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OBJECTIVE@#To explore the role of long non-coding RNA (lncRNA) X inactive specific transcript (XIST) in modulating cisplatin (DDP) resistance of human nasopharyngeal carcinoma cells and investigate the possible mechanism.@*METHODS@#Realtime PCR was performed to detect the expression of XIST in cisplatin-resistant human nasopharyngeal carcinoma cell line HNE1/DDP. The effects of up-regulation and down-regulation of XIST on DDP resistance, proliferation and apoptosis of HNE1/ DDP cells were assessed using MTT assay, EdU assay and flow cytometry. Western blotting was used to detect the changes in the expressions of programmed cell death 4 (PDCD4) and Fas ligand (Fas-L) proteins in the cells in response to up-regulation or down-regulation of XIST.@*RESULTS@#The expression of XIST was significantly up-regulated in HNE1/DDP cells in comparison with HNE1 cells (0.57±0.06 0.1±0.02, < 0.05). Down-regulation of XIST significantly decreased while up-regulation of XIST obviously increased DDP resistance of HNE1/DDP cells ( < 0.05). Down-regulation of XIST significantly reduced the proliferation (6.17 ± 1.93 16.59 ± 4.86, < 0.05) and enhanced apoptosis [(18.04 ± 4.72)% (4.22 ± 1.65)%, < 0.05], while upregulating XIST enhanced the proliferation (25.40±7.21 13.16±3.95, < 0.05) and inhibited apoptosis [(2.82±0.88)% (6.46± 1.75)%, < 0.05] in HNE1/DDP cells. Down-regulation of XIST significantly increased the protein expressions of PDCD4 and Fas-L ( < 0.05) in HNE1/DDP cells, and up-regulation of XIST resulted in reverse changes in PDCD4 and Fas-L expressions ( < 0.05).@*CONCLUSIONS@#XIST is up-regulated in HNE1/DDP cells, and down-regulation and up-regulation of XIST expression reduce and increase DDP resistance of the cells, respectively, possibly as a result of changes in the expressions of PDCD4 and Fas-L.
Subject(s)
Humans , Antineoplastic Agents , Apoptosis , Apoptosis Regulatory Proteins , Cell Line, Tumor , Cell Proliferation , Cisplatin , Drug Resistance, Neoplasm , Nasopharyngeal Carcinoma , RNA, Long Noncoding , RNA-Binding Proteins , fas ReceptorABSTRACT
Objective: To construct the rat Pdcd4 knockdown model of programmed cell death 4 (Pdcd 4) in vivo for further study of the gene's functions. Methods: E3 rats were intranasally instilled with saline or plasmids during antigen induced pulmonary inflammation model induction. The rats were anaesthetized and killed on day 21. The lungs in each group were lavaged by instillation and withdrawal of 2 mL ice-cold PBS for three times through the tracheal route, and the bronchoalveolar lavage fluid (BALF) was collected. After centrifugation, the BALF cell pellet was used to isolate the total RNA. And the mRNA level of Pdcd4 was assessed by real-time quantitative PCR. Results: Pdcd4 expression decreased significantly in BALF cells at the mRNA level after Pdcd4 RNAi in vivo. Conclusion: Rat Pdcd4 knockdown model in vivo was constructed successfully, which can lay foundation for further studies on the functions and mechanism of Pdcd4 gene.
