Programmed Cell Death 4 Protein Expression Research Articles

Exosomal microRNA-342-5p from human umbilical cord mesenchymal stem cells inhibits preeclampsia in rats.

We aimed to investigate the inhibitory effect of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes (hucMSC-Exos) transmitting microRNA-342-5p (miR-342-5p) on the development of preeclampsia (PE) by targeting programmed cell death 4 (PDCD4). The primary hucMSCs were cultured and transfected with miR-342-5p, and the exosomes (Exo) were extracted from the hucMSCs. PE rats were performed with an intraperitoneal injection of L-NAME from days 11 to 19 of gestation, and injection of Exo, Exo-negative control (NC), Exo-miR-342-5p agomir, Exo-miR-342-5p antagomir, and overexpressing PDCD4 (oe-PDCD4) vector into the placenta on the 16th day of pregnancy. HE staining was utilized to observe the pathological changes in placental tissues. TUNEL staining was implemented to evaluate cell apoptosis in placental tissues. Blood pressure and 24-h urinary protein in pregnant rats were measured by a non-invasive rat tail artery blood pressure measurement and protein auto-analyzer. Expressions of miR-342-5p, PDCD4, proinflammatory cytokines (TNF-α and IL-1β), and anti-inflammatory cytokines (IL-10 and TGF-β) were detected by RT-qPCR, and PDCD4 protein expression was determined by Western blot. The interaction between miR-342-5p and PDCD4 was analyzed by luciferase activity assay. MiR-342-5p was downregulated while PDCD4 was upregulated in the placental tissues of PE rats. HucMSC-Exo relieved pathology and suppressed inflammatory response, and apoptosis in the placental tissues, as well as reducing blood pressure and 24-h urinary protein of PE rats. Elevated miR-342-5p enhanced the promoting influence of hucMSC-Exo on PE rats, while inhibited miR-342-5p reversed the functions of hucMSC-Exo on PE rats. miR-342-5p targeted PDCD4. Overexpression of PDCD4 worsened the development of PE in rats. HucMSC-Exo conveying elevated miR-342-5p inhibits the development of PE in a rat model through downregulating PDCD4.

Circular RNA hsa_circ_0026344 suppresses gastric cancer cell proliferation, migration and invasion via the miR-590-5p/PDCD4 axis.

Circular RNA (CircRNA) is a class of non-coding RNA transcripts, with multiple pathophysiological functions. Instead, the mechanism and function of circRNA in gastric cancer (GC) are not fully deciphered. CircRNA_0026344 (circ_0026344), microRNA (miR)-590-5p and programmed cell death 4 (PDCD4) mRNA expression levels in GC tissues and cells were probed by quantitative real-time PCR. Cell viability, migration and aggressiveness were examined by cell counting kit-8 and transwell assays. Additionally, the interplay among circ_0026344, miR-590-5p and PDCD4 was verified with bioinformatics and dual-luciferase reporter gene assay. Western blot was conducted to probe PDCD4 protein expression. Circ_0026344 expression was underexpressed in GC tissues and cells, which was associated with clinicopathological characteristics such as tumour size, tumor-node-metastasis stage and lymph node metastasis. Circ_0026344 overexpression restrained the malignant biological behaviours of GC cells, while circ_0026344 knockdown functioned oppositely. Circ_0026344 could act as a competing endogenous RNA of miR-590-5p to negatively modulate its expression, and this miRNA could mitigate the impact of circ_0026344 on GC cells. In addition, PDCD4 was identified as the downstream target of miR-590-5p, and PDCD4 expression was positively modulated by circ_0026344. Circ_0026344 up-regulates PDCD4 expression via sponging miR-590-5p, thus inhibiting the progression of GC. This study further expounds the underlying molecular mechanism in the GC progression.

Knockdown of circ-UQCRC2 ameliorated lipopolysaccharide-induced injury in MRC-5 cells by the miR-326/PDCD4/NF-κB pathway

