PDCD4 Expression Research Articles

The mir-21 Inhibition Enhanced HUVEC Cellular Viability during Hypoxia-Reoxygenation Injury by Regulating PDCD4.

The purpose of this study was to explore the clinical value of altered plasma mir-21 expression level as a biomarker for the severity of coronary artery disease (CAD) and its molecular impact on HUVEC cellular injuries. Angiographically validated 56 patients with single-vessel CAD disease, 92 patients with double-vessel CAD, 139 complex coronary artery stenosis patients, and 56 healthy individuals (n = 343) were enrolled in this study. The expressions of plasma mir-21 were evidently and progressively higher while PDCD4 levels were significantly and steadily lower in single-, dual-, and multivessel occluded CAD patients than in healthy participants (P < 0.001). The relative expressions of mir-21 in hypoxia-reoxygenation- (HR-) exposed HUVECs were markedly upregulated, but PDCD4 concentrations were obviously downregulated as compared with normal control cells (P < 0.001). Moreover, altered circulatory mir-21 expression levels were able to significantly differentiate single- (AUC 0.893), double- (AUC 0.914), and multivessel stenosis CAD (AUC 0.933) patients from healthy subjects. Besides, the plasma mir-21 expressions in elderly (66-85 years) groups were remarkably higher than those in younger aged (25-45 years) subjects. Caspase-3 and ROS expression levels were remarkably elevated, but cellular viability noticeably declined in HR-induced HUVECs than in normoxic cells (P < 0.001). In contrast, mir-21 inhibition markedly reduced caspase-3 activity and ROS concentrations while significantly ameliorating HUVEC cellular viability in HR conditions. PDCD4 expressions in HR-exposed HUVECs were prominently decreased whereas mir-21 inhibition significantly enhanced PDCD4 levels (P < 0.001). Upregulated plasma mir-21 can be a valuable clinical biomarker for the detection of the severity of coronary artery stenosis patients. Elevated circulatory mir-21 concentrations have a positive correlation with aging. Inhibitory mir-21 evidently increased HUVEC cellular viability through upregulation of targeting PDCD4 and recommended a newer possible therapeutic molecule for the management of CAD patients.

Abstract 1480: miR-21 as a post-transcriptional regulator of pancreatic ductal adenocarcinoma (PDAC) tumorigenesis

Abstract Introduction: MicroRNAs are short non-coding RNAs that are frequently dysregulated across cancers. Specifically, microRNA-21 (miR-21) is a known oncomir overexpressed in pancreatic adenocarcinoma (PDAC) that regulates multiple gene targets downstream of KRAS, the site of the primary driver mutation in PDAC. Past efforts to target mutant KRAS have been limited by compensatory activation of other growth pathways and treatment-related toxicity. Inhibiting miR-21 expression is a novel therapeutic strategy to target KRAS effector function through post-transcriptional regulation. We previously demonstrated that systemic inhibition of miRNA-21 (miR-21) intercepts tumorigenesis in the transgenic KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice without causing overt toxicity. Our major goal was to verify the translation of the previous findings to human models and examine the mechanistic implications of miR-21 inhibition in PDAC. Experimental Procedures: Using publicly available data from a cohort of patients with PDAC in The Cancer Genome Atlas (TCGA), we performed differential expression analysis of miR-21 and KRAS-related gene targets as well as gene set enrichment analysis of oncogenic pathways identified by the Molecular Signatures Database (MSigDB). Concurrently, de-novo patient-derived organoid (PDO) models were generated from core biopsies and surgical resection specimens. To evaluate the effects of miR-21 inhibition on KRAS pathway activity in a human model system, we selected 6 PDO cell lines and determined miR-21 gene expression by quantitative PCR at baseline and after knockdown using a lentiviral construct. Results: Analysis of TCGA PDAC cohort identified heterogeneous endogenous expression of miR-21, which was validated ex vivo in our PDO model system. Gene set enrichment analysis revealed enrichment of gene sets associated with KRAS dependency, MEK, AKT, and MTOR signaling in patients with higher endogenous miR-21 expression. MiR-21 knockdown in PDO cell lines was stable at multiple intervals following lentiviral transduction. Further, expression of PDCD4, a tumor suppressor gene and target of miR-21 downstream of KRAS, was enhanced in PDO lines following miR-21 inhibition. Conclusions: We previously demonstrated that miR-21 appears to be an early and reliable molecular marker of pancreatic neoplasia and that systemic inhibition in a murine model intercepts PDAC tumorigenesis. We now demonstrate in human models that higher miR-21 expression is associated with enrichment of gene sets downstream of KRAS. Additionally, knocking down miR-21 expression enhances the expression of gene targets with tumor suppressor function, notably PDCD4. This suggests that modulating miR-21 expression subsequently modulates the expression of gene targets with critical cell regulatory functions and provides additional insight into novel therapeutic targets. Citation Format: Jacquelyn W. Zimmerman, Daniel H. Shu, Richard A. Burkhart, Joseph Tandurella, Elana J. Fertig, Elizabeth M. Jaffee. miR-21 as a post-transcriptional regulator of pancreatic ductal adenocarcinoma (PDAC) tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1480.

