexpression partially reversed these effects. Finally, miR-487a levels were found to be negatively correlated with BTG2 expression in OS clinical samples. Collectively, our data suggest that miR-487a is an oncogenic miRNA in OS and it lowers BTG2 expression.

Tumor suppressors BTG1 and BTG2: Beyond growth control.

Since the identification of B-cell translocation gene 1 (BTG1) and BTG2 as antiproliferation genes more than two decades ago, their protein products have been implicated in a variety of cellular processes including cell division, DNA repair, transcriptional regulation and messenger RNA stability. In addition to affecting differentiation during development and in the adult, BTG proteins play an important role in maintaining homeostasis under conditions of cellular stress. Genomic profiling of B-cell leukemia and lymphoma has put BTG1 and BTG2 in the spotlight, since both genes are frequently deleted or mutated in these malignancies, pointing towards a role as tumor suppressors. Moreover, in solid tumors, reduced expression of BTG1 or BTG2 is often correlated with malignant cell behavior and poor treatment outcome. Recent studies have uncovered novel roles for BTG1 and BTG2 in genotoxic and integrated stress responses, as well as during hematopoiesis. This review summarizes what is currently known about the roles of BTG1 and BTG2 in these and other cellular processes. In addition, we will highlight the molecular mechanisms and biological consequences of BTG1 and BTG2 deregulation during cancer progression and elaborate on the potential clinical implications of these findings.