Programmed Cell Death 4 Levels Research Articles

Silencing PDCD4 Mediates Transcription Factor EB Overexpression Promoting Proliferation, Migration, and Invasion of Cervical Cancer Hela Cells

Cervical cancer is a common gynecologic malignant tumor, the occurrence and development of which are related to multiple genetic and environmental factors. Recent studies have shown that Programmed Cell Death 4 (PDCD4) plays a crucial role in cervical cancer, and that silencing PDCD4 mediates Transcription Factor EB (TFEB) overexpression, promoting cell proliferation, migration, and invasion in this disease. This study utilized the Hela cell line as a cervical cancer model to investigate the changes in TFEB expression levels and the proliferation, migration, invasion, and EMT processes of cervical cancer cells through the silencing of PDCD4. Real-time quantitative PCR and Western blot were employed to assess the expression levels of PDCD4 and TFEB, while CCK-8, scratch assay, Transwell invasion assay, and Western blot were used to evaluate changes in cell proliferation, migration, invasion capabilities, and EMT processes. The experimental results demonstrated that silencing PDCD4 significantly increased the expression level of TFEB. Simultaneously, silencing PDCD4 also significantly accelerated the proliferation rate of Hela cells, enhanced the cells’ migration, invasion capabilities, and promoted the EMT processes. Further experimental results showed that silencing TFEB could partially reverse the promoting effects of PDCD4 silencing on cell proliferation, migration, and invasion. In cervical cancer, silencing PDCD4 can lead to TFEB overexpression, thereby promoting the proliferation, migration, and invasion of Hela cells. These findings provide crucial clues for the in-depth study of molecular mechanisms in cervical cancer and indicate that the PDCD4-TFEB pathway could potentially serve as a target for the treatment and prevention of this disease.

Programmed cell death 4 blocks autophagy and promotes dopaminergic neuronal injury in Parkinson's disease.

Dysregulation of autophagy has previously been associated with the formation of toxic proteins, such as α-synuclein, in patients with Parkinson's disease (PD). In addition, it has been indicated that programmed cell death 4 (PDCD4) can inhibit autophagy in certain conditions, such as diabetic nephropathy, atherosclerosis and cardiac hypertrophy. Therefore, the hypothesis that PDCD4 can promote dopaminergic neuron damage through autophagy was proposed. To explore this hypothesis, the present study treated human neuroblastoma SK-N-SH cells with 1-methyl-4-phenylpyridinium (MPP+) to establish an in vitro model of PD. The potential effects of PDCD4 knockdown on lactate dehydrogenase (LDH) release, cell apoptosis, inflammatory response, oxidative stress and autophagy were then evaluated in this model of PD using an LDH assay kit, flow cytometry, western blotting, ELISA and immunofluorescence. The autophagy inhibitor 3-methyladenine (3-MA) was also applied to treat these cells, and its effects on these aforementioned parameters following PDCD4 knockdown were assessed. MPP+ was shown to increase the expression levels of PDCD4 in SK-N-SH cells. PDCD4 knockdown was revealed to suppress LDH release, cell apoptosis, secretion of inflammatory factors and oxidative stress. In addition, PDCD4 knockdown was demonstrated to enhance autophagy in cells treated with MPP+. By contrast, 3-MA treatment reversed the aforementioned effects of PDCD4 knockdown on cells, suggesting autophagy to be among the processes regulated by PDCD4 in SK-N-SH cells. The results of the present study suggested the existence of regulatory effects mediated by PDCD4 on autophagy in MPP+-induced SK-N-SH cells, offering potential future targets for PD therapy.

PDCD4 silencing alleviates KA‑induced neurotoxicity of HT22 cells by inhibiting endoplasmic reticulum stress via blocking the MAPK/NF‑κB signaling pathway.