LncRNA NR2F2-AS1 functions as a tumor suppressor in gastric cancer through targeting miR-320b/PDCD4 pathway.

Gastric cancer is among the most frequently occurring gastrointestinal malignancies with a high mortality rate worldwide. Long non-coding RNAs (lncRNAs) are defined as core regulators in the occurrence and progression of multiple cancers, including gastric carcinoma. Mounting evidence has indicated that NR2F2-AS1 can inhibit several malignant tumors. However, the function and potential mechanism of NR2F2-AS1 remain unclear. In the current study, we found that NR2F2-AS1 was weakly expressed in gastric cancer cells in comparison with normal cells. The study has further disclosed that ectopic of NR2F2-AS1 repressed cell proliferation, migration, invasion and EMT whereas it promoted cell apoptosis in gastric carcinoma. Subsequently, our results confirmed that miR-320b was negatively regulated and that suppression of miR-320b alleviated the malignant behaviors of GC cells. More importantly, PDCD4 was a target of miR-320b. Mechanistically, NR2F2-AS1 modulated the expression level of PDCD4 by sponging miR-320b. Finally, rescue assays demonstrated that NR2F2-AS1 down-regulated PDCD4 expression to restrain the development of gastric cancer by competitively binding to miR-320b. On the whole, our study revealed the role of NR2F2-AS1/miR-320b/PDCD4 regulatory network in gastric cancer, suggesting NR2F2-AS1 may represent a novel therapeutic target for patients with gastric carcinoma.

Apoptosis Related PDCD4: Promising Novel Biomarker Early Detection of Oral Cancer

Aim: This study aimed to investigate the potential role of the PDCD4 gene, involved in the progression of the oral potentially malign disorder (OPMD) and oral squamous cell carcinoma cancer (OSCC). Material and Methods: The study was conducted on saliva and blood samples that were collected from OSCC (n=6), OPMD (n=6) patients, and healthy (n=6) individuals. The expression of PDCD4 was determined by using qRT-PCR. Kruskal Wallis test was performed to estimate the relationship between PDCD4 expression and clinical characteristics of OSCC and OPMD patients. The correlation between PDCD4 expression and patients groups was tested with the Rank Spearman test. Results: PDCD4 mRNA expression levels were assessed in 6 OSCCs, 6 OPMD patients, and 6 healthy individuals saliva and blood. In both blood and saliva, PDCD4 mRNA expression levels were 6/6 (100%) underexpressed in OSCC, while it was underexpressed in 5/6 (83%) of OPMD. The mean value of PDCD4 was significantly downregulated in OPMD and OSCC than in healthy (p&amp;lt;0.05). The Spearman rank test obtained a p-value for the 2 tail test of 0.024 (p &amp;lt; 0.05), which showed a significant correlation between both, while the correlation coefficient of -0.530 showed a strong negative correlation of PDCD4 downregülation and OPMD, OSCC. Conclusion: PDCD4 expression level is correlated with OSCC and OPMD. This study indicates that PDCD4 expression levels in liquid biopsies may have potential as a diagnostic biomarker in OSCC and OPMD.