BackgroundCircular RNAs (circRNAs) have been shown as important modulators in the pathogenesis of pediatric pneumonia. In this paper, we focused on the molecular basis of circRNA ubiquinol-cytochrome c reductase core protein 2 (circ-UQCRC2, circ_0038467) in lipopolysaccharide (LPS)-induced cell injury. MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to gauge the levels of circ-UQCRC2, microRNA (miR)-326 and programmed cell death 4 (PDCD4) mRNA. PDCD4 protein expression and the activation of the NF-κB signaling pathway were evaluated by western blot. Ribonuclease R (RNase R) assay was performed to assess the stability of circ-UQCRC2. Cell viability and apoptosis were detected by the Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6 were measured by the enzyme-linked immunosorbent assay (ELISA). Targeted relationship between miR-326 and circ-UQCRC2 or PDCD4 was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. ResultsOur data showed the up-regulation of circ-UQCRC2 level in pneumonia serum and LPS-treated MRC-5 cells. The silencing of circ-UQCRC2 attenuated LPS-induced MRC-5 cell injury. Mechanistically, circ-UQCRC2 directly targeted miR-326, and circ-UQCRC2 regulated PDCD4 expression through miR-326. MiR-326 was a downstream effector of circ-UQCRC2 function, and PDCD4 was a functional target of miR-326 in regulating LPS-induced MRC-5 cell injury. Additionally, circ-UQCRC2 knockdown inactivated the NF-κB signaling pathway by regulating the miR-326/PDCD4 axis. ConclusionOur findings demonstrated a novel regulatory network, the miR-326/PDCD4/NF-κB pathway, for the function of circ-UQCRC2 in LPS-induced cell injury in MRC-5 cells.

Downregulation of PDCD4 induced by progesterone is mediated by the PI3K/AKT signaling pathway in human endometrial cancer cells.

Programmed cell death4 (PDCD4) has been identified as a tumor‑suppressor gene that inhibits neoplastic transformation, tumor progression, and protein translation. It has been reported that multiple factors participate in the regulation of PDCD4 mRNA and protein. The endometrium is regulated by changing concentrations of ovarian hormones, such as estrogen and progesterone, and shows periodical changes. However, whether ovarian hormones regulate PDCD4 expression remains unclear. This study aimed to explore the effect and mechanism of estrogen or progesterone on PDCD4 mRNA and protein expression in human endometrial cancer cells. The expression of PDCD4 mRNA and protein in Ishikawa and HEC‑1‑A cells was detected by quantitative real‑time PCR and western blot analysis. The signaling pathway‑related proteins were detected by western blot analysis. The results showed that PDCD4 mRNA levels exhibited no significant changes after treatment with estrogen or progesterone in both Ishikawa and HEC‑1‑A cell lines. Estrogen also had no obvious effect on PDCD4 protein expression. However, progesterone effectively decreased the expression of PDCD4 protein and the PI3K/AKT pathway may be involved in the downregulation of PDCD4 protein induced by progesterone. These results suggest that the downregulation of PDCD4 induced by progesterone affects the therapeutic efficacy of progesterone in human endometrial cancer or endometriosis, which may have important implications for progesterone treatment in the clinic.

Identifying Involvement of H19-miR-675-3p-IGF1R and H19-miR-200a-PDCD4 in Treating Pulmonary Hypertension with Melatonin

Non-coding RNAs play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). The aim of this study was to characterize the therapeutic role of melatonin as well as the underlying molecular mechanism (its effects on the expression of H19 and its downstream signaling pathways) in the treatment of PAH. Real-time PCR and western blot analysis were performed to evaluate the expression of H19, miR-200a, miR-675, insulin-like growth factor-1 receptor (IGF1R), and programmed cell death 4 (PDCD4). The value of systolic pulmonary artery pressure (SPAP) and the ratio of medial thickening in the monocrotaline (MCT) group were increased, whereas the melatonin treatment could decrease these values to some extent. The weights of RV (right ventricle), LV (left ventricle) + IVS (interventricular septal), and RV/(LV + IVS) in the MCT group were much higher than those in the MCT + melatonin and control groups. In addition, the expression of H19, miR-675, IGF1R mRNA, and IGF1R protein in the MCT group was the highest, whereas their expression in the control group was the lowest. The expression of miR-200, PDCD4 mRNA, and PDCD4 protein in the MCT group was the lowest, whereas their expression in the control group was the highest. Furthermore, H19 directly suppressed the expression of miR-200a, whereas miR-675-3p and miR-200a directly inhibited the expression of IGF1R and PDCD4, respectively. Finally, melatonin treatment inhibited cell proliferation; upregulated the expression of H19, miR-675-3p, and PDCD4; and downregulated the expression of miR-200a and IGF1R. This study demonstrated the role of H19-miR-675-3p-IGF1R- and H19-miR-200a-PDCD4-signaling pathways in the melatonin treatment of PAH.

MiR-1260b inhibitor enhances the chemosensitivity of colorectal cancer cells to fluorouracil by targeting PDCD4/IGF1.