Human programmed cell death 4 (PDCD4) has been reported to participate in multiple neurological diseases. However, the role of PDCD4 in epilepsy, as well as its underlying mechanism, remains unclear. To induce excitotoxicity, 100 µM kainic acid (KA) was applied for the stimulation of HT22 cells for 12 h. Initially, the mRNA and protein expression levels of PDCD4 were evaluated using reverse transcription-quantitative PCR and western blotting. A lactate dehydrogenase assay was performed to detect cell injury. Cell apoptosis was assessed using flow cytometry and western blotting was performed to determine the expression levels of apoptosis-related proteins. Oxidative stress was detected using dichlorodihydrofluorescein diacetate staining, and malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) assay kits. Furthermore, the expression levels of MAPK/NF-κB signaling-related proteins and endoplasmic reticulum (ER) stress-related proteins C/EBP homologous protein, glucose-regulated protein 78, activating transcription factor 4 and phosphorylated-eukaryotic initiation factor-2α were assessed by western blotting. It was revealed that PDCD4 expression was markedly elevated in KA-induced HT22 cells, whereas PDCD4 silencing alleviated KA-induced neurotoxicity of HT22 cells by alleviating cell injury and inhibiting apoptosis. In addition, PDCD4 silencing reduced the levels of reactive oxygen species and MDA, but elevated those of SOD and GSH-Px. PDCD4 silencing also suppressed ER stress by blocking the MAPK/NF-κB signaling pathway. By contrast, the MAPK agonist phorbol myristate acetate reversed the effects of PDCD4 silencing on KA-induced neurotoxicity and oxidative stress in HT22 cells. In conclusion, PDCD4 silencing alleviated KA-induced neurotoxicity and oxidative stress in HT22 cells by suppressing ER stress through the inhibition of the MAPK/NF-κB signaling pathway, which may provide novel insights into the treatment of epilepsy.

Higher expression of programmed cell death 4 (PDCD4) in acute myeloid leukemia is associated with better prognosis after chemotherapy.

Acute myeloid leukemia (AML) is a common heterogeneous malignancy. Novel molecular markers aid diagnosis, patient sub-categorization, and optimal clinical decisions. Here, we explored the prognostic implications associated with the expression of the programmed cell death (PDCD) family of molecules in AML patients. Based on the findings from the TCGA and OHSU cohorts, we observed that the mRNA abundance of PDCD4 is significantly higher compared to other molecules within the PDCD family. Furthermore, high expression of PDCD4 was associated with predicted long-term patient survival in diagnosed AML patients. In the chemotherapy group, patients with high PDCD4 expression showed a tendency toward longer overall survival (OS) (P = 0.0266) and event-free survival (EFS) (P = 0.0008). High PDCD4 levels served as a favorable independent predictor for both OS and EFS in AML patients. However, subgroup analyses in the hematopoietic stem cell transplantation (HSCT) group revealed no significant difference in OS or EFS between individuals with high and low PDCD4 expression. Furthermore, in the low PDCD4 expression group, AML patients who underwent HSCT experienced improved survival outcomes (P = 0.0015), helping to mitigate the unfavorable prognosis associated with PDCD4 downregulation. Conversely, in the high PDCD4 expression group, HSCT offered a notable short-term survival advantage, while patients with high PDCD4 expression responded favorably to long-term survival through chemotherapy. Biological function enrichment showed that the expression of PDCD4 was correlated with complement and coagulation cascades, cell receptor signaling pathways, and cholesterol metabolism. The findings from this study will aid in better categorizing heterogeneous AML patients and guiding more appropriate clinical decision-making.

PRMT5 knockdown enhances cell viability and suppresses cell apoptosis, oxidative stress, inflammation and endothelial dysfunction in ox-LDL-induced vascular endothelial cells via interacting with PDCD4.

Atherosclerosis (AS) is an important pathological basis of cardiovascular disease (CVD). The development of AS commences with endothelial dysfunction due to vascular endothelial cell injury. It is well documented that protein arginine methyltransferase 5 (PRMT5) is highly related to cardiovascular events. BioGRID database analysis indicates that PRMT5 may interact with programmed cell death 4 (PDCD4), which is reported to be involved in AS progression. This present research was formulated to elucidate the biological roles of PRMT5/PDCD4 in vascular endothelial cell injury during AS. In this current work, HUVECs were stimulated with 100mg/L ox-LDL for 48h to construct an in vitro AS model. Expression levels of PRMT5 and PDCD4 were analyzed by performing RT-qPCR and western blot. The viability and apoptosis of HUVECs were determined using CCK-8, flow cytometry and western blot assays. The status of oxidative stress and inflammation was assessed via commercial detection kits and ELISA assay, respectively. Besides, biomarkers of endothelial dysfunction were detected via commercial detection kit and western blot assay. In addition, the interacting relationship between PRMT5 and PDCD4 was verified by Co-IP assay. Highly expressed PRMT5 was observed in ox-LDL-stimulated HUVECs. Knockdown of PRMT5 enhanced the viability and inhibited the apoptosis of ox-LDL-induced HUVECs as well as alleviated ox-LDL-triggered oxidative stress, inflammation and endothelial dysfunction in HUVECs. PRMT5 interacted and bound with PDCD4. Furthermore, the enhancing effect on cell viability as well as the suppressing effects on cell apoptosis, oxidative stress, inflammation and endothelial dysfunction of PRMT5 knockdown in ox-LDL-induced HUVECs were partially abolished upon up-regulation of PDCD4. To conclude, down-regulation of PRMT5 might exert protective effects against vascular endothelial cell injury during AS by suppressing PDCD4 expression.

