Circular RNA circNF1 siRNA Silencing Inhibits Glioblastoma Cell Proliferation by Promoting the Maturation of miR-340.
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ABSTRACT:
SUBMITTER: Liu L
PROVIDER: S-EPMC8475906 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Deregulation of microRNAs (miRs) contributes to tumorigenesis. Down-regulation of miR-340 is observed in multiple types of cancers. However, the biological function of miR-340 in glioblastoma multiforme (GBM) remains largely unknown. In the present study, we demonstrated that expression of miR-340 was downregulated in both glioma cell lines and tissues. Survival of GBM patients with high levels of miR-340 was significantly extended in comparison to patients expressing low miR-340 levels. Biological functional experiments showed that the restoration of miR-340 dramatically inhibited glioma cell proliferation, induced cell-cycle arrest and apoptosis, suppressed cell motility and promoted autophagy and terminal differentiation. Mechanistic studies disclosed that, miR-340 over-expression suppressed several oncogenes including p-AKT, EZH2, EGFR, BMI1 and XIAP. Furthermore, ROCK1 was validated as a direct functional target miR-340 and silencing of ROCK1 phenocopied the anti-tumor effect of mR-340. Our findings indicate an important role of miR-340 as a glioma killer, and suggest a potential prognosis biomarker and therapeutic target for GBM.
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Project description:Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.
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Project description:Alterations in cellular metabolism and bioenergetics are vital for cancer cell growth and motility. Here, the role of the mitochondrial protein voltage-dependent anion channel (VDAC1), a master gatekeeper regulating the flux of metabolites and ions between mitochondria and the cytoplasm, in regulating the growth of several cancer cell lines was investigated by silencing VDAC1 expression using small interfering RNA (siRNA). A single siRNA specific to the human VDAC1 sequence at nanomolar concentrations led to some 90% decrease in VDAC1 levels in the lung A549 and H358, prostate PC-3, colon HCT116, glioblastoma U87, liver HepG2, and pancreas Panc-1 cancer cell lines. VDAC1 silencing persisted 144 hours post-transfection and resulted in profound inhibition of cell growth in cancer but not in noncancerous cells, with up to 90% inhibition being observed over 5 days that was prolonged by a second transfection. Cells expressing low VDAC1 levels showed decreased mitochondrial membrane potential and adenoside triphosphate (ATP) levels, suggesting limited metabolite exchange between mitochondria and cytosol. Moreover, cells silenced for VDAC1 expression showed decreased migration, even in the presence of the wound healing accelerator basic fibroblast growth factor (bFGF). VDAC1-siRNA inhibited cancer cell growth in a Matrigel-based assay in host nude mice. Finally, in a xenograft lung cancer mouse model, chemically modified VDAC1-siRNA not only inhibited tumor growth but also resulted in tumor regression. This study thus shows that VDAC1 silencing by means of RNA interference (RNAi) dramatically inhibits cancer cell growth and tumor development by disabling the abnormal metabolic behavior of cancer cells, potentially paving the way for a more effective pipeline of anticancer drugs.
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Project description:BackgroundThe dysregulation of oncogenes and tumor suppressor genes plays an important role in many cancers, including hepatocellular carcinoma (HCC), which is one of the most common cancers in the world. In a previous microarray experiment, we found that DLGAP5 is overexpressed in HCCs. However, whether the up-regulation of DLGAP5 contributes to hepatocarcinogenesis remains unclear.Methodology/principal findingsIn this study, we showed that DLGAP5 was significantly up-regulated in 76.4% (168 of 220) of the analyzed HCC specimens when compared with adjacent liver tissue. DLGAP5 overexpression was evident in 25% (22 of 88) of the HCC specimens without AFP expression, suggesting that DLGAP5 may be a novel biomarker for HCC pathogenesis. The silencing of DLGAP5 gene expression by RNA interference significantly suppressed cell growth, migration and colony formation in vitro. The expression level of DLGAP5 was also found to be related to the methylation level of its promoter in the HCC specimens.Conclusions/significanceTaken together, these data suggest that the expression of DLGAP5 is regulated by methylation and that the up-regulation of DLGAP5 contributes to HCC tumorigenesis by promoting cell proliferation.
| S-EPMC3851768 | biostudies-literature
Project description:BackgroundBladder cancer (BC) has high mortality due to distant metastasis. Previous works suggested that microRNA (miRNA)-340 is a critical regulator for the development and progression of various cancers. The specific biological function of miR-340 in BC is little known.MethodsIn the present study, RT-qPCR was performed to measure the expression of miR-340 in paired BC tissues and adjacent non-tumor tissues. Next, the target gene of miR-340 was identified using dual-luciferase reporter assay and its level was also tested in tissues. Moreover, cell proliferation and apoptosis were analyzed by CCK-8 and flow cytometry. Finally, the expression of PCNA, Bax was detected by RT-qPCR and western blotting, as well as PI3K/AKT signaling measured by western blotting.ResultThe results demonstrated that miR-340 expression was downregulated and its target Glut-1 level was upregulated in BC tissues. Functionally, overexpression of miR-340 suppressed the proliferation and induced apoptosis in BC cells, while Glut-1 reversed the suppression of proliferation or induction of apoptosis induced by miR-340. Additionally, miR-340 repressed PCNA, p-PI3K and p-AKT levels but enhanced Bax level, while Glut-1 rescued the effects.ConclusionIn conclusion, miR-340 functions as a tumor suppressor of BC, which inhibited proliferation and induced apoptosis by targeting Glut-1 partly through regulating PCNA, Bax expression and PI3K/AKT pathway. This study suggested that miR-340 is a potential target for the treatment of BC.