Colorectal cancer (CRC) is the most common malignant tumor type and has become resistant to 5-fluorouracil (5-FU) in recent decades, which is one of the most popular therapies. Recently, microRNA (miRNA or miR) has been investigated as a potential therapeutic strategy for CRC. However, there has been little investigation of the underlying mechanism of the association between expression of miRNA and chemosensitivity. The present study aimed to investigate the effect of miR-1260b inhibitor on CRC cells, and their chemosensitivity to 5-FU, by treating them with the miR-1260b inhibitor. miR-1260b inhibitor was demonstrated to significantly promote the proliferation and invasion of the CRC cell line, HCT116, and to increase the apoptotic rate. Furthermore, it was validated that programmed cell death 4 (PDCD4) was a direct target of miR-1260b inhibitor in CRC with bioinformatics tools and a luciferase assay. Western blot analysis revealed that miR-1260b inhibitor could significantly decrease PDCD4 expression, and downregulate the expression of phosphorylated-Akt (p-Akt) and phosphorylated-extracellular-signal-regulated kinase (p-ERK). In conclusion, it was confirmed that the anti-tumor effect of the miR-1260b inhibitor was conducted by blocking the phosphorylated 3-kinase/Akt pathway as dysregulated protein expression induced by miR-1260b inhibitor was rescued by insulin-like growth factor. Notably, miR-1260b inhibitor could significantly enhanced the chemoresponse of HCT116 cells to 5-FU via reduced proliferation, increased apoptosis, and downregulation of PDCD4, p-Akt and p-ERK protein expression. In summary, the present study may provide a novel direction for future clinical therapy to enhance the chemosensitivity of tumor cells.

Prognostic significance of miR-21 and PDCD4 in patients with stage II esophageal carcinoma after surgical resection.

Many studies have shown that randomized clinical trial with long-term follow-up found no improvement in stage II esophageal carcinoma (EC) patients receiving preoperative neoadjuvant chemoradiotherapy or chemotherapy treatment, this limitation underscored the urgent need for novel and reliable biomarkers for prognosis and prediction in stage II EC. miR-21 is frequently over-expressed while programmed cell death 4 (PDCD4) is often down-regulated in solid tumors. This study aimed to investigate the clinicopathological and prognostic significance of miR-21 and PDCD4 expression and to elucidate any correlation between miR-21 and PDCD4 expression in stage II EC patients. The expression level of miR-21 was up-regulated while the PDCD4 protein was down-regulated in stage II EC tissues compared with the adjacent non-cancerous tissues. Analyses of the clinicopathological parameters indicated that miR-21 expression was associated with differentiation grade, T stage, and N stage. PDCD4 protein expression was associated with T stage, N stage, and tumor size. The univariate linear regression analysis suggested a significant negative correlation between miR-21 and PDCD4 expression. The Kaplan-Meier curve showed that high miR-21 expression or low PDCD4 expression predicted poor progression-free survival (PFS) and overall survival (OS) of patients with stag II EC. In conclusion, both up-regulated miR-21 and down-regulated PDCD4 expression were associated with the aggressive progression and poor prognosis of stage II EC. miR-21 and PDCD4 might be potential biomarkers of tumor progression and indicators of prognosis of stag II EC.

Berberine regulates the microRNA-21-ITGΒ4-PDCD4 axis and inhibits colon cancer viability.

Berberine is sourced from multiple medicinal herb resources and is easy to extract. With the advantages of low price, safety and convenience, berberine may have potential for wide clinical use. The present study aimed to investigate whether berberine inhibited the viability of colon cancer and whether it regulated the three-gene network microRNA (miR)-21-integrin β4 (ITGβ4)-programmed cell death 4 (PDCD4). It was demonstrated that berberine treatment suppressed colon cancer cell viability, and induced apoptosis and activated caspase-3 activity in the human colon carcinoma HCT116 cell line. Berberine inhibited miR-21 expression and promoted ITGβ4 and PDCD4 protein expression in the HCT116 cell line. Overexpression of miR-21 reduced the anti-cancer effects of berberine on cell viability, apoptosis rate and caspase-3 activity of the HCT116 cell line. However, it was revealed that the overexpression of miR-21 also suppressed ITGβ4 and PDCD4 protein expression in the HCT116 cell line. In conclusion, miR-21, ITGβ4 and PDCD4 are involved in the anti-cancer effects of berberine on cell viability and apoptosis in the HCT116 cell line.