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Objective To observe and analyze the expression of programmed cell death 4 (PDCD4) gene and apoptosis inhibitor Livin in triple negative breast cancer (TNBC) tissues and its relationship with prognosis.Methods One hundred cases of TNBC tumor tissue,50 cases of adjacent carcinoma tissue,50 cases of normal breast tissue were selected as the research data.The immunohistochemical technique was applied to detect and compare the expression positive rates of PDCD4 and Livin protein in three kinds of tissues.The patients were followed up.The overall survival (OS) and the progression free survival (PFS) were observed and compared.Results The expression positive rate of PDCD4 in TNBC tissue was significantly lower than that in adjacent carcinoma tissue or normal breast tissue,the differences were statistically significant (x2=26.613,32.000,P＜0.05).The expression was correlated with the clinical pathological features of tumor size,lymph node metastasis,clinical stage,axillary lymph node metastasis and cancer embolus (x2=26.936,13.210,22.774,27.463,5.803,P＜0.05);the expression positive rate of Livin protein in TNBC tissue was significantly higher than that in adjacent carcinoma tissue or normal breast tissue and the expression positive rate of Livin protein in adjacent carcinoma tissue was significantly higher than that in normal breast tissue,the differences were statistically significant (x2 =14.614,57.353,19.048,P＜0.05).The expression was correlated with the clinical pathological features of lymph node metastasis,clinical stage,axillary lymph node metastasis and cancer embolus (x2 =10.788,6.160,27.350,8.914,P＜0.05);OS,PFS in the patients with PDCD4 negative expression were significantly lower than those in the patients with PDCD4positive expression.OS,PFS in the patients with Livin positive expression were significantly lower than those in the patients with Livin negative expression,the above differences were statistically significant (x2 =23.931,19.163,22.649,17.213,P＜0.05).OS in the TNBC patients was correlated with age (RR=1.405),clinical stage (RR =2.897),tumor diameter (RR=2.722),axillary lymph node metastasis (RR=2.516),vascular invasion (RR=3.020),PDCD4 Expression (RR=1.752) and Livin expression (RR=2.051) (P＜0.05).PFS in the patients was correlated with clinical stage (RR =2.756),axillary lymph node metastasis (RR =2.437),PDCD4 expression (RR =1.649) and Livin expression (RR=1.804) (P＜0.05).Conclusion The PDCD4 low expression and Livin protein over-expression exist in TNBC tissues.Their abnormal expressions are correlated with the clinicopathological features of tumor and the prognosis of patient,and could be used as the auxiliary indexes in evaluation of progression and prognosis of TNBC.
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Objective To investigate the role of nicorandil pretreatment on protecting myocardium after coronary microembolization (CME) and on the PDCD4/NF-κB/TNF-α signaling pathway in miniature pigs. Methods Fifteen Bama miniature pigs were randomly(random number) divided into the sham operation group (sham group), microembolization group (CME group) and CME plus nicorandil group, with 5 pigs in each group. The CME model was constructed by injecting polyethylene microspheres via microcatheter into the left anterior descending artery, and pigs in the sham group were injected with the same amount of saline. Pigs in the CME plus nicorandil group were injected intravenously with nicorandil (150 μg/kg) via ear vein 30 min before CME. Cardiac function indexes were measured using cardiac ultrasonography. The expression of PDCD4 and TNF-α mRNA in myocardium were detected by fluorescence quantitative PCR, and the protein expression of PDCD4 and TNF-α in myocardium were detected by Western blotting. NF-κB activation was evaluated by electrophoretic mobility shift assay. Results (1) Cardiac function was significantly lower and the level of serum cTnI was significantly higher in the CME group compared with the sham group. CME reduced myocardial systolic dysfunction and left ventricular dilatation. The CME plus nicorandil group showed improved CME-induced cardiac function and reduced serum cTnI level when compared with the CME Group (P < 0.05). (2) Compared with the CME group, the CME plus nicorandil group showed lower PDCD4 and TNF-α expression and NF-κB activity as well as improved cardiac function (P < 0.05). Conclusions The pretreatment of nicorandil effectively reduced the myocardial damage caused by CME, mainly through inhibiting the PDCD4/NF-κB/TNF-α pathway in cardiomyocytes.
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Programmed cell death 4 (PDCD4) is a newly discovered tumor suppressor gene, whose expression is regulated by methylation of PDCD4 gene and many kinds of microRNAs.Protein of PDCD4 enco-ding can improve the sensitivity of tumor cells to anticancer agents, inhibit tumor development and metastasis process, and play an important role in a variety of tumors.It is expected to be a clinical indicator to determine the prognosis of tumor.
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Objective To investigate the effect and mechanism of programmed cell death 4 ( PDCD4 ) on radiosensitivity of pancreatic cancer cells. Methods Pancreatic cancer tissues and corresponding adjacent tissues were collected, the expression level of PDCD4 was detected by RT-PCR and Western blot. Human pancreatic cancer cells Sw1990 were transfected with PDCD4 overexpression vector ( group pIRES2-PDCD4 ) , empty vector ( pIRES2 group ) , and treated with transfection reagent, respectively. The expression level of PDCD4 was detected by RT-PCR and Western blot. After radiation treatment, cell apoptosis was detected by flow cytometry, cell survival was detected by clone assay, and the expression levels ofβ-catenin, c-myc and Cleaved Caspase-3 were detected by Western blot. Results The expression of PDCD4 mRNA and protein in pancreatic cancer tissues was significantly lower than that in adjacent tissues (t=4. 869, 9. 208, P<0. 05). The expression of PDCD4 mRNA and protein in pIRES2-PDCD4 group was significantly lower than that in the non-transfection group ( t =9. 074, 18. 927, P <0. 05). After radiation, the apoptosis rate and Cleaved Caspase-3 level in the pIRES2-PDCD4 group were significantly higher than those in the non-transfection group (t =3. 670, 4. 086, P <0. 05), while the expression levels of β-catenin and c-myc in the cells were significantly lower than those in the non-transfection group (t =9. 242, 17. 644, P <0. 05). The radiosensitivity of pIRES2-PDCD4 group was higher than that of non-transfection group, and the sensitization ratio was 1. 843. Conclusions PDCD4 can increase radiosensitivity and promote apoptosis of pancreatic cancer cells, to which the Wnt signaling pathway may be related.
