RESUMO
Objective To investigate the impact of long non-coding RNA(LncRNA)taurine up-regulated gene 1(TUG1)on high glucose-induced cardiomyocyte apoptosis by regulating miR-181b-5p/programmed cell death protein 4(PDCD4)axis.Methods Diabetic cardiomyopathy(DCM)cell model was established in vitro with high glucose(HG,25 mmol/L glucose).AC16 cells were divided into the NG(5.5 mmol/L glucose)group,the HG group,the HG+sh-NC group,the HG+sh-TUG1 group,the HG+miR-NC group,the HG+miR-181b-5p group,the HG+sh-TUG1+anti-miR-NC group,the HG+sh-TUG1+anti-miR-181b-5p group,the HG+miR-181b-5p+pcDNA group and HG+miR-181b-5p+pc-PDCD4 group.The Cell Counting Kit-8(CCK-8)method was applied to detect cell viability.Lactate dehydrogenase(LDH)assay was applied to detect LDH release.Quantitative real-time polymerase chain reaction(qRT-PCR)was applied to detect expression levels of TUG1,miR-181b-5p and PDCD4 mRNA.Flow cytometry was applied to detect apoptosis.Western blot assay was applied to detect levels of B-cell lymphoma 2-associated X(Bax),activated caspase 3(cleaved caspase 3)and PDCD4 proteins.Caspase-Glo3 assay was applied to assess caspase 3 activity.Dual-luciferase reporter assay was applied to verify the targeting relationship between TUG1 or PDCD4 and miR-181b-5p.Results Compared with the NG group,the cell activity decreased in the HG group,and LDH release,apoptosis rate,Bax,cleaved caspase 3 expression and caspase 3 activity increased(P＜0.05),which could be antagonized by TUG1 knockdown or miR-181b-5p overexpression(P＜0.05).Inhibition of miR-181b-5p was able to alleviate the impact of TUG1 silencing on cardiomyocyte viability and apoptosis under high glucose treatment(P＜0.05).The overexpression of PDCD4 attenuated the promotion effect of miR-181b-5p up-regulation on the viability of cardiomyocytes treated with high glucose and the inhibitory effect on apoptosis.TUG1 was able to increase the expression of PDCD4 through adsorption of miR-181b-5p(P＜0.05).Conclusion TUG1 promotes high glucose-induced cardiomyocyte apoptosis by down-regulating miR-181b-5p and up-regulating PDCD4.
RESUMO
Objective Gastric cancer is the most common cancer in the world. In China, Patients with gastric cancer are mostly treated with platinum-based chemotherapy. Programmed cell death 4 (PDCD4) was found as an important proapoptosis recently, the aim of the present study was to investigate the role of PDCD4 reversed the apoptosis induced by cisplatin in gastric cancer cell. The study will provide the target marker for treatment and diagnosis of cisplatin resistance in gastric cancer.Methods Stable transfection with pCMV-PDCD4 vector into human cisplatin resistance gastric cancer cell line-SGC7901/DDP; the cells were divided into control group, over-expression group, control with cisplatin group, over-expression with cisplatin group for following experiments. Hoechst dying with immunofluorescence and flow cytometry were used to measure the cell apoptosis in vitro; Real-time PCR was used to detect the mRNA expression levels of PDCD4, and the protein levels of PDCD4, pAK, pGSK3β, BCL-2 and Bak were detected by Western blot. The cells were divided into vector group, PDCD4 group, PDCD4 with activator group for detect the level of PARP(C) by Western blot.Results Compared with control group, the Results of real-time PCR and western blot were showed the level of PDCD4 was augmented in over-expression group (also in the over-expression with cisplatin group), which was indicated stable transfection with PDCD4 was successful. Immunofluorescence (with hoechst dying) and flow cytometry demonstrated that PDCD4 facilitated cell apoptosis exposed to cisplatin. PDCD4 overexpression attenuated the protein levels of pAkt, pGSK3β and BCL-2, but increased the protein levels of BAK. Furthermore, incubation with SC-79 (the activator of Akt) reversed cell apoptosis induced by PDCD4.Conclusion Overexpression of PDCD4 promotes the apoptosis induced by cisplatin through pAKT/pGSK3β pathway, which is favorable to reverse cisplatin resistance in gastric cancer.