Validation of miRNAs as diagnostic and prognostic biomarkers, and possible therapeutic targets in medullary thyroid cancers.

Medullary thyroid cancer (MTC) is a rare type of neuroendocrine tumor that produces a hormone called calcitonin (CT). Thyroidectomy is the preferred treatment for MTC, as chemotherapy has been shown to have limited effectiveness. Targeted therapy approaches are currently being used for patients with advanced, metastatic MTC. Several studies have identified microRNAs, including miR-21, as playing a role in the development of MTC. Programmed cell death 4 (PDCD4) is a tumor suppressor gene that is an important target of miR-21. Our previous research has shown that high levels of miR-21 are associated with low PDCD4 nuclear scores and high CT levels. The aim of this study was to investigate the potential of this pathway as a novel therapeutic target for MTC. We used a specific process to silence miR-21 in two human MTC cell lines. We studied the effect of this anti-miRNA process alone and in combination with cabozantinib and vandetanib, two drugs used in targeted therapy for MTC. We analyzed the effect of miR-21 silencing on cell viability, PDCD4 and CT expression, phosphorylation pathways, cell migration, cell cycle, and apoptosis. Silencing miR-21 alone resulted in a reduction of cell viability and an increase in PDCD4 levels at both mRNA and protein levels. It also led to a reduction in CT expression at both mRNA and secretion levels. When combined with cabozantinib and vandetanib, miR-21 silencing did not affect cell cycle or migration but was able to enhance apoptosis. Silencing miR-21, although not showing synergistic activity with TKIs (tyrosine kinase inhibitors), represents a potential alternative worth exploring as a therapeutic target for MTC.

Differential regulation of miR-21-5p delays wound healing of melanocyte-deprived vitiligo skin by modulating the expression of tumor-suppressors PDCD4 and Maspin.

The loss of melanocytes in vitiligo is associated with architectural, transcriptional, and cellular perturbations of keratinocytes and manifests as a reduced proliferation potential in vitro and delayed re-epithelialization in vivo. To understand the molecular mechanisms underlying this delay, microRNA (miRNA) profiling was performed on split skin biopsies collected on Day 1 (basal level) and Day 14 (wound re-epithelialization) from nonlesional (NL) and lesional (L) skin of five subjects with stablenonsegmental vitiligo and 129 miRNAs were found to be differentially regulated between the NL and L healed epidermis. miR-21-5p, expressed at comparable levels on NL and L Day 1 samples, demonstrated significant upregulation during re-epithelialization. However, the extent of its upregulation was relatively lower in L (10 times compared to Day 1) as compared to NL skin (17 times compared to Day 1). The overexpression of miR-21 in keratinocytes led to a significant increase in the expression of proliferation markers (Ki67 and MCM6 messenger RNA, Ki67 positivity), along with an increase in keratinocyte migration. Using a smallinterferingRNAmediated knockdown approach, we further demonstrated that miR-21-5p mediates its effects by suppressing the expression of programmed cell death 4 (PDCD4) and mammary serine protease inhibitor (Maspin), both tumor-suppressor genes. Investigation of clinical samples corroborated the lower miR-21 levels and a higher expression of PDCD4 and Maspin in L Day 14 compared to the NL Day 14 epidermis. In conclusion, this study revealed that a relatively lower upregulation of miR-21-5p in L skin leads to significantly higher levels of PDCD4 and Maspin, delaying wound re-epithelialization by reducing the proliferation and migration of keratinocytes.

Clinical Significance of PDCD4 in Melanoma by Subcellular Expression and in Tumor-Associated Immune Cells.