Role of miR-21 in the growth and metastasis of human salivary adenoid cystic carcinoma.

Aberrant microRNA (miRNA/miR) expression has been reported in various cancer types. miR-21, which is considered to be a proto-oncogene and is frequently overexpressed in certain cancer types, has been implicated in tumorigenesis. The aim of the present study was to investigate the effect of miR-21 degradation on tumor progression and its potential mechanisms in human salivary adenoid cystic carcinoma (SACC) development. Results of reverse transcription-quantitative polymerase chain reaction analysis indicated that SACC cells with high metastatic potential (SACC-LM cells) exhibited a significantly higher expression of miR-21 compared with SACC cells with a lower metastatic potential (SACC-83 cells). In addition, following transfection of SACC-LM cells with miR-21 inhibitor, cell viability was reduced, which may be a result of reduced cell proliferation and metastasis, and the induction of apoptosis, as determined by Cell Counting Kit-8, wound healing, Matrigel invasion and flow cytometry assays. Furthermore, bioinformatics analysis indicated that programmed cell death 4 (PDCD4), phosphatase and tensin homolog deleted on chromosome ten (PTEN) and B-cell lymphoma (Bcl)-2 are potential target genes of miR-21. Therefore, western blotting was performed to investigate the expression of these proteins, and the results demonstrated that miR-21 expression level was negatively associated with PDCD4 and PTEN protein expression, and positively associated with Bcl-2 protein expression, in SACC-LM cells, indicating that miR-21 may promote SACC progression via PDCD4, PTEN and Bcl-2. In conclusion, the present study indicates that miR-21 may be a novel target for SACC therapy and provide a novel basis for the clinical treatment of SACC.

Absence of nuclear Programmed cell death 4 as an indicator of poor prognosis in uveal melanoma patients

PurposeUveal melanoma (UM) is the most frequently occurring intraocular malignancy in adults. Arising from the uncontrolled proliferation of melanocytes in the choroid, ciliary body and iris, UMs have a strong propensity to metastasise to the liver, where they are fatal. In a previous study, proteomic examination of UMs at either a high or low risk of developing metastasis identified the down regulation of Programmed Cell Death 4 (PDCD4) levels with increasing metastatic risk.MethodsThis study examined PDCD4 protein expression and subcellular localisation in UM patient samples by immunohistochemistry (IHC). 165 primary (PUM) and 18 metastatic UM (MUM) samples with full clinical, histological and genetic data were included in the study. Slides were assessed and scored according to (A) percentage of tumour cells with cytoplasmic PDCD4 expression; (B) intensity of cytoplasmic staining and (C) percentage of PDCD4 positive tumour nuclei.ResultsIn PUM and MUM samples, PDCD4 was localised primarily to the cytoplasm. An equal percentage of primary and metastatic samples expressed nuclear PDCD4 (28%). Loss of nuclear PDCD4 was strongly associated with factors associated with a poor prognosis, in particular, monosomy 3 (p = 0.014) and loss of nuclear BAP1 (p = 0.012). PUMs with nuclear PDCD4 in ≥10% of tumour cells had an improved survival time compared with PUMs with <10% nuclear expression of PDCD4 (p = 0.105).ConclusionsThe tumour suppressor PDCD4 shuttles between the nucleus and cytoplasm to exert its effects in normal cells. In PUM samples, an absence of nuclear PDCD4 localisation was associated with a poor outcome. Further functional studies are necessary to determine how PDCD4 localisation influences UM cell behaviour.

Downregulation of PDCD4 by miR-21 suppresses tumor transformation and proliferation in a nude mouse renal cancer model.

