Apoptosis Expression Research Articles

MiR‐486a‐5p Exerts Cardioprotection Against Ischemia Repefusion via Suppression of PDCD4

Extracellular vesicle (EV) derived from mesenchymal stem cells (MSCs) have been demonstrated to be a critical component for cardioprotection against myocardial infarction triggered by stem cell therapy. EVs are nano‐size phospholipid bilayer particles that carry proteins, lipids, RNA, including microRNAs (miRs). Because EVs can deliver their cargo to other cells, they hold potential application for development as clinical therapeutics. Herein we tested the hypothesis that a specific MSC‐EV miR, miR‐486a‐5p, mediates some aspect of cardioprotective effect against ischemia/reperfusion injury (I/R).Extracellular vesicles were isolated from B6/129‐derived MSCs using differential ultracentrifugation method. To characterize EV RNA, total RNA was isolated from MSC cells and EVs and used to synthesize libraries for small RNA deep sequencing. EVs and cell miRs were ranked based on enrichment in EV RNA (EV/cell levels). We found that miR‐486a‐5p is the most highly enriched miR in MSC‐derived EV RNA at level of 5853‐fold higher than in cells (Padj=1.52E‐59). The differential level of miR‐486‐5p between EVs and cell was validated by Taqman Advanced small RNA qRT‐PCR. We searched for miR‐486a‐5p gene targets using Targetscan7.0 and found 64 gene targets involving in cell death and survival pathways (Pathway Studio). Programmed cell death 4 (PDCD4) was chosen for further study based on weighted accumulative score++ (Targetscan7.0). To test whether miR‐486 can directly target PDCD4, luciferase assay was performed using a reporter vector containing PDCD4 3′‐UTR. 3′‐UTR of PTEN, a known target of miR‐486 was used as a control. When H9c2 cells transfected with the reporter vector were incubated with a range of miR‐486 mimic doses, luciferase activity was significantly reduced in a dose‐specific manner. Next, we determined whether transfection of H9c2 cells with miR‐486 mimic affected PDCD4 expression. The mimic transfection was efficient with a 200‐fold expression of miR‐486 mimic relative to the control. Whereas miR‐486 mimic transfection did not influence the expression of PDCD4 mRNA, PDCD4 protein was significantly suppressed (~60% downregulation, P<0.001 vs. control).In H9c2 cardiomyocytes, in vitro experiments showed that treatment with miR‐486 mimic did not change cell survival in normoxic condition. However, treatment with 50 and 100 nM of mimic significantly reduced I/R mediated injury (increased cell viability (Hoechst‐PPI assay) and decreased lactate dehydrogenase leakage (P<0.01 vs. control) after simulated I/R (12h hypoxia with ischemia mimetic solution and 2h reperfusion). Moreover, TUNEL assay revealed miR‐486 mimic treatment significantly suppressed apoptosis. Western blotting analysis of cell lysates after simulated I/R showed that improved I/R outcome was associated with the inhibition of PDCD4 protein levels, and levels of its downstream effectors, cleaved caspase‐3 and Bax. In conclusion, these findings indicate that miR‐486‐5p can ameliorate I/R mediated damage through suppression of PDCD4 protein levels and inhibition of apoptosis in cardiomyocytes. Whether miR‐486a‐5p contributes to exosome‐mediated cardioprotection in vivo will be determined using our in vivo I/R model of coronary occlusion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiRNA‑26a‑5p and miR‑26b‑5p inhibit the proliferation of bladder cancer cells by regulating PDCD10