Simple SummaryWhile targeting programmed cell death (PDCD) 1 is a central treatment against melanoma, little is known about the related protein PDCD4. We defined differences in melanoma PDCD4 subcellular localization (either total cellular or nuclear-only) during oncogenesis, evaluated its presence on tumor-infiltrating immune cells, and determined its impact on survival. High PDCD4 expression resulted in improved survival in patients with primary and intracranial but not extracranial metastatic melanoma. High PDCD4 levels in surrounding tumor tissue were also associated with increased infiltrating immune cells. PDCD4 may be a potentially useful biomarker in melanoma to help guide our understanding of patient prognosis. Methods to increase PDCD4 in those with melanoma brain metastases may also help improve disease response.Little is known about the subcellular localization and function of programmed cell death 4 (PDCD4) in melanoma. Our past studies suggest PDCD4 interacts with Pleckstrin Homology Domain Containing A5 (PLEKHA5) to influence melanoma brain metastasis outcomes, as high intracranial PDCD4 expression leads to improved survival. We aimed to define the subcellular distribution of PDCD4 in melanoma and in the tumor microenvironment during neoplastic progression and its impact on clinical outcomes. We analyzed multiple tissue microarrays with well-annotated clinicopathological variables using quantitative immunofluorescence and evaluated single-cell RNA-sequencing on a brain metastasis sample to characterize PDCD4+ immune cell subsets. We demonstrate differences in PDCD4 expression during neoplastic progression, with high tumor and stromal PDCD4 levels associated with improved survival in primary melanomas and in intracranial metastases, but not in extracranial metastatic disease. While the expression of PDCD4 is well-documented on CD8+ T cells and natural killer cells, we show that it is also found on B cells and mast cells. PDCD4 expression in the tumor microenvironment is associated with increased immune cell infiltration. Further studies are needed to define the interaction of PDCD4 and PLEKHA5 and to evaluate the utility of this pathway as a therapeutic target in melanoma brain metastasis.

Tumor-derived exosomal microRNA-106b-5p activates EMT-cancer cell and M2-subtype TAM interaction to facilitate CRC metastasis

Epithelial-mesenchymal transition (EMT) is reported to involve in the crosstalk between tumor cells and tumor-associated macrophages (TAMs). Exosomes are considered as important mediators of orchestrating intercellular communication. However, the underlying mechanisms by which EMT-colorectal cancer (CRC) cells promote the M2 polarization of TAMs remain less understood. In this study, we found that EMT-CRC cells promoted the M2-like polarization of macrophages by directly transferring exosomes to macrophages, leading to a significant increase of the microRNA-106b-5p (miR-106b) level in macrophages. Mechanically, an increased level of miR-106b activated the phosphatidylinositol 3-kinase (PI3K)γ/AKT/mammalian target of rapamycin (mTOR) signaling cascade by directly suppressing programmed cell death 4 (PDCD4) in a post-transcription level, contributing to the M2 polarization of macrophages. Activated M2 macrophages, in a positive-feedback manner, promote EMT-mediated migration, invasion, and metastasis of CRC cells. Clinically, miR-106b was significantly elevated in CRC tissues and negatively correlated with the levels of PDCD4 in CRC specimens, and high expression of exosomal miR-106b in plasma was significantly associated with the malignant progression of CRC. Taken together, our results indicate that exosomal miR-106b derived from EMT-CRC cells has an important role in intercellular communication for inducing M2 macrophage polarization, illuminating a novel mechanism underlying CRC progression and offering potential targets for prevention of CRC metastasis.

Human esophageal fibroblast-derived exosomal miR-21 reduced the cisplatin sensitivity to esophageal carcinoma EC9706 cells.

The objective of this study was to investigate the effect of human esophageal fibroblast-derived exosomal miR-21 on cisplatin sensitivity against esophageal squamous EC9706 cells. EC9706 cells were co-cultured indirectly with human esophageal fibroblasts (HEF) or miR-21 mimics transfected-HEF in the transwell system. The exosomes in HEF-culture conditioned medium were extracted by differential ultracentrifugation. EC9706 cells were co-cultured with HEF-derived exosomes directly. The cisplatin sensitivity against EC9706 cells was revealed via half maximal inhibitory concentration (IC50) values using MTT assay. The expressions of miR-21, programmed cell death 4 (PDCD4) mRNA, and gene of phosphate and tension homology deleted on chromosome ten (PTEN) mRNA were determined by qRT-PCR. The changes of the protein level were detected using western blot assay. IC50 values of cisplatin against EC9706 cells were increased after EC9706 cells were co-cultured with either HEF or exosomes derived from miR-21 mimics-transfected HEF. Following the increased level of miR-21, the mRNA expression and protein levels of PTEN and PDCD4 were decreased in EC9706 cells. The cisplatin sensitivity to EC9706 cells was reduced by HEF-derived exosomal miR-21 through targeting PTEN and PDCD4. This study suggested that non-tumor cells in the tumor micro-environment increased the tumor anti-chemotherapy effects through their exosomes.

Down-Regulation of PDCD4 Promotes Proliferation, Angiogenesis and Tumorigenesis in Glioma Cells.