Programmed cell death 4 (PDCD4) is known to suppress neoplastic transformation, cell proliferation and metastasis, and to be downregulated by microRNA-21 (miR-21) in renal cell carcinoma (RCC) cell lines and tissues. The aim of the present study was to investigate the roles of and association between PDCD4 and miR-21 in a nude mouse renal cancer model. A total of 24 BALB/c male nude mice were randomly assigned into the following three groups: Negative control (NC; n=8), miR-21 inhibitor (n=8) and miR-21 mimic (n=8). Subsequently, renal cell adenocarcinoma 786-O cells were subcutaneously transplanted into the armpits of the mice, which were then injected daily with NC small interfering (si)RNA, precursor-miR-21 (mimic) or anti-miR-21 (inhibitor). Tumors were removed from the mice and weighed 16 days following 786-O cell transplantation. In addition, the expression of miR-21 and PDCD4 mRNA in cancer tissues was analyzed using reverse transcription-quantitative PCR. The expression of PDCD4 protein in cancer tissues was also examined using immunohistochemistry and western blotting. Furthermore, 786-O cells were transfected with PDCD4 siRNA or NC siRNA, and the effects of silencing PDCD4 on tumor cell growth, proliferation and invasion were investigated using soft agar colony formation, EdU cell proliferation assay and Transwell migration and invasion assays. Another 16 BALB/c male nude mice were randomly assigned into two groups as follows: NC (n=8) and PDCD4 siRNA (n=8). The 786-O cells were subcutaneously transplanted into the armpits of the mice, which were subsequently injected daily with NC siRNA or PDCD4 siRNA. The tumors were removed and weighed 16 days following transplantation. Compared with the NC group, tumor weight in the miR-21 mimic group was significantly increased. By contrast, tumor weight in the miR-21 inhibitor group was significantly decreased. Similar to the results observed in human renal cancer tissue and cell lines, miR-21 expression in the nude mouse renal cancer models was significantly upregulated in the miR-21 mimic group compared with the NC group, while it was significantly lower in the miR-21 inhibitor group. Furthermore, there was a significant reduction in PDCD4 protein levels in the miR-21 mimic group and a significant increase in the miR-21 inhibitor group compared with the NC, whereas PDCD4 mRNA expression was not significantly altered. In the EdU proliferation assay, the mean percentage of new cells that incorporated EdU was 28.6% in the NC siRNA group and significantly increased to 44.7% in PDCD4 siRNA transfected cells. In the soft agar colony formation assay, Transwell and migration and invasion assays, a significant increase in colony formation, migration and invasion capacity in PDCD4 siRNA-transfected cells was observed compared with the NC. Furthermore, compared with the NC group, tumor weight in the PDCD4 siRNA group was significantly increased. Similar to the results observed in human renal cancer tissue and cell lines, miR-21 promoted cancer cell hyperplasia and proliferation, and post-transcriptionally downregulated PDCD4 protein expression, in the nude mouse renal cancer model. The results of the present study and previous studies indicate that PDCD4 and miR-21 serve an important role in renal cancer. Thus, increasing PDCD4 expression or inhibiting miR-21 expression may constitute effective novel therapeutic strategies for the treatment of renal cancer.

Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4.

Pulmonary hypertension (PH) is a progressive disorder whose cellular pathogenesis involves enhanced smooth muscle cell (SMC) proliferation and resistance to apoptosis signals. Existing evidence demonstrates that the tumor suppressor programmed cell death 4 (PDCD4) affects patterns of cell growth and repair responses in the systemic vasculature following experimental injury. In the current study, the regulation PDCD4 and its functional effects on growth and apoptosis susceptibility in pulmonary artery smooth muscle cells were explored. We previously demonstrated that pharmacological activation of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) attenuated hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMCs) by inhibiting the expression and mitogenic functions of microRNA-21 (miR-21). In the current study, we hypothesize that PPARγ stimulates PDCD4 expression and HPASMC apoptosis by inhibiting miR-21. Our findings demonstrate that PDCD4 is reduced in the mouse lung upon exposure to chronic hypoxia (10% O2 for 3 wk) and in hypoxia-exposed HPASMCs (1% O2). HPASMC apoptosis was reduced by hypoxia, by miR-21 overexpression, or by siRNA-mediated PPARγ and PDCD4 depletion. Activation of PPARγ inhibited miR-21 expression and resultant proliferation, while restoring PDCD4 levels and apoptosis to baseline. Additionally, pharmacological activation of PPARγ with rosiglitazone enhanced PDCD4 protein expression and apoptosis in a dose-dependent manner as demonstrated by increased annexin V detection by flow cytometry. Collectively, these findings demonstrate that PPARγ confers growth-inhibitory signals in hypoxia-exposed HPASMCs through suppression of miR-21 and the accompanying derepression of PDCD4 that augments HPASMC susceptibility to undergo apoptosis.

Role of miR‑21 on vascular endothelial cells in the protective effect of renal delayed ischemic preconditioning.