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Project description:Zinc fingers and homeoboxes 1 (ZHX1) is a transcription repressor that has been implicated in the tumorigenesis and progression of diverse tumors. The functional role and regulating mechanism of ZHX1 has not been elucidated in glioblastoma (GBM). Previous reports have suggested that a large number of non-coding RNAs play a vital role in glioma initiation and progression. This study aimed to investigate the functional role and co-regulatory mechanisms of the metastasis-associated lung adenocarcinoma transcript-1 (MALAT1)/ microRNA-199a (miR-199a)/ZHX1 axis in GBM. We analyzed the expression of the MALAT1/miR-199a/ZHX1 axis and its correlation with patients' overall survival using two different glioma gene-expression datasets. A series of in vitro and in vivo studies including dual luciferase reporter assay, fluorescence in situ hybridization (FISH), RNA immunoprecipitation, and pull-down experiments were completed to elucidate the biological significance of the MALAT1/miR-199a/ZHX1 axis in promoting glioma proliferation and progression. Elevated ZHX1 expression correlated with poor prognosis in GBM patients, and in vitro studies demonstrated that ZHX1 attenuated GBM cell apoptosis by downregulation of pro-apoptotic protein (Bax) and upregulation of anti-apoptotic protein (Bcl-2). Furthermore, knockdown of MALAT1 inhibited GBM proliferation and progression in vitro and reduced tumor volume and prolonged survival in an orthotopic GBM murine model. Finally, we demonstrated that MALAT1 promoted ZHX1 expression via acting as a competing endogenous RNA by sponging miR-199a. The MALAT1/miR-199a/ZHX1 axis promotes GBM cell proliferation and progression in vitro and in vivo, and its expression negatively correlates with GBM patient survival. Blocking the MALAT1/miR-199a/ZHX1 axis can serve as a novel therapeutic strategy for treating GBM.
| S-EPMC6819876 | biostudies-literature
Project description:BackgroundmiR-340-5p, localized to 5q35, is a tumor suppressor miRNA implicated in multiple cancers. As a CpG island is present at the putative promoter region of its host gene, RNF130, we hypothesized that the intronic miR-340-5p is a tumor suppressor miRNA epigenetically silenced by promoter DNA methylation of its host gene in multiple myeloma.ResultsBy pyrosequencing-confirmed methylation-specific PCR, RNF130/miR-340 was methylated in 8/15 (53.3%) myeloma cell lines but not normal plasma cells. Methylation correlated inversely with the expression of both miR-340-5p and RNF130. In completely methylated WL-2 and RPMI-8226R cells, 5-AzadC treatment led to demethylation and re-expression of miR-340-5p. In primary samples, RNF130/miR-340 methylation was detected in 4 (22.2%) monoclonal gammopathy of undetermined significance, 15 (23.8%) diagnostic myeloma, and 7 (23.3%) relapsed myeloma. RNF130/miR-340 methylation at diagnosis was associated with inferior overall survival (median 27 vs. 68 months; P = 3.944E-5). In WL-2 cells, overexpression of miR-340-5p resulted in reduced cellular proliferation [MTS, P = 0.002; verified in KMS-12-PE (P = 0.002) and RPMI-8226R (P = 2.623E-05) cells], increased cell death (trypan blue, P = 0.005), and enhanced apoptosis by annexin V-PI staining. Moreover, by qRT-PCR, overexpression of miR-340-5p led to repression of both known targets (CCND1 and NRAS) and bioinformatically predicted targets in MAPK signaling (MEKK1, MEKK2, and MEKKK3) and apoptosis (MDM4 and XIAP), hence downregulation of phospho-ERK1/2 and XIAP by Western blot. Furthermore, by qRT-PCR, in CD138-sorted primary samples (n = 37), miR-340-5p and XIAP were inversely correlated (P = 0.002). By luciferase assay, XIAP was confirmed as a direct target of miR-340-5p via targeting of the distal but not proximal seed region binding site.ConclusionsCollectively, tumor-specific methylation-mediated silencing of miR-340-5p is likely an early event in myelomagenesis with adverse survival impact, via targeting multiple oncogenes in MAPK signaling and apoptosis, thereby a tumor suppressive miRNA in myeloma.