The programmed cell death 4 (PDCD4) tumor-suppressor gene regulates cell apoptosis, protein translation, signal transduction, and induction of mediators of inflammation. However, the mechanism by which PDCD4 is down-regulated and regulates tumor growth remains elusive. In this study, we showed that PDCD4 is down-regulated in glioma cells and acts as a tumor suppressor. Based on the TCGA data, we confirmed that AKT2, but not AKT1 or AKT3, interacts with PDCD4, thus leading to the suppression of PDCD4 in glioma cells. Moreover, the analysis suggested that PDCD4 regulates the expression of IL-5, CCL-5, VEGF, and CXCL10 via the NF-kB pathway. Additionally, depletion of levels of PDCD4 promoted angiogenic activity of glioma cells via the VEGF-STAT3 pathway. When tumor cells over-expressing PDCD4 were injected into nude mice, the increased expression of PDCD4 blocked tumorigenesis and prolonged overall survival. Our study indicates the need to develop drugs that can modulate the expression of PDCD4 and test their efficacy in clinical trials.

Diagnostic and prognostic value of P53 and the human programmed cell death 4 genes in colorectal cancer

Objective The aim was to study the human programmed cell death 4 (PDCD4) and p53 as diagnostic and prognostic markers in patients with colorectal cancer (CRC). Background CRC is one of the most common cancers. Novel molecules and pathways continue to emerge in the search for improved therapeutic strategies. Patients and methods A total of 120 patients diagnosed with CRC were included and classified based on stage to group 1 (early-stage patients) and group 2 (advanced-stage patients). Moreover, 60 patients diagnosed with benign colorectal polyps were included as a control group (group 3). P53 and PDCD4 antigen levels were measured at presentation, and their relations to patient demographics and clinical features were estimated. Results For PDCD4, area under the curve was 0.889 at cutoff more than 3 ng/ml, with a diagnostic sensitivity of 85.0% and specificity 93.33%, whereas for P53, area under the curve was 1.000 at cutoff greater than 295 pg/ml, with a sensitivity and specificity of 100%. PDCD4 was significantly elevated in group 1 patients compared with group 2 (mean ± SD: 6.9 ± 3.4 vs 1.6 ± 1), with P value less than 0.001. PDCD4 levels were significantly related to tumor grade in groups 1 and 2 (P = 0.041 and 0.011, respectively). P53 levels were higher among patients in group 1 than group 2 (mean ± SD: 2422 ± 692.5 vs 2392.7 ± 680.3, respectively). P53 levels had statistically significant relation with tumor grade in group 2 patients (P = 0.004). Conclusion Both P53 and PDCD4 had diagnostic value. Elevated PDCD4 levels had a positive prognostic value, whereas elevated P53 levels had a negative prognostic value.

Nuclear PDCD4 Expression Defines a Subset of Luminal B-Like Breast Cancers with Good Prognosis.

The hormone receptor-positive (estrogen and/or progesterone receptor (PR)-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that includes luminal A-like (LumA-like) and luminal B-like (LumB-like) subtypes. Decreased PR levels is a distinctive biological feature of LumB-like tumors. These tumors also show reduced sensitivity to endocrine therapies and poorer prognosis than LumA-like tumors. Identification of biomarkers to accurately predict disease relapse in these subtypes is crucial in order to select effective therapies. We identified the tumor suppressor PDCD4 (programmed cell death 4), located in the nucleus (NPDCD4), as an independent prognostic factor of good clinical outcome in LumA-like and LumB-like subtypes. NPDCD4-positive LumB-like tumors presented overall and disease-free survival rates comparable to those of NPDCD4-positive LumA-like tumors, indicating that NPDCD4 improves the outcome of LumB-like patients. In contrast, NPDCD4 loss increased the risk of disease recurrence and death in LumB-like compared with LumA-like tumors. This, along with our results showing that LumB-like tumors present lower NPDCD4 positivity than LumA-like tumors, suggests that NPDCD4 loss contributes to endocrine therapy resistance in LumB-like BCs. We also revealed that PR induces PDCD4 transcription in LumB-like BC, providing a mechanistic explanation to the low PDCD4 levels in LumB-like BCs lacking PR. Finally, PDCD4 silencing enhanced BC cell survival in a patient-derived explant model of LumB-like disease. Our discoveries highlight NPDCD4 as a novel biomarker in LumA- and LumB-like subtypes, which could be included in the panel of immunohistochemical markers used in the clinic to accurately predict the prognosis of LumB-like tumors.