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Objective To investigate the expression of miR-21 in renal clear cell carcinoma and its clinical significance as well as how miR-21 regulates the proliferation and apoptosis of 786-O renal clear cell carcinoma cell line through regulating programmed cell death 4 (PDCD4).Methods By analyzing the data of renal clear cells cancer in The Cancer Genome Atlas (TCGA)database,we compared the expression of miR-21 in renal cancer tissues and adjacent normal tissues and explored the differences in miR-21 level in renal cancer at different clinicopathological stage,T stage,N stage and M stage.We also analyzed the association between miR-21 level and survival of patients by Kaplan-Meier method and Log-rank test.786-O cells were transfected with AS-miR-21 to deplete miR-21. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis, respectively.We then measured the mRNA and protein levels of PDCD4 in 786-O cells depleted for miR-21 by qRT-PCR and Western blot,respectively,and performed a dual-luciferase assay to detect the direct regulation of PDCD4 by miR-21.Results Expression of miR-21 was significantly higher in renal cancer tissues than in adjacent tissues(P <0.0001).The expression levels of miR-21 at stage Ⅲ and stage Ⅳ renal cancer were significantly higher than that at stage Ⅰ (both P <0.0001).Moreover,miR-21 expression was positively correlated with clinicopathological stages of renal cancer by correlation analysis (r =0.262,P <0.0001 ).The correlation test indicated that miR-21 level was also positively correlated with T stage of renal cancer (r =0.250,P <0.0001 ),lymph node metastasis (N1)and distant metastasis (all P <0.0002).Patients with high miR-21 expression had significantly shorter median survival time than those with low miR-21 expression (Log-rank P < 0.001 ).Compared with control cells,786-O cells depleted for miR-21 showed significantly decreased cell proliferation (P <0.05 )and increased cell apoptosis rate (P =0.005 ).PDCD4 mRNA (P = 0.002 )and protein levels were significantly elevated in 786-O cells with down-regulated miR-21 levels.In addition,the dual-luciferase reporter assay showed that the relative luciferase intensity of PDCD4 reporter in cells transfected with AS-miR-21 was significantly higher than that of control cells (P =0.003).Conclusion miR-21 expression was up-regulated in renal cancer and correlated with clinicopathological stage and survival of patients.miR-21 promoted 786-O cell proliferation and inhibited apoptosis probably through regulating PDCD4 expression. These results indicate that miR-21 plays an important role in formation and development of renal cancer.
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Objective To investigate the expression of miR-21 in renal clear cell carcinoma and its clinical significance as well as how miR-21 regulates the proliferation and apoptosis of 786-O renal clear cell carcinoma cell line through regulating programmed cell death 4 (PDCD4).Methods By analyzing the data of renal clear cells cancer in The Cancer Genome Atlas (TCGA)database,we compared the expression of miR-21 in renal cancer tissues and adjacent normal tissues and explored the differences in miR-21 level in renal cancer at different clinicopathological stage,T stage,N stage and M stage.We also analyzed the association between miR-21 level and survival of patients by Kaplan-Meier method and Log-rank test.786-O cells were transfected with AS-miR-21 to deplete miR-21. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis, respectively.We then measured the mRNA and protein levels of PDCD4 in 786-O cells depleted for miR-21 by qRT-PCR and Western blot,respectively,and performed a dual-luciferase assay to detect the direct regulation of PDCD4 by miR-21.Results Expression of miR-21 was significantly higher in renal cancer tissues than in adjacent tissues(P <0.0001).The expression levels of miR-21 at stage Ⅲ and stage Ⅳ renal cancer were significantly higher than that at stage Ⅰ (both P <0.0001).Moreover,miR-21 expression was positively correlated with clinicopathological stages of renal cancer by correlation analysis (r =0.262,P <0.0001 ).The correlation test indicated that miR-21 level was also positively correlated with T stage of renal cancer (r =0.250,P <0.0001 ),lymph node metastasis (N1)and distant metastasis (all P <0.0002).Patients with high miR-21 expression had significantly shorter median survival time than those with low miR-21 expression (Log-rank P < 0.001 ).Compared with control cells,786-O cells depleted for miR-21 showed significantly decreased cell proliferation (P <0.05 )and increased cell apoptosis rate (P =0.005 ).PDCD4 mRNA (P = 0.002 )and protein levels were significantly elevated in 786-O cells with down-regulated miR-21 levels.In addition,the dual-luciferase reporter assay showed that the relative luciferase intensity of PDCD4 reporter in cells transfected with AS-miR-21 was significantly higher than that of control cells (P =0.003).Conclusion miR-21 expression was up-regulated in renal cancer and correlated with clinicopathological stage and survival of patients.miR-21 promoted 786-O cell proliferation and inhibited apoptosis probably through regulating PDCD4 expression. These results indicate that miR-21 plays an important role in formation and development of renal cancer.