An innovative systematic approach introduced the involved lncRNA-miR-mRNA network in cell cycle and proliferation after conventional treatments in breast cancer patients

ABSTRACT The present study aimed to explore the involved lncRNA-miRNA-mRNA network in the cell cycle and proliferation after conventional treatments in Luminal A breast cancer patients.The candidate miRNAs (miRs), lncRNAs, and mRNAs were first taken from the Gene Expression Omnibus and TCGA databases. The lncRNA–miR–mRNA network was then constructed using the high-throughput sequencing data. The expression levels of selected targets were measured in the breast cancer and healthy samples by the Real-Time PCR technique and compared with the clinical outcomes by the Kaplan-Meier method.Our analysis revealed a group of differentially expressed 3 lncRNAs, 9 miRs, and 14 mRNAs in breast cancer patients. A significant expression decrease of the selected tumor suppressor lncRNAs, miRs, and genes and a substantial expression increase of the selected onco-lncRNAs, oncomiRs, and oncogenes were obtained in the patients compared to the healthy group. The plasma levels of the lncRNAs, miRs, and mRNAs were more significant after the operation, chemotherapy, and radiotherapy than the pre-treatment. The Kaplan-Meier analysis indicated that the patients with a high expression of miR-21, miR-20b, IGF1R, and E2F2 and a low expression of miR-125a, PDCD4, and PTEN had exhibited a shorter overall survival rate.Our results suggested that the underlying mechanisms of the lncRNA, miRs, and mRNAs and relevant signaling pathways may be considered predictive and therapeutic targets for breast cancer.

LncRNA SLC16A1-AS1 regulates the miR-182/PDCD4 axis and inhibits the triple-negative breast cancer cell cycle

Purpose Although SLC16A1-AS1 is involved in lung cancer, its function in breast cancer is still elusive. We observed downregulation of SLC16A1-AS1 expression in triple-negative breast cancer (TNBC) by analyzing TCGA dataset. Therefore, we analyzed the function of SLC16A1-AS1 in TNBC. Methods We observed downregulation of SLC16A1-AS1 expression in TNBC by analyzing TCGA dataset. Therefore, we analyzed the function of SLC16A1-AS1 in TNBC. Results SLC16A1-AS1 expression was downregulated in TNBC tissues. SLC16A1-AS1 interacted with miR-182, whereas SLC16A1-AS1 and miR-182 overexpression failed to affect their expression. SLC16A1-AS1 overexpression upregulated the expression of PDCD4, a downstream target of miR-182. SLC16A1-AS1 and PDCD4 overexpression suppressed cell cycle progression from the G1 phase to the G2 phase. MiR-182 and silencing of PDCD4 played the opposite role. Additionally, miR-182 overexpression inhibited the role of SLC16A1-AS1 overexpression on cell cycle progression in both BT-549 and BT20 cells. The cell proliferation assay showed that SLC16A1-AS1 and PDCD4 overexpression decreased the cell proliferation rate. Conclusion SLC16A1-AS1 may inhibit cell cycle progression and restrain TNBC cell proliferation by regulating the miR-182/PDCD4 axis.

Gender Differential Expression of AR/miR-21 Signaling Axis and Its Protective Effect on Renal Ischemia-Reperfusion Injury.

Objective: The aim of this study was to investigate gender differences after renal ischemia-reperfusion injury in mice and the effects of androgen receptor (AR) and microRNA-21 (miR-21) on apoptosis in renal ischemia-reperfusion injury. Methods: Renal ischemia-reperfusion injury model was induced by 45 min of bilateral renal artery ischemia and reperfusion. BALB/c mice were randomly divided into groups according to different experimental protocols. The levels of renal function were evaluated by serum creatinine and blood urea nitrogen. TUNEL staining was used to analyze the pathological changes and apoptosis levels of renal tissue, and western blotting and qPCR were used to detect the expressions of miR-21, AR, PDCD4 and caspase3. Results: After renal ischemia-reperfusion injury in mice with different genders, the levels of plasma urea nitrogen and creatinine in female and male mice increased, the histopathological score increased, and TUNEL staining in renal tissue indicated increased apoptosis. The expressions of miR-21, PDCD4, and active caspase-3 protein were up-regulated. The above trend was more pronounced in male mice, and a significant decrease in AR mRNA expression was detected. Silencing the expression of AR aggravated the decline of renal function and renal tubular injury after renal ischemia in mice. The expression of PDCD4 and active caspase-3 increased, while the level of miR-21 was correspondingly decreased. Up-regulation of miR-21 expression by pre-miR-21 could negatively regulate PDCD4, reduce the expression level of active caspase3, and yet induce AR expression accordingly. MiR-21 alleviated renal ischemia-reperfusion injury by inhibiting renal tubular epithelial cell apoptosis. The effect of antagomiR-21 was the opposite, which aggravated renal ischemia-reperfusion injury. Conclusion: There are gender differences in renal ischemia-reperfusion injury. Male mice are more susceptible to renal ischemia-reperfusion injury than female. Silencing AR expression or down-regulating the level of miR-21 can promote the expression of PDCD4 and apoptosis protein caspase3, thereby aggravating ischemia-reperfusion injury in mice. The protective effect of AR and miR-21 in renal ischemia-reperfusion injury has a certain synergy.