SUN-122 Nuclear PDCD4 Expression Predicts Good Clinical Outcome in Luminal A-Like and Luminal B-Like Breast Cancer Subtypes

Hormone receptor-positive (HR+, estrogen and/or progesterone receptor-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that comprises 70% of BCs. This subtype includes both luminal (Lum) A- and B-like subtypes, which have differences in prognosis and sensitivity to endocrine therapies. The development of biomarkers guiding treatment decisions in these settings is required. Tumor suppressor PDCD4 (programmed cell death 4), which can be found both in the nucleus (NPDCD4) or the cytoplasm (CPDCD4), inhibits tumor growth and metastasis, and its loss is associated with poor prognosis in solid tumors. To explore the clinical relevance of PDCD4 in BC, we analyzed its expression by immunohistochemistry in a cohort of 619 patients with primary invasive BC. We found that 34.7% of patients showed NPDCD4 and 21.3% showed CPDCD4. NPDCD4 positivity, but not CPDCD4, was associated with lower clinical stage (P = 0.0003), with presence of more differentiated tumors (P = 6.4x10-6), and with estrogen and progesterone receptor (PR) expression (P = 9.2x10-9 and P = 2.8x10-9, respectively). Kaplan-Meier analysis revealed that NPDCD4 expression was associated with a longer overall survival (OS) and disease-free survival (DFS) in LumA-like (P = 0.008 and P = 0.028, respectively) and LumB-like (P = 0.004 and P = 0.012, respectively) subtypes. Interestingly, patients with LumB-like tumors displaying NPDCD4 presented estimated OS and DFS rates similar to the ones observed in patients with LumA-like tumors also expressing NPDCD4, indicating that its presence improves the clinical outcome of LumB-like patients. Multivariate Cox regression analysis identified NPDCD4 as an independent predictor of good clinical outcome in both LumA-like (HR: 0.45, 95% CI 0.22-0.96, P = 0.038) and LumB-like (HR: 0.28, 95% CI 0.10-0.80, P = 0.018) subtypes. We validated our results by in silico analysis using expression data from the METABRIC cohort. Bioinformatics analysis of BC cells from the Cancer Cell Line Encyclopedia revealed a positive correlation between PDCD4 and PR expression (P = 0.015). Since LumB-like tumors present a higher risk of resistance to endocrine therapy and both PR and PDCD4 levels in this subtype are lower than in the LumA-like one, we postulated that the presence of PR may modulate PDCD4 expression. Silencing of PR expression in HR+ cells decreased PDCD4 protein levels while reconstitution of PR in a PR-null cell line increased them, confirming PR requirement for PDCD4 modulation. In line with PDCD4 physiological function, its knockdown increased cell migration capability of HR+ BC cells, whereas its restoration led to a decrease in cell migration of HR-negative BC models. Our findings identified NPDCD4 positivity as a novel biomarker of clinical outcome in LumA- and B-like subtypes and revealed PDCD4 reconstitution as a novel therapeutic strategy in BC.

MicroRNA‑21 serves an important role during PAOO‑facilitated orthodontic tooth movement.

Periodontal accelerate osteogenesis orthodontics (PAOO) is an extension of described techniques that surgically alter the alveolar bone; however, the specific mechanism underlying the technique is not completely understood. The aim of the present study was to evaluate the roles of microRNA (miR)-21 during PAOO. Sprague-Dawley rats were divided into the following four groups: i) Group tooth movement (TM), underwent TM and were administered normal saline (NS); ii) Group PAOO, underwent PAOO + TM and were administered NS; iii) Group agomiR-21, underwent PAOO + TM and were administered agomiR-21; and iv) Group antagomiR-21, underwent PAOO + TM and were administered antagomiR-21. To validate the rat model of PAOO, morphological analyses were performed and measurements were collected. Reverse transcription-quantitative PCR, western blotting and immunohistochemical staining were performed to examine the expression levels of programmed cell death 4 (PDCD4), activin A receptor type 2B (ACVR2b), receptor activator of NF-κΒ ligand (RANKL) and C-Fos. Dual-luciferase reporter assays were performed to validate PDCD4 as a target of miR-21 in vitro. Following 7 days of treatment, the TM distance of group PAOO was longer compared with groups TM and antagomiR-21 (P<0.05), but shorter compared with group agomiR-21 (P<0.05). Tartrate-resistant acid phosphatase staining indicated that following treatment with agomiR-21, osteoclast activity was notably increased, whereas the mRNA and protein expression levels of PDCD4 were notably decreased compared with group PAOO. The mRNA and protein expression levels of RANKL and C-Fos in group agomiR-21 were notably increased compared with group PAOO, whereas group antagomiR-21 displayed the opposite pattern (P<0.05). With regard to ACVR2b, no significant differences were observed among the group agomiR-21 and antagomiR-21 compared with group PAOO. Bioinformatics analysis predicted that PDCD4 was a potential target gene of miR-21, and dual-luciferase reporter assays demonstrated that miR-21 directly targeted PDCD4. In conclusion, the present study demonstrated that miR-21 serves an important role during PAOO-mediated orthodontic TM.