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OBJECTIVE@#To study the inhibition effect of miR-106a inhibitor on tumor growth of ovarian cancer xenografts mice.@*METHODS@#BALB/c mice were selected as experimental animals, ovarian cancer SKOV-3 cells transfected with miR-106a inhibitor and its negative control were inoculated subcutaneously, intratumoral injection of miR-106a inhibitor and its negative control were continued after tumor formation, and they were enrolled as treatment group and model group, respectively. Tumor volume and weight as well as Ki-67 and programmed cell death 4 (PDCD4) expression were determined; miR-106a inhibitor and its negative control as well as miR-106a mimic and its negative control were transfected into SKOV-3 cells, and expression of PDCD4 in cells was determined.@*RESULTS@#Tumor tissue volume and weight as well as mRNA expression and protein expression of Ki-67 in treatment group were significantly lower than those in the model group while mRNA expression and protein expression of PDCD4 were significantly higher than those in the model group; transfection of miR-106a mimic could decrease mRNA expression and protein expression of PDCD4 in SKOV-3 cells, and transfection of miR-106a inhibitor could increase mRNA expression and protein expression of PDCD4 in SKOV-3 cells.@*CONCLUSIONS@#Transfection of miR-106a inhibitor can inhibit the growth of tumor in ovarian cancer xenografts mice through increasing the expression of PDCD4.
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Objective To study the inhibition effect of miR-106a inhibitor on tumor growth of ovarian cancer xenografts mice. Methods BALB/c mice were selected as experimental animals, ovarian cancer SKOV-3 cells transfected with miR-106a inhibitor and its negative control were inoculated subcutaneously, intratumoral injection of miR-106a inhibitor and its negative control were continued after tumor formation, and they were enrolled as treatment group and model group, respectively. Tumor volume and weight as well as Ki-67 and programmed cell death 4 (PDCD4) expression were determined; miR-106a inhibitor and its negative control as well as miR-106a mimic and its negative control were transfected into SKOV-3 cells, and expression of PDCD4 in cells was determined. Results Tumor tissue volume and weight as well as mRNA expression and protein expression of Ki-67 in treatment group were significantly lower than those in the model group while mRNA expression and protein expression of PDCD4 were significantly higher than those in the model group; transfection of miR-106a mimic could decrease mRNA expression and protein expression of PDCD4 in SKOV-3 cells, and transfection of miR-106a inhibitor could increase mRNA expression and protein expression of PDCD4 in SKOV-3 cells. Conclusions Transfection of miR-106a inhibitor can inhibit the growth of tumor in ovarian cancer xenografts mice through increasing the expression of PDCD4.
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Objective To investigate the expression of DNM T1 and PDCD4 in Tongue Squamous cell carcinoma and their signif‐icance to Clinical .Methods The protein expression of DNM T1 and PDCD4 in 40 cases of TSCC tissues and The adjacent no tumor tissues were measured by Elivision Two Step immunohistochemical method(IHC) ,the relationship between DNM T1 ,PDCD4 and clinicopathological parameters were analyzed .Results There was positively over expression of DNM T1 while the expression of PD‐CD4 was at a low lever or lost in TSCC tissues ,the expression of two genes DNM T1 and PDCD4 in the adjacent no tumor tissues were in contrast ;there was a significant negative correlation between DNM T1 and PDCD4 expression in TSCC(r = - 0 .452 ,P<0 .05) .The expression of DNM T1 protein were associated with histopathological differentiation types ,regional lymph node metasta‐sis and TNM staging(P< 0 .05) ,it had nothing to do with age and gender ;the expression of PDCD4 protein were associated with regional lymph node metastasis and TNM staging(P< 0 .05) ,and it had nothing to do with age ,gender ,histopathological differenti‐ation types .Conclusion Implying the abnormal expression of DNM T1 and PDCD4 protein might be closely related to the develop‐ment and progression of TSCC .DNM T1 may be participates in the inactivation of PDCD4 expression ,and led to the development of the cancer at last .