MicroRNA (miR)‑26a‑5p and miR‑26b‑5p consistently play an antitumor role in many types of cancers, but the underlying mechanism remains unclear in bladder cancer (BC). In the present study, we found that, in BC tissues, the levels of miR‑26a‑5p and miR‑26b‑5p were lower than in paired normal tissues. The upregulation of miR‑26‑5p significantly inhibited the proliferation of BC cell lines (T24 and 5637). Bioinformatics analysis indicated that Programmed Cell Death10 (PDCD10) was the downstream target gene of miR‑26a‑5p/miR‑26b‑5p, and this was ascertained by western blotting and quantitative real‑time reverse transcription PCR (RT‑qPCR). In addition, in the 3'‑UTR of PDCD10, the binding site was identified using a luciferase reporter assay. We determined that clinical BC tissues presented higher PDCD10 levels than adjacent normal tissues and that PDCD10 promoted proliferation of BC cell lines. Overexpression of miR‑26a‑5p/miR‑26b‑5p inhibited the stimulatory effect on proliferation of BC cells induced by PDCD10. In addition, in vivo experiments and clinical data revealed that the prognosis of BC patients with high expression of miR‑26a‑5p/miR‑26b‑5p and low expression of PDCD10 was better than that of patients with low miR‑26‑5p and high PDCD10 expression. These data revealed that miR‑26a‑5p and miR‑26b‑5p were pivotal regulators in BC progression by targeting the proliferation‑related protein, PDCD10. The miR‑26‑5p/PDCD10 interaction may provide important insight into the pathway of BC progression and present novel opportunities for future diagnosis and treatment strategies, especially for patients with high levels of PDCD10.

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.

Long non-coding RNA CASC15 promotes melanoma progression by epigenetically regulating PDCD4

BackgroundLong non-coding RNAs (LncRNAs) have been identified as critical regulators in a variety of cancer types. Cancer susceptibility candidate 15 (CASC15), a lncRNA located at chromosome 6p22.3, has been discovered to participate in melanoma progression and phenotype switching. Nevertheless, the roles and molecular mechanisms of CASC15 in melanoma are far from being understood.ResultsWe found that CASC15 expression was up-regulated in melanoma tissues and associated with advanced pathological stages. Function experiments displayed that CASC15 knockdown hindered proliferation, facilitated apoptosis and suppressed invasion, while CASC15 overexpression facilitated proliferation and invasion in melanoma cells. Further mechanistic analysis showed that CASC15 epigenetically silenced the expression of programmed cell death 4 (PDCD4) by recruiting EZH2 and increasing H3K27me3 level at the promoter region of PDCD4. Additionally, PDCD4 overexpression inhibited proliferation, enhanced apoptosis and decreased invasion of melanoma cells. Moreover, CASC15-knockdown-induced anti-cancer effects were abated by PDCD4 down-regulation. Furthermore, depletion of CASC15 blocked tumor growth of melanoma by up-regulating PDCD4 in vivo.ConclusionsCASC15 acts as an oncogene by negatively regulating PDCD4 expression via recruiting EZH2 and subsequently increasing H3K27me3 level. Together, our study indicates that CASC15/EZH2/PDCD4 may serve as a promising therapeutic target for melanoma intervention.

Clinical significance of decreased programmed cell death 4 expression in patients with giant cell tumors of the bone

Programmed cell death 4 (PDCD4) has been recognized as a novel tumor suppressor gene, which inhibits the activation and translation of activator protein (AP)-1. Dysregulated expression of PDCD4 is also involved in various human tumors and is linked to tumor progression and development. However, the function and clinical implication of PDCD4 in giant cell tumors of the bone (GCTBs) has not been previously investigated. In the present study, PDCD4 expression was determined in 83 samples of GCTBs at mRNA and protein levels by quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemistry. The results demonstrated that PDCD4 mRNA expression was reduced in 63% of GCTB samples (17/27) and protein expression was decreased in 65% of samples (54/83), compared with adjacent non-tumor tissues. Furthermore, decreased expression of PDCD4 was significantly associated with certain clinicopathological characteristics, including the Campanacci grade and recurrence. A strong negative correlation was determined between PDCD4 expression and the Ki-67 positive rate in GCTBs (r=−0.6392; P<0.001). The results of the present study suggest that PDCD4 may serve a role in the malignant progression of human GCTBs and may be an important prediction factor for prognosis.