Degradation of the Tumor Suppressor PDCD4 Is Impaired by the Suppression of p62/SQSTM1 and Autophagy.

PDCD4 (programmed cell death 4) is a tumor suppressor that plays a crucial role in multiple cellular functions, such as the control of protein synthesis and transcriptional control of some genes, the inhibition of cancer invasion and metastasis. The expression of this protein is controlled by synthesis, such as via transcription and translation, and degradation by the ubiquitin-proteasome system. The mitogens, known as tumor promotors, EGF (epidermal growth factor) and TPA (12-O-tetradecanoylphorbol-13-acetate) stimulate the degradation of PDCD4 protein. However, the whole picture of PDCD4 degradation mechanisms is still unclear, we therefore investigated the relationship between PDCD4 and autophagy. The proteasome inhibitor MG132 and the autophagy inhibitor bafilomycin A1 were found to upregulate the PDCD4 levels. PDCD4 protein levels increased synergistically in the presence of both inhibitors. Knockdown of p62/SQSTM1 (sequestosome-1), a polyubiquitin binding partner, also upregulated the PDCD4 levels. P62 and LC3 (microtubule-associated protein 1A/1B-light chain 3)-II were co-immunoprecipitated by an anti-PDCD4 antibody. Colocalization particles of PDCD4, p62 and the autophagosome marker LC3 were observed and the colocalization areas increased in the presence of autophagy and/or proteasome inhibitor(s) in Huh7 cells. In ATG (autophagy related) 5-deficient Huh7 cells in which autophagy was impaired, the PDCD4 levels were increased at the basal levels and upregulated in the presence of autophagy inhibitors. Based on the above findings, we concluded that after phosphorylation in the degron and ubiquitination, PDCD4 is degraded by both the proteasome and autophagy systems.

P1626MicroRNA-21 deteriorates left ventricular reverse remodeling by promoting cardiac fibrosis in non-ischemic cardiomyopathy

Abstract Background Left ventricular reverse remodeling (LVRR) contributes to better outcomes in patients with non-ischemic cardiomyopathy (NICM). It is reported that LVRR is associated with progression of cardiac fibrosis. MicroRNAs (miRs) have emerged as powerful regulators of post-transcriptional gene expression. We focused on miR-21, which plays a key role in pathogenesis of fibrosis in multiple organs. The aim of this study was to clarify the effect of miR-21 on cardiac fibrosis and LVRR in patients with NICM. Methods We measured plasma miR-21 levels in 16 patients with NICM. LVRR was defined as increased LVEF by ≥10% and decreased LV end-diastolic diameter index by ≥10% from baseline data after optimal medication treatment at 1-year of follow-up. Further, we examined miR-21 expression and its potential role in cardiac fibrosis induced by transverse aortic constriction (TAC) in mice and angiotensin II (Ang II) stimulation in neonatal rat cardiomyocytes (NRCMs). Results There were 12 patients without LVRR and 4 patients with LVRR. Plasma miR-21 levels were significantly higher in patients without LVRR compared with those with LVRR. In TAC mice heart, miR-21 levels were significantly increased and programmed cell death 4 (PDCD4), a main target of miR-21, was decreased. In vitro, miR-21 levels were significantly increased and its upstream transcriptional factor, activator protein 1 (AP-1), was activated by Ang II stimulation in NRCMs. After transfection of miR-21 specific inhibitor, PDCD4 levels were upregulated. Furthermore, AP-1 activity, expression of collagen type I, and α-smooth muscle actin levels were significantly decreased after miR-21 inhibition. Conclusions These findings suggested that miR-21/PDCD4/AP-1 feedback loop pathway was involved in LVRR in patients with NICM by promoting cardiac fibrosis. MiR-21 can be the therapeutic target in NICM.

MiRNA-182 regulates the cardiomyocyte apoptosis in heart failure.