B-cell receptor signaling induces proteasomal degradation of PDCD4 via MEK1/2 and mTORC1 in malignant B cells

B-cell receptor (BCR) signaling plays a major role in the pathogenesis of B-cell malignancies and is an established target for therapy, including in chronic lymphocytic leukemia cells (CLL), the most common B-cell malignancy. We previously demonstrated that activation of BCR signaling in primary CLL cells downregulated expression of PDCD4, an inhibitor of the translational initiation factor eIF4A and a potential tumor suppressor in lymphoma. Regulation of the PDCD4/eIF4A axis appeared to be important for expression of the MYC oncoprotein as MYC mRNA translation was increased following BCR stimulation and MYC protein induction was repressed by pharmacological inhibition of eIF4A. Here we show that MYC expression is also associated with PDCD4 down-regulation in CLL cells in vivo and characterize the signaling pathways that mediate BCR-induced PDCD4 down-regulation in CLL and lymphoma cells. PDCD4 downregulation was mediated by proteasomal degradation as it was inhibited by proteasome inhibitors in both primary CLL cells and B-lymphoma cell lines. In lymphoma cells, PDCD4 degradation was predominantly dependent on signaling via the AKT pathway. By contrast, in CLL cells, both ERK and AKT pathways contributed to PDCD4 down-regulation and dual inhibition using ibrutinib with either MEK1/2 or mTORC1 inhibition was required to fully reverse PDCD4 down-regulation. Consistent with this, dual inhibition of BTK with MEK1/2 or mTORC1 resulted in the strongest inhibition of BCR-induced MYC expression. This study provides important new insight into the regulation of mRNA translation in B-cell malignancies and a rationale for combinations of kinase inhibitors to target translation control and MYC expression.

Circ_0060731 mediated miR-21–5p-PDCD4/ESR1 pathway to induce apoptosis of placental trophoblasts in intrahepatic cholestasis of pregnancy

PurposeIntrahepatic cholestasis of pregnancy (ICP) is a pregnancy complication. However, the pathogenesis of ICP is currently unclear. MethodsWe analyzed the placenta samples of 10 normal and 10 ICP pregnant women. the expressions of circ0060731, miR-21–5p, and their downstream target genes PDCD4, ESR1, and apoptotic protein cleaved-caspase3 were detected in the cell model. ResultsThe expression of Circ_0060731, PDCD4, ESR1, and caspase-3 was higher in the ICP placenta tissue than in the control group, and the expression of miR-21–5p was lower in the ICP group than in the control group. In HTR8/Svneo cells treated with TCA, the expression/levels of Circ_0060731, PDCD4, ESR1, and caspase-3 were significantly higher in the ICP group than in the control group, and miR-21–5p was significantly lower in the ICP group than in the control group. Lentiviral knockdown of miR-21–5p significantly increased the expressions of its downstream genes of PDCD4 and ESR1, and also increased cell apoptosis. Overexpression of miR-21–5p significantly reduced the expression of PDCD4 and ESR1 and reduced cell apoptosis. The dual-luciferase experiment showed that both PDCD4 and ERS1 were the target genes of miR-21–5p. ConclusionCirc_0060731 mediated miR-21–5p-PDCD4/ESR1 pathway could induce apoptosis of placental trophoblasts in intrahepatic cholestasis of pregnancy.