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Project description:BackgroundMicroRNAs (miRNAs) function as important regulators of gene expression involved in tumor pathogenesis, including retinoblastoma. However, the expression profiles and potential roles in retinoblastoma are still largely unclear.Material and methodsDifferentially expressed miRNAs (DEmiRs) and genes (DEGs) in retinoblastoma were extracted from Gene Expression Omnibus (GEO) repository. Expression levels of miR-340 and WIF1 were detected in retinoblastoma tissues and cell lines by qRT-PCR. Both gain-of-function and loss-of-function experiments were performed to explore the effects of miR-340 on cell proliferation, migration and invasion. Bioinformatics analysis and luciferase reporter assay were used to explore the interaction between miR-340 and WIF1.ResultsA total of 11 DEmiRs were identified in retinoblastoma tissue and blood samples. Among them, we validated that miR-340 was the most highly expressed miRNA and correlated with tumor size, ICRB stage and optic nerve invasion. miR-340 was observed to enhance the proliferation, migration and invasion capacity of retinoblastoma cells. We then identified 26 DEGs from 3 retinoblastoma GEO datasets and subsequently constructed a miRNA-mRNA regulatory network. Further analysis revealed that WIF1 was a direct target of miR-340. Moreover, overexpression of WIF1 could repress retinoblastoma progression induced by miR-340 in vitro and in vivo.ConclusionCollectively, miR-340 functioned as an oncomiRNA to promote retinoblastoma cell proliferation, migration and invasion via regulating WIF1. Our data also provided multiple miRNAs and genes that may contribute to a better understanding of retinoblastoma pathogenesis.
| S-EPMC8187089 | biostudies-literature
Project description:The present study identified that fibroblast growth factor receptor 3 (FGFR3) was significantly upregulated in bone metastasis of lung adenocarcinoma. RNA interference (RNAi) is a powerful approach for treating a wide range of human diseases, including cancer, through downregulating the expression of selected genes. In the present study, the invasiveness of A549 cells cultured in vitro was altered by small interfering (si)RNA targeting FGFR3, and the regulatory effect of silencing FGFR3 on the expression levels of E-cadherin and matrix metalloproteinase (MMP)9 was investigated. Human lung adenocarcinoma A549 cells were transfected with synthetic specific siRNAs targeting a fragment of the FGFR3 gene (namely, siRNA-855, siRNA-1447 and siRNA-2076) or with negative control (NC) siRNA. Cells were divided into five groups (A, siRNA-855 group; B, siRNA-1447 group; C, siRNA-2076 group; D, NC-siRNA group; and E, blank control group). The effect of the above siRNAs targeting FGFR3 on the invasion capacity of A549 cells was detected by Transwell assay. siRNAs against FGFR3 were transfected into A549 cells with by Lipofectamine® 2000, and the expression levels of FGFR3, E-cadherin and MMP9 were measured by reverse transcription-quantitative polymerase chain reaction and western blot assay. The experimental findings indicated that the expression levels of FGFR3 and MMP9 were significantly reduced in the siRNA-FGFR3-transfected groups (A-C groups), compared with those in the D and E groups (P<0.01). In addition, the expression levels of E-cadherin were markedly elevated in the A-C groups, compared with those in the D and E groups (P<0.01). There was no significant difference in E-cadherin expression between the A-C groups, or between the D and E groups (P>0.05). These results indicated that siRNA-FGFR3 was able to decrease the invasiveness of A549 cells, inhibit the expression of MMP9 and increase the expression of E-cadherin by downregulating the expression of FGFR3. Taken together, the results of the present study indicated that the upregulation of E-cadherin expression and the downregulation of MMP9 expression are able to inhibit the migration of A549 cells, and siRNA silencing FGFR3 acts as a tumor suppressor in these cells.
| S-EPMC5228408 | biostudies-literature
Project description:Although increasing evidence indicated that deregulation of microRNAs (miRNAs) contributed to tumor initiation and progression, but little is known about the biological role of miR-340 in ovarian cancer (OC). In this study, we found that miR-340 expression was downregulated in OC tissues compared with its expression in normal ovarian epithelium and endometrium, and treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) or trichostatin A (TSA) increased miR-340 expression in OC cells. In addition, ectopic miR-340 expression inhibited OC cell growth and metastasis in vitro and in vivo. Four and a half LIM domains protein 2 (FHL2) was confirmed as a direct target of miR-340 and silencing FHL2 mimicked the effects of miR-340 in OC cells. Further mechanistic study showed that miR-340 inhibited the Wnt/β-catenin pathway by targeting FHL2, as well as downstream cell cycle and epithelial-to-mesenchymal transition (EMT) signals in OC cells. Moreover, the greatest association between miR-340 and FHL2 was found in 481 ovarian serous cystadenocarcinoma tissues via pan-cancer analysis. Finally, we revealed that lower miR-340 or higher FHL2 was associated with poor OC patient outcomes. Our findings indicate that the miR-340-FHL2 axis regulates Wnt/β-catenin signaling and is involved in tumorigenesis in OC. Therefore, manipulating the expression of miR-340 or its target genes is a potential strategy in OC therapy.
| S-EPMC6506554 | biostudies-literature