Vascular endothelial cells may serve crucial roles in the development of acute kidney injury (AKI). microRNA (miR)-21, which possesses a renal protective function has been found on vascular endothelial cells. The present study aimed to test the hypothesis that miR-21 may protect vascular endothelial cells against injury, which may contribute to the protective effects of renal delayed ischemic preconditioning (IPC). Preconditioned (15 min ischemia) or Sham mice (not clamped) were subjected to 35 min occlusion of bilateral renal pedicles 4 days following preconditioning or Sham treatment. Human umbilical vein endothelial cells (HUVECs) were treated with cobalt(II) chloride (CoCl2) to establish an in vitro hypoxia model. Locked nucleic acid-modified anti-miR-21 or scrambled control oligonucleotides were transfected into cells or delivered into mice via tail vein injection <1 h prior to IPC. Following 24 h of reperfusion or hypoxia, morphological and functional parameters, apoptosis and miR-21 and programmed cell death 4 (PDCD4) expression were assessed in vivo and in vitro. Treatment of HUVECs with CoCl2 led to an upregulation of miR-21 expression, a downregulation of PDCD4 protein expression and attenuation of apoptosis. Inhibition of miR-21 expression led to increased expression levels of PDCD4 protein and apoptosis in HUVECs. IPC attenuated renal IR injury in mice. The protective effect of IPC appeared to be dependent on upregulated miR-21 expression. IPC-induced upregulation of miR-21 expression also occurred in HUVECs, and IPC also led to reduced PDCD4 expression and vascular permeability in mouse kidneys. The effects of IPC were attenuated by the inhibition of miR-21; miR-21 expression attenuated damage in vascular endothelial cells, which may contribute to the protective effects of delayed IPC on renal IR injury. The present study suggested a novel target for the prevention and repair of AKI in the future.

The effect and mechanism of action of metformin on invitro FaDu cell proliferation.

ObjectiveTo investigate the effect and mechanism of action of metformin on proliferation of a human hypopharyngeal carcinoma cell line (FaDu).MethodsFaDu cells were treated with metformin (25–125 mmol/l). Cell proliferation was evaluated via CCK-8 assay. Real-time quantitative reverse transcription–polymerase chain reaction was used to evaluate microRNA (miR)-21-5p and PDCD4 (programmed cell death 4) expression. PDCD4 protein was quantified by Western blot.ResultsMetformin significantly inhibited FaDu cell proliferation in a dose- (25–100 mmol/l) and time-dependent manner (12 h–36 h), significantly downregulated miR-21-5p, and upregulated PDCD4 mRNA and protein expression.ConclusionsMetformin significantly inhibited FaDu cell proliferation, possibly via downregulation of miR-21-5p and upregulation of PDCD4.

Programmed cell death 4 and BCR-ABL fusion gene expression are negatively correlated in chronic myeloid leukemia.

Programmed cell death 4 (PDCD4) is a tumor suppressor that inhibits carcinogenesis, tumor progression and invasion by preventing gene transcription and translation. Downregulation of PDCD4 expression has been identified in multiple types of human cancer, however, to date, the function of PDCD4 in leukemia has not been investigated. In the present study, PDCD4 mRNA and protein expression was investigated in 50 patients exhibiting various phases of chronic myeloid leukemia (CML) and 20 healthy individuals by reverse transcription-quantitative polymerase chain reaction and western blot analysis. PDCD4 expression and cell proliferation was also investigated following treatment with the tyrosine kinase inhibitor, imatinib, in K562 cells. The results demonstrated that PDCD4 mRNA and protein expression was decreased in all CML samples when compared with healthy controls, who expressed high levels of PDCD4 mRNA and protein. No significant differences in PDCD4 expression were identified between chronic phase, accelerated phase and blast phase CML patients. In addition, PDCD4 expression was negatively correlated with BCR-ABL gene expression (r=-0.6716; P<0.001). Furthermore, K562 cells treated with imatinib exhibited significantly enhanced PDCD4 expression. These results indicate that downregulation of PDCD4 expression may exhibit a critical function in the progression and malignant proliferation of human CML.

MicroRNA-16 suppresses the activation of inflammatory macrophages in atherosclerosis by targeting PDCD4.