Linc00472 suppresses proliferation and promotes apoptosis through elevating PDCD4 expression by sponging miR-196a in colorectal cancer.

Long intergenic non-coding RNA Linc00472 has been considered as a tumor suppressor in some cancers. However, the function and mechanism of Linc00472 in colorectal cancer has not been well elucidated. In this study, we found that Linc00472 was down-regulated in colorectal cancer tissues and cells. Elevated Linc00472 expression suppressed proliferation and induced apoptosis in colorectal cancer cells. Moreover, Linc00472 acted as a competing endogenous RNA (ceRNA) of miR-196a to release programmed cell death 4 (PDCD4). Furthermore, miR-196a overexpression or PDCD4 knockdown reversed Linc00472-mediated proliferation inhibition and apoptosis induction in colorectal cancer cells. Ectopic Linc00472 expression hindered tumor growth in vivo. Our study demonstrated that Linc00472 suppressed proliferation and induced apoptosis through up-regulating PDCD4 by decoying miR-196a, which may be an effective therapeutic target for colorectal cancer.

Expression of programmed cell death 1 receptor and its downstream cytokines in peripheral blood mononuclear cells of patients with generalized pustular psoriasis

Objective To determine the expression of programmed cell death 1 receptor (PDCD1) , its ligands and downstream nuclear factor kappa B (NF-κB) in peripheral blood mononuclear cells (PBMC) of patients with generalized pustular psoriasis (GPP) . Methods The total RNA was extracted from 9 GPP patients and 10 healthy controls separately by using Trizol, and reverse transcription PCR (RT-PCR) was performed to determine the mRNA expression of PDCD1, programmed cell death 1 ligand 1 (PD-L1) , PD-L2 and NF-κB. Enzyme-linked immunosorbent assay (ELISA) was conducted to the serum levels of interleukin-27 (IL-27) , IL-22 and IL-4 in the peripheral blood of 23 GPP patients and 24 healthy controls. Results The mRNA expression of PDCD1 was significantly lower in the GPP group (3.13 ± 4.62) than in the healthy control group (23.70 ± 15.33, t= 3.40, P 0.05) . The GPP group also showed significantly higher levels of IL-27, IL-22 and IL-4 compared with the healthy control group (all P < 0.001) . Conclusion PDCD1 and its ligands were abnormally expressed in the PBMC of patients with GPP, and may play an important role in the occurrence and development of GPP. Key words: Psoriasis; Programmed cell death 1 receptor; Programmed cell death 1 ligand 2 protein; Antigens, CD274; NF-kappa B

PDCD4 suppresses proliferation, migration, and invasion of endometrial cells by inhibiting autophagy and NF-κB/MMP2/MMP9 signal pathway.

Endometriosis (EM) is a kind of estrogen-dependent disease in reproductive-age women. Ovarian EM is the most common type. Although EM is a benign disease, it shares many similar features with cancers. Programmed cell death 4 (PDCD4), a newly identified tumor suppressor, plays an important role in inhibiting tumorigenesis and tumor progression at the transcriptional and translational levels. To explore the roles of PDCD4 in EM, we detected the expression of PDCD4 in control endometrium and eutopic/ectopic endometrium of ovarian EM patients, and analyzed the effects of PDCD4 on the biological behaviors of endometrial cell lines and primary endometrial cells. The results demonstrated that PDCD4 was downregulated in eutopic and ectopic endometrium of EM patients compared with control endometrium. PDCD4 effectively inhibited the proliferation and colony-forming ability of endometrial cells maybe by inhibiting cell autophagy. In addition, PDCD4 also suppressed the migration and invasion ability of endometrial cells, the mechanism may be related to NF-κB/MMP2/MMP9 signal pathway. Taken together, these results suggest that PDCD4 could be involved in the pathogenesis of EM, and provide a novel approach to target the aberrant PDCD4 expression in EM.