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Objective To investigate the influence of atorvastatin (Lipitor) on the expression of programmed cell death 4 (PDCD4) in human CD4+T lymphocytes in vitro. Methods Human CD4+T cells obtained from healthy individuals were activated with PHA and treated with atorvastatin. The mRNA and protein expression levels of PDCD4 were detected by real-time PCR and western-blot respectively. Results The stimulation of PHA obviously increased the mRNA and protein expression of PDCD4 and the secretion of those serum cytokines. The expression of PDCD4 and the production of serum TNF-α were significantly decreased, whereas the serum levels of IL-10 were significantly increased after treated by different concentration of atorvastatin. The serum secretion of TNF-α was positive correlation with the expression of PDCD4 through the linear related analysis (r=0.782, P<0.01), and the secretion of IL-10 was negative correlation with the expression of PDCD4 (r=-0.653, P<0.05). Conclusion The anti-inflammatory effects of atorvastatin are mediated by down regulating the expression of PDCD4 in CD4+T cells.
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Objective To investigate the relationship of programmed cell death 4(PDCD4) with the invasion of astrocytic glio-mas .Methods Using the immunohistochemical method to detect the expression of PDCD4 in astrocytic gliomas in different grades . Measuring the peritumoral low-density area on MRI scan ,then compared with the results of immunohistochemical expression .Re-sults The downregulation of PDCD4 was with the increasing of the malignant grade of astrocytic gliomas .The tumor grade malig-nancy was positively correlated with the grade of the peritumoral low-density area on MRI scan(P<0 .05) ,while the expression of PDCD4 was negatively correlated with the grade of astrocytic gliomas (P<0 .01) .Conclusion PDCD4 might serve as one of the in-dicators of invasion and malignant phenotype for astrocytic gliomas .
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Objective To study the relationship between the expression of cytochrome c ( Cyt c) and programmed cell death 4 (PDCD4) in pancreatic cancer, and investigate the pathway of PDCD4 inducing the apoptosis of pancreatic cancer cells. Methods Pancreatic cancer specimens from 69 patients who received pancreatic resection from 1990 to 2002 in First Affiliated Hospital of China Medical University were collected. The expression of Cyt c in the 69 paraffin specimens of pancreatic cancer was detected by immunohistochemistry, and the expression of Cyt c in 8 samples of cold-preserved fresh pancreatic cancer and normal pancreatic tissues were detected by Western blot. The expression of PDCD4 and Cyt c in pancreatic cancer was analyzed by paired t test and chi-square test. Results Compared with normal pancreatic tissues, the expression of Cyt c in pancreatic cancer was significantly decreased. The positive expression rate of Cyt c in 69 samples of pancreatic cancer was 41% (28/69). The expression of Cyt c was positive in most patients with positive expression of PDCD4, and the expression of PDCD4 was negative in most patients with negative expression of Cyt c. The expression of PDCD4 and Cyt c was closely correlated with each other (χ2= 10.52, P < 0.05). Conclusions There is a close relationship between the expression of PDCD4 and Cyt c in pancreatic cancer. PDCD4 may induce the apoptosis of pancreatic cancer cells through mitochondrial pathway.
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Objective:To establish a glioma cell line U251 stably expressing programmed cell death 4(PDCD4)gene,and to observe the influence of exogenous PDCD4 gene on the proliferation and cell cycle of U251 cells.Methods:Recombinant eukaryotic expression vector pEGFP-PDCD4 was transfected into human glioma cell line U251 by Lipofectamine 2000,and the U251 cells stably expressing PDCD4 were established by G418 selection.Reverse transcription polymerase chain reaction(RT-PCR)and Western blotting were employed to detect the expression of PDCD4 mRNA and protein.Furthermore,cell proliferation and colony forming ability were determined by cell counting and colony formation assay;the cell cycle was detected by FACS.Results:High expression of PDCD4 mRNA and protein was observed in U251 cells transfected with pEGFP-PDCD4,whereas no PDCD4 mRNA and protein expression was detected in the non-transfected and vector-transfected cells.Further,cells transfected with pEGFP-PDCD4 grew more slowly and had lower colony formation rate than cells of the other two control groups(P