Programmed cell death 4 (PDCD4) is involved in a number of bioprocesses, such as apoptosis and inflammation. However, its regulatory mechanisms in atherosclerosis remain unclear. In this study, we investigated the role and mechanisms of action of PDCD4 in high-fat diet-induced atherosclerosis in mice and in foam cells (characteristic pathological cells in atherosclerotic lesions) derived from ox-LDL-stimulated macrophages. MicroRNA (miR)-16 was predicted to bind PDCD4 by bioinformatics analysis. In the mice with atherosclerosis and in the foam cells, PDCD4 protein expression (but not the mRNA expression) was enhanced, while that of miR-16 was reduced. Transfection with miR-16 mimic decreased the activity of a luciferase reporter containing the 3′ untranslated region (3′UTR) of PDCD4 in the macrophage-derived foam cells. Conversely, treatment with miR-16 inhibitor enhanced the luciferase activity. However, by introducing mutations in the predicted binding site located in the 3′UTR of PDCD4, the miR-16 mimic and inhibitor were unable to alter the level of PDCD4, suggesting that miR-16 is a direct negative regulator of PDCD4 in atherosclerosis. Furthermore, transfection wtih miR-16 mimic and siRNA targeting PDCD4 suppressed the secretion and mRNA expression of pro-inflammatory factors, such as interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), whereas it enhanced the secretion and mRNA expression of the anti-inflammatory factor, IL-10. Treatment with miR-16 inhibitor exerted the opposite effects. In addition, the phosphorylation of p38 and extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) expression were altered by miR-16. In conclusion, our data demonstrate that the targeting of PDCD4 by miR-16 may suppress the activation of inflammatory macrophages though mitogen-activated protein kinase (MAPK) and NF-κB signaling in atherosclerosis; thus, PDCD4 may prove to be a potential therapeutic target in the treatment of atherosclerosis.

Abstract B41: Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity

Abstract Conventional wisdom holds that only one of the two strands in a microRNA (miRNA) precursor duplex is selected as the active guide strand. The complementary passenger strand is thought to be inactive. In triple negative breast cancer (TNBC), high levels of the miRNA guide strand called miR-17-5p inhibits ribosomal translation of tumor suppressor genes, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, we previously reported that antisense DNA-LNA knockdown of miR-17-5p guide strand reduced PDCD4 and PTEN proteins in human MDA-MB-231 TNBC cells, instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of the miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by the miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and the miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3′UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knockdown miR-17-5p reduced PDCD4 and PTEN protein expression, instead of raising them. Transfection of miR-17-5p or miR-17-3p RNA mimics into TNBC cells also reduced PDCD4 and PTEN protein levels. To test the hypothesis that miR-17-3p passenger strand can also modulate the same mRNA targets as miR-17-5p, we performed luciferase assays using reporter constructs harboring predicted miR-17-5p and/or miR-17-3p binding sites from the 3′UTR of PDCD4 and PTEN mRNAs. We found that the miR-17-5p mimic lowered the expression of luciferase vectors containing one predicted binding site from the PDCD4 or PTEN 3′UTR domains. However, the miR-17-3p mimic lowered the expression of luciferase vectors containing all four 3′UTR target sites of PDCD4, and two of the six predicted PTEN 3′UTR target sites. In addition, we used luciferase assays to test the hypothesis that antisense DNA-LNA against miR-17-5p mimicked passenger strand miR-17-3p. We found that anti-miR-17-5p DNA-LNA lowered the expression of luciferase reporter vectors containing two of the four predicted PDCD4 3′UTR target sites for miR-17-3p, and one of the six predicted PTEN 3′UTR target sites for miR-17-3p. From these results, we concluded that the miR-17-3p passenger strand might regulate the translation of PDCD4 and PTEN mRNAs, just like the miR-17-5p guide strand. Moreover, the antisense DNA-LNA against miR-17-5p guide strand mimicked the miR-17-3p passenger strand, effectively raising the miR-17-3p concentration in TNBC cells. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand. Supported by the Department of Biochemistry and Molecular Biology. Conflict of interest: pending PCT/US2015/015681 patent application licensed to Nikita RNA LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Specific blocking of miR-17-5p guide strand in triple negative breast cancer cells, without amplifying passenger strand activity. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B41.

Non-Specific Blocking of miR-17-5p Guide Strand in Triple Negative Breast Cancer Cells by Amplifying Passenger Strand Activity.