MicroRNA-22-3p and MicroRNA-149-5p Inhibit Human Hepatocellular Carcinoma Cell Growth and Metastasis Properties by Regulating Methylenetetrahydrofolate Reductase.

microRNAs are small endogenous noncoding RNAs that have emerged as key negative regulators that target gene expression through RISC. Our previous study showed that the methylenetetrahydrofolate reductase gene (MTHFR) plays a key role in one carbon metabolism, which is downregulated by miR-22-3p and miR-149-5p, and that it could exert a potential anti-cancer effect. Whether miR-22-3p/miR-149-5p can regulate MTHFR to exert anti-cancer effects has become the focus of our research. Normal (HL-7702 cells) and cancerous (QGY-7703/HepG2 cells) human hepatocellular cells were transfected with 100 nM hsa-miR-22-3p/hsa-miR-149-5p mimic or controls. After 24, 48, and 72 h, cell proliferation ability was tested using CCK-8. The changes in MTHFR expression at both the transcriptional and translational levels were determined by RT-qPCR and Western blotting, respectively. Cancerous cell invasion and migration ability were confirmed by means of a transwell assay. We found that ectopic miR-22-3p/miR-149-5p inhibits hepatocellular carcinoma cell proliferation but does not inhibit normal human hepatocyte proliferation. The transfection of ectopic miR-22-3p/miR-149-5p downregulated the MTHFR expression in QGY-7703 and HepG2 but not in HL-7702. QGY-7703 and HepG2 migration and invasion were inhibited by ectopic miR-22-3p/miR-149-5p. Additionally, we found that ectopic miR-22-3p/miR-149-5p significantly increased the expression of TP53INP1 and PDCD4 in QGY-7703. The results of the study suggest that miRNA-22-3p and miRNA-149-5p inhibit tumor growth and metastasis properties may be by regulating MTHFR and that they exert anticancer effects in hepatocellular carcinoma cells.

A Double-Negative Feedback Interaction between miR-21 and PPAR-α in Clear Renal Cell Carcinoma.

Simple SummaryClear cell renal cell carcinoma (ccRCC) is the main histotype of kidney cancer, which is typically highly resistant to conventional systemic therapies and also known for abnormal lipid accumulation. Identifying the actors and deciphering the molecular mechanisms that lead to tumor progression is an important step in the development of new therapeutic strategies to cure ccRCC. In this context, we focused our attention on miR-21, an oncogenic miRNA upregulated in many solid tumors, and peroxysome proliferator-activated receptor-α (PPAR- α), the master regulator of lipid metabolism and one target of miR-21. In this study, our data show a double-negative feedback interaction between PPAR-α and miR-21. Thus, miR-21 silencing could be therapeutically exploited to restore PPAR-α expression and consequently inhibit the oncogenic events mediated by the aberrant lipid metabolism of ccRCC.Clear cell renal cell carcinoma (ccRCC) is the main histotype of kidney cancer, which is typically highly resistant to conventional therapies and known for abnormal lipid accumulation. In this context, we focused our attention on miR-21, an oncogenic miRNA overexpressed in ccRCC, and peroxysome proliferator-activated receptor-α (PPAR- α), one master regulator of lipid metabolism targeted by miR-21. First, in a cohort of 52 primary ccRCC samples, using RT-qPCR and immunohistochemistry, we showed that miR-21 overexpression was correlated with PPAR-α downregulation. Then, in ACHN and 786-O cells, using RT-qPCR, the luciferase reporter gene, chromatin immunoprecipitation, and Western blotting, we showed that PPAR-α overexpression (i) decreased miR-21 expression, AP-1 and NF-κB transcriptional activity, and the binding of AP-1 and NF-κB to the miR-21 promoter and (ii) increased PTEN and PDCD4 expressions. In contrast, using pre-miR-21 transfection, miR-21 overexpression decreased PPAR-α expression and transcriptional activity mediated by PPAR-α, whereas the anti-miR-21 (LNA-21) strategy increased PPAR-α expression, but also the expression of its targets involved in fatty acid oxidation. In this study, we showed a double-negative feedback interaction between miR-21 and PPAR-α. In ccRCC, miR-21 silencing could be therapeutically exploited to restore PPAR-α expression and consequently inhibit the oncogenic events mediated by the aberrant lipid metabolism of ccRCC.