Sipi soup inhibits cancer‑associated fibroblast activation and the inflammatory process by downregulating long non‑coding RNA HIPK1‑AS

Sipi soup (SPS), the aqueous extract derived from the root bark of Sophora japonical L, Salix babylonica L., Morus alba L., as well as Amygdalus davidiana (Carr.) C. de Vos, is a traditional Chinese medicine frequently used to prevent and treat infection and inflammation. However, the role of SPS in cancer-associated fibroblasts (CAFs) require further investigation. In the present study, the effects of SPS on fibroblast inactivation and the underlying mechanism were investigated. Reverse transcription-quantitative polymerase chain reaction was used to analyze the mRNA expression levels of fibroblast activation protein (FAP), interleukin (IL)-6, α-smooth muscle actin (α-SMA) and programmed cell death 4 (PDCD4). Flow cytometry was used to evaluate cell apoptosis. Immunofluorescence was used to determine the number of activated fibroblasts. The present study reported that SPS treatment did not affect the proliferative apoptotic potential of fibroblasts. Treatment with HeLa cell culture medium (CM) induced a significant increase in the expression levels of FAP, IL-6 and α-SMA, but reduced the expression of PDCD4. SPS reversed the effects of HeLa CM on the expression of these genes. Analysis with a long non-coding (lnc)RNA array of numerous differentially expressed lncRNAs revealed that the expression levels of the lncRNA homeodomain-interacting protein kinase 1 antisense RNA (HIPK1-AS) were increased in cervicitis tissues and cervical squamous cell carcinoma tissues compared with in normal cervical tissues. HIPK1-AS expression levels were upregulated in response to HeLa CM, but were decreased under SPS treatment. The downregulation of HIPK1-AS expression via short hairpin RNA abolished the effects of HeLa CM on the expression of inflammation-associated genes. The findings of the present study suggested that SPS may prevent the progression of cervical cancer by inhibiting the activation of CAF and the inflammatory process by reducing HIPK1-AS expression.

MiR-103 Functions as a Tumor Suppressor by Directly Targeting Programmed Cell Death 10 in NSCLC.

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases. Absence of miR-103 has recently been identified to be associated with metastatic capacity of primary lung tumors. However, the exact role of miR-103 in NSCLC and the molecular mechanism are unclear. In the present study, we showed that miR-103 expression was reduced in NSCLC tissues and cells. miR-103 expression was negatively correlated with tumor size and stage. The overall survival was longer in patients with higher miR-103 level than in those with lower miR-103 expression. miR-103 inhibited cell proliferation in A549 cells, decreased tumor weight and volume, and prolonged survival of tumor-implanted nude mice. miR-103 increased apoptotic cell death in A549 cells. Furthermore, miR-103 decreased the invasion and migration abilities in A549 cells, as evidenced by Transwell and wound healing results. Downregulation of miR-103 significantly reduced the level of programmed cell death 10 (PDCD10). We found a significant decrease in the relative luciferase activity of the reporter gene in A549 cells cotransfected with the miR-103 mimic and pGL3-PDCD10 WT 3′-UTR, but not pGL3-PDCD10 mut 3′-UTR. We showed that overexpression of PDCD10 significantly inhibited miR-103-induced inhibition of cell proliferation, increased apoptosis, and decreased invasion and migration in A549 cells. Moreover, we found that PDCD10 expression was increased in NSCLC tissues and cells. PDCD10 expression was positively correlated with tumor size and stage. Overexpression of PDCD10 increased cell proliferation and inhibited apoptosis in A549 cells. The data demonstrated that dysregulation of the miR-103/PDCD10 signal may be a novel therapeutic target for the treatment of NSCLC.

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.

