Résumé
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.
Résumé
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.
Résumé
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.