Pdcd4 promotes lipid deposition by attenuating PPARα-mediated fatty acid oxidation in hepatocytes

BackgroundNonalcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. The involvement of programmed cell death 4 (Pdcd4) in inflammation and metabolic diseases has been widely reported. However, the precise regulatory role of Pdcd4 in hepatocytic lipid metabolism and NAFLD is not well known. Research design and methodsWe established a high-fat diet–induced NAFLD (HFD-NAFLD) rat model and a free fatty acids (FFAs)-treated cell model, and analyzed the expression and distribution of PDCD4. The lentivirus for Pdcd4 knockout and the vector for Pdcd4 overexpression were used to alter Pdcd4 expression in BRL 3A cells. Thereafter, lipid accumulation, FA metabolic gene expression, and peroxisome proliferator-activated receptor alpha (Pparα)-dependent peroxisomal β-oxidation-related gene expression, especially that of the critical transcription factors and enzymes acyl-CoA oxidases 1–3 (Acox1-3), were detected both at the mRNA and protein levels. ResultsPDCD4 expression increased and it was mainly distributed in hepatocyte nuclei of the HFD-NAFLD rats. as well as the FFAs-treated CBRH-7919 and BRL 3A cell lines. Pdcd4 knockout significantly suppressed FFAs-induced lipid accumulation, and Pdcd4 overexpression accelerated FFAs-induced lipid accumulation in hepatocytes. Mechanistically, Pdcd4 negatively regulated the expression Pparα and Acox1-3. In addition, rescue experiments confirmed that Pparα knockdown could attenuate the expression of Acox1-3 in Pdcd4 knockout cells, which ultimately restored lipid deposition to normal levels. PPARα expression decreased in the liver of the HFD-NAFLD rats. The enrichment of PDCD4 in hepatocyte nuclei correlated with lower PPARα expression after FFAs treatment in vitro. ConclusionOur results indicate that the abundance of PDCD4 under high-fat conditions facilitates hepatocellular lipid accumulation by decreasing PPARα-dependent FA peroxisomal β-oxidation.

Arsenic activates STAT3 signaling during the transformation of the human bronchial epithelial cells

Arsenic (As3+), a metalloid abundant in environment, is classified as a group I carcinogen associated with several common human cancers, including cancers in lung, skin, bladder, liver, and prostate (Wei et al., 2019). The mechanisms of As3+-induced carcinogenesis had been extensively studied, and different mechanisms might be involved in different types of cancer (Wei et al., 2019). Recent studies showed that exposure to a high dose of arsenic is able to induce lung cancer. Meanwhile, prolonged exposure to a low concentration of arsenic can increase the risk of lung cancer also (Liao et al., 2009; Fernández et al., 2012). Emerging evidence indicated that prolonged exposure to arsenic promotes malignant transformation and some of the transformed cells have cancer-stem-like properties (Ngalame et al., 2014). In the present report, we revealed that exposure to As3+ for short time period inhibited tyrosine-705 phosphorylation of signal transducer and activator of transcription 3 (pSTAT3Y705) and induced Src homology region 2 domain-containing phosphatase-1 (SHP-1) in bronchial epithelial cell line, BEAS-2B. In addition, we found that long term exposure of the cells to As3+ activates phosphorylation of STAT3 at serine 727 (pSTAT3S727) as well as pSTAT3Y705. Moreover, As3+ is able to induce the expression of miRNA-21 (miR-21) and decrease the expression of PDCD4. Taken together, our data suggest that activation of STAT3 and induction of miR-21 are important contributing factors to the reduced expression of PDCD4, which may play significant role in As3+-induced transformation of BEAS-2B cells.

Self-assembly of DNA nanogels with endogenous microRNA toehold self-regulating switches for targeted gene regulation therapy.

Herein, a smart nanohydrogel with endogenous microRNA-21 toehold is developed to encapsulate gemcitabine-loaded mesoporous silica nanoparticles for targeted pancreatic cancer therapy. This toehold mediated strand displacement method can simultaneously achieve specific drug release and miRNA-21 silencing, resulting in the up-regulation of the expression of tumor suppressor genes PTEN and PDCD4.

Hsa_circ_0008259 modulates miR-21-5p and PDCD4 expression to restrain osteosarcoma progression.