MiR-21 over-expression and Programmed Cell Death 4 down-regulation features malignant pleural mesothelioma

BackgroundDifferential diagnosis between malignant pleural mesothelioma (MPM) and benign mesothelial conditions is still challenging and there is a lack of useful markers. Programmed cell death 4 (PDCD4) is a well-known tumor suppressor gene in several cancers, its post-transcriptional activity is directly controlled by miR-21, whose over-expression has been recently reported in MPM compared to normal mesothelium. Aim of this study was to test this suppressor gene as a possible new marker of malignant transformation in mesothelial cells, as well as a new prognostic marker.MethodsPDCD4 nuclear expression was assessed by immunohistochemistry (IHC) in 40 non-neoplastic pleural (NNP) and 40 MPM formalin-fixed and paraffin-embedded specimens. PDCD4 and miR-21 expressions were analyzed by qRT-PCR in all cases. In situ hybridization (ISH) of miR-21 was performed in 5 representative cases of both groups. The prognostic relevance of PDCD4 was assessed in a public available gene expression dataset.ResultsIHC showed that PDCD4 nuclear expression was significantly lower in MPM than in NNP. PDCD4 was down-regulated, whereas miR-21 was over-expressed in MPM cases compared to NNP ones. ISH detected miR-21 only in MPM specimens. Down-expression of PDCD4 was found significantly associated with short overall survival in publicly available data.ConclusionsThese findings highlighted a switch between PDCD4 and miR-21 expression in MPM. Further studies should assess the diagnostic reliability of these two markers for MPM in biopsy and effusion specimens.

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.

Dual function of programmed cell death 10 (PDCD10) in drug resistance

Drug resistance, a major challenge in cancer chemotherapy, is a result of several mechanistic alterations including resistance to apoptosis. Apoptosis is a well-controlled cell death mechanism which is regulated by several signaling pathways. Alterations in structure, function, and expression pattern of the proteins involved in the regulation of apoptosis have been linked to drug resistance. Programmed Cell Death 10 (PDCD10) protein is recently associated with the regulation of cell survival and apoptosis. However, the role of PDCD10 in drug resistance has not been clearly established. Here, we aimed to figure out the role of PDCD10 in resistance to anti-cancer agents in different cell lines. We found that PDCD10 expression was cell- and anti-cancer agent-specific; down-regulated in doxorubicin- and docetaxel-resistant MCF7 cells while up-regulated in doxorubicin-resistant HeLa cells. Down-regulation of PDCD10 expression by siRNA in parental MCF7 cells increased the resistance while it increased sensitivity in doxorubicin-resistant HeLa cells. Similarly, over-expression of PDCD10 in parental HeLa cells increased the resistance to doxorubicin while it re-sensitized doxorubicin-resistant MCF7 cells. Moreover, the alterations in PDCD10 expression led to changes in caspase 3/7 activity and the levels of apoptosis-related genes. Our results point out a possible dual role of PDCD10 in drug resistance for the first time in the literature and emphasize PDCD10 as a novel target for reversal of drug resistance in cancer.

Thioridazine upregulates programmed cell death 4 to induce apoptosis in nasopharyngeal carcinoma through the PI3K/Akt signalling pathway.