Conventional wisdom holds that only one of the two strands in a micro ribonucleic acid (miRNA) precursor duplex is selected as the active miRNA guide strand. The complementary miRNA passenger strand, however, is thought to be inactive. High levels of the oncogenic miRNA (oncomiR) guide strand called miR-17-5p is overexpressed in triple negative breast cancer (TNBC) and can inhibit ribosomal translation of tumor suppressor gene mRNAs, such as programmed cell death 4 (PDCD4) or phosphatase and tensin homolog (PTEN). We hypothesized that knocking down the oncogenic microRNA (oncomiR) miR-17-5p might restore the expression levels of PDCD4 and PTEN tumor suppressor proteins, illustrating a route to oligonucleotide therapy of TNBC. Contrary to conventional wisdom, antisense knockdown of oncomiR miR-17-5p guide strand reduced PDCD4 and PTEN proteins by 1.8±0.3 fold in human TNBC cells instead of raising them. Bioinformatics analysis and folding energy calculations revealed that mRNA targets of miR-17-5p guide strand, such as PDCD4 and PTEN, could also be regulated by miR-17-3p passenger strand. Due to high sequence homology between the antisense molecules and miR-17-3p passenger strand, as well as the excess binding sites for the passenger strand on the 3’UTR of PDCD4 and PTEN mRNAs, introducing a miR-17-3p DNA-LNA mimic to knock down miR-17-5p reduced PDCD4 and PTEN protein expression instead of raising them. Our results imply that therapeutic antisense sequences against miRNAs should be designed to target the miRNA strand with the greatest number of putative binding sites in the target mRNAs, while minimizing affinity for the minor strand.

MiR-21 regulates tumor progression through the miR-21-PDCD4-Stat3 pathway in human salivary adenoid cystic carcinoma

miR-21, which is a putative tumor onco-miR and frequently overexpressed microRNA in various tumors, has been linked to tumor progression through targeting of tumor-suppressor genes. In this study, we sought to determine whether miR-21 has any role on tumor progression of salivary adenoid cystic carcinoma (SACC) and the possible mechanisms. We found that the level of miR-21 expression was significantly higher in SACC than that in normal salivary tissues, and it is also higher in tumors with metastasis than that without metastasis. Using an anti-miR-21 inhibitor in an in vitro model, downregulation of miR-21 significantly decreased the capacity of invasion and migration of SACC cells, whereas a pre-miR-21 increased the capacity of invasion and migration of SACC cells. To explore the potential mechanisms by which miR-21 regulate invasion and migration, we identified one direct miR-21 target gene, programmed cell death 4 (PDCD4), which has been implicated in invasion and metastasis. The suppression of miR-21 in metastatic SACC-LM cells significantly increased the report activity of PDCD4 promoter and the expression of PDCD4 protein. This subsequently resulted in downregulation of the p-STAT3 protein. The level of miR-21 expression positively related to the expression of PDCD4 protein and negatively related to the expression of p-STAT3 protein in SACC specimens, respectively, indicating the potential role of the STAT3-miR-21-PDCD4 pathway in these tumors. Dysregulation of miR-21 has an important role in tumor growth and invasion by targeting PDCD4. Therefore, suppression of miR-21 may provide a potential approach for the treatment of advanced SACC patients.

MiR-183 regulates biological behavior in papillary thyroid carcinoma by targeting the programmed cell death 4.

Numerous studies have demonstrated that microRNAs (miRNAs) play vital roles in papillary thyroid carcinoma (PTC). The aim of the present study was to examine the expression levels of miR-183 in PTC and investigate whether its potential roles involved targeting the programmed cell death 4 (PDCD4). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to examine the expression levels of miR-183 in 38 PTC specimens and 4 PTC cell lines. MTT, colony formation, wound-healing and Transwell invasion assays, and flow cytometry were conducted to explore the potential functions of miR-183 in human TPC1 papillary thyroid carcinoma cells. The dual-luciferase reporter assay was performed to validate whether PDCD4 was a direct target of miR-183. The effects of modulating miR-183 on endogenous levels of PDCD4 were subsequently confirmed via RT-qPCR and western blotting. Functional assays were used to indicate the roles of endogenous PDCD4 in TPC1. The results showed the miR-183 expression levels were significantly upregulated in PTC specimens and cell lines (P<0.05). Overexpression of miR-183 in TPC1 promoted cell proliferation, migration, invasion and decreased apoptosis. The dual-luciferase reporter assay confirmed that PDCD4 was a direct target of miR-183. RT-qPCR and western blotting showed that miR-183 negatively regulated PDCD4 protein expression but had no impact on mRNA expression of PDCD4. Knockdown of PDCD4 expression in TPC1 cells significantly enhanced cell proliferation, migration, invasion and inhibited apoptosis. The results of the present study suggested that miR-183 acts as a papillary thyroid carcinoma oncogene through the negative regulation of PDCD4 protein expression at the post-transcriptional levels. Therefore, targeting miR-183 provides a novel strategy for the diagnosis and treatment of patients with PTC.