Background: Osteosarcoma (OS) is one of the most common primary bone tumors in children and adolescents. However, the molecular mechanism of OS tumorigenesis is still little known. Circular RNA (circRNA) is a key player in the progression of many cancers. This study is performed to decipher the role and mechanism of circ_0008259 in the progression of OS.Methods: A differentially expressed circRNA, circ_0008259, was screened out by analyzing the expression profile of circRNA in OS tissue. Circ_0008259, miR-21-5p and programmable cell death 4 (PDCD4) mRNA expression levels in OS tissues and cells were detected by qRT-PCR. Cell viability, metastatic potential and apoptosis were evaluated by cell counting kit-8 assay, Transwell and flow cytometry. The targeting relationship between circ_0008259 and miR-21-5p, and miR-21-5p and PDCD4 mRNA was analyzed and probed by bioinformatics analysis and dual-luciferase reporter assay, RNA-binding protein immunoprecipitation assay and RNA-pull down assay. The regulatory effects of circ_0008259 and miR-21-5p on PDCD4 protein expression in OS cells were detected by Western blot assay.Results: Circ_0008259 expression and PDCD4 expression were down-regulated and miR-21-5p expression was elevated in the OS tissues and cells. Functional experiments showed that circ_0008259 overexpression significantly inhibited the proliferation and metastatic potential of OS cells and promoted the apoptosis. Besides, PDCD4 was validated as the target gene of miR-21-5p, and circ_0008259 could competitively bind to miR-21-5p, thus up-regulating PDCD4 expression in OS cells.Conclusions: Circ_0008259 suppresses OS progression via regulating miR-21-5p/PDCD4 axis.

The brain targeted delivery of programmed cell death 4 specific siRNA protects mice from CRS-induced depressive behavior

Depression is one of the most common psychiatric disorders. Recently, studies demonstrate that antidepressants generating BDNF not only maintain synaptic signal transmission but also repress neuroinflammatory cytokines such as IL-6 and IL-1β. Therefore, promoting BDNF expression provides a strategy for the treatment of depression. Our recent research has indicated that programmed cell death 4 (Pdcd4) is a new target for antidepressant treatment by facilitating BDNF. Herein, we modified Pdcd4 specific small interfering RNA (siPdcd4) with the rabies virus glycoprotein peptide (RVG/siPdcd4) which enables it cross the blood-brain barrier (BBB). We found that RVG/siPdcd4 complex was selectively delivered to neurons and microglia and silenced the expression of Pdcd4, thereby up-regulating the level of BDNF and down-regulating IL-6 and IL-1β expression. More importantly, RVG/siPdcd4 injection attenuated synaptic plasticity impairment and protected mice from CRS-induced depressive behavior. These findings suggest that RVG/siPdcd4 complex is a potential therapeutic medicine for depression.

Abstract MP219: Hnrnpa2b1-dependent Selective Sorting Of Mir-486a-5p Into Msc-derived Exosomes Contributes To Cardioprotection

Exosomes (Exo) are a class of extracellular vesicles and involvement of stem cell-derived Exo in cardiac repair and cardioprotection is thought to be an important in the heart. Our HYPOTHESIS Is that specific microRNAs (miRs) from mesenchymal stem cell (MSC)-derived exosomes are actively and selectively sorted into Exo by RNA binding proteins and motifs on the miR, to serve specific functions of the Exo, including Cardioprotection. Methods: We characterized the miR populations of parental MSCs and their Exo via RNA Seq and confirmed by QRT-PCR the subpopulation of miRs that is increased in Exo vs . MSC cells. We then used Multiple Em for Motif Elicitation (MEME) Version 5.3.3 and determined the predicted conserved motifs. From these, we predicted RNA binding protein sites from the literature. In parallel, we performed mass spectrometry and western blot analyses to determine RNA binding proteins in MSC and Exo. Predicting that hnRNPA2B1 was a likely RNA binding protein for the new motif, we knocked out the cognate gene (CRISPR) in MSC and evaluated the KO Exo vs. the WT Exo by RNA Seq and QRT-PCR. We performed protein and RNA pulldowns, and EMSA to validate binding of hnRNPA2B1 to several of the miRs, and investigated the effects of these miRs on cell survival after simIR and in an in vivo mouse model of MI. Results: We found a set of eight miRs that are selectively concentrated in the MSC Exo. MEME software predicted a conserved binding motif of gAGu, which is close to canonical sites for binding of hnRNPA2B1 and hnRNPA1. We determined hnRNPA2B1 was in MSC and Exo and showed KO hnRNPA2B1 cells and Exo had no compensatory perturbation of other RNA binding proteins. The KO MSC Exo show reduction of the selective sorting of the miRs of interest. Pulldowns and binding assay results verify binding of hnRNPA2B1 to both miR-486a-5p and miR-122a. Finally, we showed that miR-486a-5p is protective in H9C2 cells submitted to simIR and results in significant 68% reduction of infarct size (n=7, P=0.0175) in vivo in association with repression of PDCD4 expression and apoptosis. Conclusions: We determined that a set of miRs is selectively concentrated in MSC Exo and demonstrated the necessity of hnRNPA2B1 in that process. This appears to involve a conserved RNA sequence motif (mutational analysis underway). A major miR affected is miR-486a-5p, which is strongly cardioprotective. Our results support that miR-486a-5p is selectively concentrated in MSC Exo and contributes to cardioprotection by reducing PDCD4 activity in apoptosis.

Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Retraction Note to: CiRS-126 inhibits proliferation of ovarian granulosa cells through targeting the miR-21-PDCD4-ROS axis in a polycystic ovarian syndrome model.

Polycystic ovarian syndrome (PCOS) is considered to be one of the most prevalent endocrine disorders affecting women of reproductive age. CiRS-126, an innovative circular microRNA, has previously been proven to be a promising miR-21 sponge. However, a proper understanding of the impact of ciRS-126 on PCOS is needed. Circular RNA (CiRS) profiles were initially evaluated in ovarian cortex samples obtained from 18 women with PCOS as well from 9 women without PCOS. Insulin-induced ovarian granulosa cells isolated from mice were utilized for the functional study. CiRS microarray analysis and quantitative real-time PCR indicated that ciRs-126 expression was downregulated while miR-21 expression was upregulated in PCOS samples and insulin-induced granulosa cells as compared with non-PCOS samples and non-insulin-induced granulosa cells. Furthermore, ectopic overexpression of ciRS-126 was associated with a reduction in proliferation and increased apoptosis in insulin-treated granulosa cells. Meanwhile, bioinformatic prediction and the results of the dual-luciferase reporter assay indicated the presence of consecutive binding in the ciRS-126-miR-21-programmed cell death protein 4 (PDCD4) axis. Moreover, overexpression of miR-21 blocked ciRS-126 repression of proliferation and triggered the death of insulin-induced granulosa cells. Excessive PDCD4 expression counteracted the influence of miR-21 on cell death and proliferation. The data indicated that PDCD4 played a regulatory role in ROS generation, which is reportedly involved in apoptosis. Therefore, ciRS-126 reduction in PCOS granulosa cells targeted the miR-21-PDCD4 axis to reduce proliferation and promote apoptosis. CiRS-126 shows potential as a promising predictor of clinical outcome as well as a therapeutic target in PCOS.

Abstract 2160: Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression

Abstract Programmed Cell Death Protein 4 (PDCD4), a well-known tumor suppressor, acts to inhibit translation initiation and cap-dependent translation by inhibiting the helicase activity of EIF4A. EIF4A tends to target mRNAs with structured 5'-UTR. PDCD4 can also prevent tumorigenesis by inhibiting tumor promoter-induced neoplastic transformation, and studies indicate that PDCD4 may inhibit translation of certain mRNAs via directly binding. A previous study has demonstrated that PDCD4 inhibits translation of p53 mRNA and treatment with DNA-damaging agents down-regulates PDCD4 expression but activates p53 expression. The study further demonstrated that treatment with DNA-damaging agents resulted in down-regulation of PDCD4 expression and an increase in p53 expression, suggesting a potential mechanism in which p53 regulates expression of Pdcd4. However, whether p53 directly regulates PDCD4 remains unknown. Herein, we demonstrate for the first time that p53 regulates PDCD4 expression. First, we found that overexpression of p53 in p53-null cells (H1299 and Saos2 cells) decreased PDCD4 protein level. Secondly, p53 dose-dependently decreased PDCD4 promoter activity in gene reporter assays. Moreover, we demonstrated that mutations on p53 (R273H: contact mutant, hotspot and R175H: conformational mutant, hotspot) abolished p53-mediated PDCD4 repression. Furthermore, using Tet-on expression system, PDCD4 protein level was decreased when WT p53, but not hotspot mutants of p53, was expressed in H1299 cells. Finally, mutations on DNA-binding domain, but not C-terminal regulatory domain, of p53 disrupted p53-mediated PDCD4 repression. Taken together, our results suggest that p53 functions as a novel regulator of PDCD4 and the relationship between of p53 and PDCD4 may be involved in tumor development and progression. Citation Format: Wei-Hsiung Yang, Andrew P. George, William H. Yang, Chiung-Min Wang, Richard H. Yang. Tumor suppressor p53 down-regulates programmed cell death protein 4 (PDCD4) expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2160.