Thioridazine (THZ) has been identified as a potential regulator of tumour progression, and programmed cell death 4 (PDCD4) has been reported as a novel tumour suppressor. This study aimed to investigate the link between PDCD4 and THZ in the regulation of nasopharyngeal cancer (NPC) cell proliferation. The effect of THZ on NPC cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Then, the involvement of apoptosis and cell cycle in the THZ-mediated regulation of cell viability was assessed by flow cytometry. Related mRNAs and proteins were subsequently examined by real-time PCR and western blot, respectively. After transfection with the PDCD4-siRNA, pGC-FU-GFP-PDCD4 vector and phosphoinositide 3-kinase (PI3K) inhibitor Ly294002, we investigated the antagonistic effects of THZ and PDCD4 on NPC-related protein expression. MTT assays showed that THZ treatment suppressed cell viability. THZ-treated cells were arrested at the G1/G0 phase and showed a significantly increased apoptotic fraction. Furthermore, PDCD4-siRNA antagonized THZ treatment and promoted NPC cell proliferation. Western blot analysis showed that PDCD4 overexpression or PI3K inhibition by LY294002 significantly reduced the expression of phospho-PI3K, phospho-Akt, phospho-mammalian target of rapamycin and phospho-p70s6k, but not their total protein levels. In conclusion, our findings show that THZ and PDCD4 exert antagonistic effects on NPC cell proliferation, probably through the PI3K/Akt pathway. Moreover, these results provide an insight into the mechanism by which THZ targets PDCD4 in NPC cell lines and suggest that the ectopic expression of PDCD4 is a potential therapeutic strategy.

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.

Association Between the Deletion Allele of Ins/Del Polymorphism (Rs145204276) in the Promoter Region of GAS5 with the Risk of Atherosclerosis

Background/Aims: LncRNA is a growth arrest-specific transcript 5 (GAS5) with tumor suppressor activities in some cancers, but its role in atherosclerosis is unclear. Methods: Bioinformatics algorithm analysis was utilized to search the target of GAS5 and miR-21, followed by luciferase assay to confirm these targets. Real-time PCR and western-blot were utilized to verify the connection among GAS5, miR-21 and Programmed cell death 4 (PDCD4). MTT assay and flow cytometry analysis were performed to explore the mechanism of GAS5 in the regulation of atherosclerosis. Results: GAS5 directly targets miR-21 and functions as a competing endogenous RNA to suppress miR-21 expression. We also observed that rs145204276 polymorphism, including INS/INS and DEL/DEL, on GAS5 promoter increased transcription activity of GAS5, but the presence of rs145204276 DEL/DEL allele significantly promoted the transcription of GAS5 promoter compared with rs145204276 INS/INS allele. PDCD4 was predicted as a direct target gene of miR-21 with a binding site on PDCD4 3’UTR. It was further confirmed by luciferase assay that miR-21 significantly reduced the luciferase activity of wild-type PDCD4 3’UTR but not that of mutant PDCD4 3’UTR. In addition, high glucose significantly inhibited the growth rate of EC genotyped as DEL/DEL or INS/ INS, and apparently promoted the apoptotic rate of either DEL/DEL or INS/INS genotype ECs. Furthermore, the effect of high glucose was stronger in the INS/INS group, while the expression of GAS5 was dramatically upregulated with the presence of GAS5 DEL/DEL, while GAS5 positively regulated PDCD4 expression via inhibiting miR-21 expression. GAS5 siRNA and miR-21 mimics significantly decreased GAS5 and PDCD4 expressions, and the inhibitory effects of GAS5 siRNA or miR-21 mimics on GAS5 and PDCD4 expressions in the INS/INS group was stronger. Moreover, GAS5 siRNA and miR-21 mimics remarkably triggered cells proliferation and suppressed cell apoptosis, and the inhibition effects of GAS5 siRNA or miR-21 mimics on either cell viability and apoptosis in the INS/INS group was stronger. In this study, we enrolled 1,306 subjects with or without atherosclerosis and found that the INS/DEL or DEL/DEL genotypes significantly decreased the risk of atherosclerosis compared with the ins/ins genotype (adjusted odds ratio: 0.74 and 0.40, respectively). Conclusion: In summary, rs145204276 was associated with the risk of atherosclerosis by affecting the proliferation and apoptosis of endothelial cells via regulating the GAS5/miR-21/PDCD4 signaling pathway.