To investigate the effect of miRNA-182 on cardiomyocyte apoptosis of heart failure (HF). HF model in rats was established by rapid ventricular pacing. AAV-miRNA-182 (adeno-associated virus) vector was constructed to upregulate miRNA-182 level and its negative control AAV-NC was prepared. Rats undergoing rapid pacing (pacing group) and sham operation (sham group) were injected with AAV-miRNA-182 or AAV-NC, respectively. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was conducted to determine the miRNA-182 level in rats. Cardiac function test was carried out after the HF model establishment. Western blot was performed to examine the protein levels of human programmed cell death4 (PDCD4) and phosphoacidic cluster sorting protein (PACS2) in rats. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay was performed to evaluate the apoptotic rate of cardiomyocytes. HF model in rats was successfully established by rapid pacing. Injection of AAV-miRNA-182 markedly upregulated miRNA-182 level in rats. Compared with rats in the sham group, left ventricle ejection fraction (LVEF; 81.8% ± 2.4% vs. 64.3% ± 2.2%, p<0.05) and left ventricular fractional shortening (LVFS; 44.7% ± 2.4% vs. 29.1% ± 0.9%, p<0.05) markedly decreased in the pacing + NC group, whereas heart rate (HR) and left ventricular end-diastolic pressure (LVEDP) increased. Compared with rats in the pacing + NC group, HR [(441.6 ± 22) /min vs. (368.4 ± 27)/min, p<0.05] and LVEDP [(34.8 ± 11.4) mmHg vs. (19.4 ± 10.3) mmHg, p<0.05] were reduced in the pacing + miRNA-182 group. The protein levels of PDCD4 and PACS2 were downregulated in the pacing + miRNA-182 group relative to the pacing + NC group. Rapid pacing stimulation induced cardiac structural remodeling and cardiomyocyte apoptosis, which were alleviated by injection of AAV-miRNA-182. MiRNA-182 inhibits cardiomyocyte apoptosis induced by non-ischemic HF via downregulating PDCD4 and PACS2.

Influence of exosome-derived miR-21on chemotherapy resistance of esophageal cancer.

To explore the influence of exosome-derived micro-ribonucleic acid (miR)-21 on chemotherapy resistance of esophageal cancer and its mechanism. Human esophageal cancer TE-1 and Eca109/DDP cell lines and human normal esophageal Het-1A cells were cultured, and the exosomes were extracted from cells. After miR-21 was inhibited with an inhibitor and overexpressed with miRNA mimics combined with cisplatin, the cell viability was detected via cell counting kit-8 (CCK-8), the interaction between miR-21 and programmed cell death 4 (PDCD4) was detected via dual-luciferase reporter gene assay, and the changes in the protein level were detected via Western blotting. The expression level of exosome-derived miR-21 in esophageal cancer cells was higher than that in normal esophageal cells, and it was the highest in cisplatin-resistant esophageal cancer cells. After treatment with cisplatin, miR-21 overexpression significantly reduced the invasion ability of esophageal cancer cells. After miR-21 overexpression, the sensitivity of esophageal cancer cells to cisplatin was lowered. MiR-21 interacted with the 3'-untranslated region (UTR) of PDCD4. Moreover, the miR-21 overexpression significantly down-regulated the mRNA and protein levels of PDCD4 in cells. MiR-21 affects the sensitivity of esophageal cancer to cisplatin through targeting PDCD4.

Expression of tumor suppressor programmed cell death 4 in endometrioid endometrial carcinomas and clinicopathological significance.

Programmed cell death 4 (PDCD4), as a novel tumor suppressor, serves important roles in the pathogenesis of tumors. The expression of PDCD4 is downregulated or lost in various human tumors. However, the expression of PDCD4 in endometrial cancer and the clinicopathological significance remain unclear. The aim of the present study was to investigate the expression of PDCD4 in endometrioid endometrial carcinoma (EEC) and the association with clinicopathological parameters. The expression of PDCD4 in EEC tissues and control endometrium was detected by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. PDCD4 expression was also investigated in control endometrial glandular epithelial cells and the endometrial cancer KLE cell line by immunocytochemistry, and the association between PDCD4 expression and clinicopathological parameters of patients with EEC was analyzed. The results demonstrated that PDCD4-positive staining was mainly located in the cytoplasm of endometrial glandular epithelial cells and EEC cells. The staining index of PDCD4 in the proliferative phase was significantly increased compared with that in the secretory phase of control endometrium (P<0.001). There was significantly decreased PDCD4 expression in grade (G) 2/3 EEC tissues compared with the proliferative phase of control endometrium (P<0.001). PDCD4 expression was significantly associated with tumor grade. The PDCD4 levels in G1 EEC tissues were higher compared with the G2/3 EEC group (P<0.01). The results indicated that PDCD4 is associated with the histological grade of EEC, and that PDCD4 may be a valuable indicator of the degree of tumor malignancy in patients with EEC.