Effect of microRNA-21 on proliferation and regulation expression of programmed cell death 4 in prostate cancer cell PC-3

Objective To explore effect of microRNA (miRNA, miR)-21 on proliferation and regulation expression of programmed cell death 4 (PDCD4) in prostate cancer cell PC-3. Methods Expression of miR-21 in LNCap, PC-3, DU145 prostate cancer cell and human normal prostate epithelial cell RWPE-1 was examed by reverse transcriptase-polymerase chain reaction (RT-PCR) method. miR-21 inhibitor and miR-21 NC were transfected into PC-3 cell by liposome Lipofectamine™ 2000. Cell viability was measured by MTT assay. Cell apoptosis and cell cycle was measured by flow cytometry. The expression of PDCD4 protein and mRNA was measured by Western blotting and RT-PCR. Results The expression of miR-21 in LNCap, PC-3, DU145 cell (0.36±0.03, 0.85±0.08, 1.09±0.10) was higher than that in RWPE-1 cell (0.17±0.02) (P=0.013, P=0.007, P=0.001). Compared with miR-21 NC, the expression of miR-21 was down-regulated (0.20±0.02 vs. 1.07±0.10) (P=0.006), cell viability (0.37±0.04) vs. (0.75±0.07) was reduced (P=0.006), cell early apoptotic rate [(20.40±1.97)% vs. (2.53±0.24)%] and late apoptotic rate [(16.56±1.65)% vs. (2.53±0.24)%] was increased (P=0.002, P=0.003), cell cycle was arrested in the G1 phase [(59.58±3.26)% vs. (47.33±4.35)%, P=0.000], the expression of PDCD4 protein (1.67±0.16 vs. 0.25±0.02) and mRNA (1.02±0.10 vs. 0.24±0.02) was increased in miR-21 inhibitor group (P=0.005, P=0.007). Conclusion Down-regulation of miR-21 could inhibit the proliferation and cell cycle progression in PC-3 cells via up-regulation of expression of PDCD4. Key words: MicroRNA-21; Prostate cancer cell PC-3; Proliferation; Programmed cell death 4

MicroRNA‑206 contributes to the progression of steroid‑induced avascular necrosis of the femoral head by inducing osteoblast apoptosis by suppressing programmed cell death 4.

The expression of microRNA-206 (miR-206) is aberrantly induced in steroid-induced avascular necrosis of femoral head (SANFH). Therefore, investigating the function of miR-206 in SANFH and uncovering the functional mechanism associated with the condition will promote the understanding and treatment of the disease. The purpose of the present study was to investigate the pro-osteoclasteogenic effect of miR-206 that occurs through regulation of programmed cell death 4 (PDCD4). The expression of miR-206 and PDCD4 was analyzed in the clinical SANFH specimens. The level of miR-206 and PDCD4 was regulated in human osteoblast lineage hFOB1.19 and the effect of different treatments on cell viability, proliferation, apoptosis and differentiation potential of osteoblasts were analyzed with a Cell Counting kit-8, 5-ethynyl-2′-deoxyuridine staining, flow cytometry and Hoechst staining. The expression of miR-206 was upregulated while PDCD4 was downregulated in the SANFH specimens. Induced expression of miR-206 decreased cell viability and proliferation, while apoptosis was induced. At the molecular level, overexpression of miR-206 inhibited the expression of PDCD4, alkaline phosphatase (ALP) and B-cell lymphoma 2 (Bcl-2), and increased the expression of apoptosis regulator Bcl2-X-associated protein (Bax). Inhibiting the expression of miR-206 increased cell viability and proliferation but had no effect on cell apoptosis, as detected by flow cytometry and Hoechst staining. However, at the molecular level, inhibiting the expression of miR-206 induced expression of PDCD4, ALP and Bcl-2, while it decreased the expression of Bax. Additionally, knockdown of PDCD4 blocked the effect of miR-206 inhibition on hFOB1.19 cells, representing a PDCD4-dependent manner of miR-206 in inducing apoptosis of osteoblasts. Therefore, miR-206 promoted the onset of SANFH by inducing apoptosis and suppressed the proliferation of osteoblasts, which was dependent on the inhibition of PDCD4.